Introduction: Hypogammaglobulinemia is common in patients with chronic lymphocytic leukemia (CLL). Per consensus guidelines, Intravenous Immunoglobulin (IVIG) prophylaxis is generally reserved for patients who have had recurrent, serious infections (e.g., those requiring IV antibiotics or hospitalization) and who also have serum IgG < 500 mg/dL. While prior studies demonstrated reduced infections with IVIG without survival benefit, these were conducted prior to the era of contemporary CLL therapies. We conducted an analysis of infectious outcomes in CLL patients who received IVIG between 2005 and 2022. Methods: This was a retrospective cohort study of CLL patients at a single institution. Patients who consented to CLL research were identified based on receipt of IVIG. Patients were excluded if they received IVIG for alternative indications (e.g., autoimmune hemolytic anemia). Infection frequency and severity were assessed during the year prior to and the year following IVIG initiation. Additional clinical parameters, including treatment history, concurrent therapy during IVIG use, and baseline immunoglobulin levels were also collected. Results: Of 79 patients identified with IVIG treatment, 48 met inclusion criteria of IVIG use for hypogammaglobulinemia and infections, based on charts available for close review. The median age at IVIG initiation was 58 years, with an average time from CLL diagnosis to IVIG initiation of 10.5 years. At initiation, 52% had Rai stage 0-II disease and 48% had Rai stage III - IV disease. Baseline mean IgG prior to IVIG was 411 mg/dL (range 93 – 894 mg/dL) and baseline mean IgA levels was 42 mg/dL (range 4 - 177 mg/dL). Pre-IVIG: 73% of patients experienced at least one grade 2 or 3 infection in the year prior to initiation of IVIG. Most patients had recurrent infections; the average number of grade 2 or higher infections was 1.6 (range 0 - 4). Two patients had a grade 3 infection in the year prior to IVIG initiation, though notably 15 others had prior grade 3 infections since they were diagnosed with CLL. In total, 17 patients (35%) had a grade 3 infection at any time prior to IVIG. Post-IVIG: Only 12 patients (25%) experienced a grade 2 or higher infection in the year following IVIG initiation, with a mean of 0.375 infections (range 0 – 4). Two of these patients (4%) had grade 3 infections, which were multifocal pneumonia and cellulitis. This represents a 66% relative risk reduction, and a Number Needed to Treat (NNT) of 2.1 to prevent grade 2 or higher infections in the year following IVIG initiation (RR 0.34, P = 0.0001, 95% CI: 0.20 - 0.58). No significant differences in prior CLL treatment (75% vs. 83%, p = 0.57), baseline IgG (p = 0.91) or IgA levels (p = 0.93) were observed between patients with and without post-IVIG infections. Conclusion: In this intra-patient pre/post analysis, IVIG prophylaxis significantly reduced both moderate (grade 2 or higher) infections, consistent with prior literature. While IVIG is recommended primarily for patients with severe infections (per 2025 NCCN guidelines), our findings suggest IVIG may also yield meaningful improvements in patient quality of life by reducing grade 2 infections. Future IVIG studies should incorporate quality of life metrics to define the role of IVIG in modern CLL care.

Receptor tyrosine kinase-like orphan receptors 1 and 2 (ROR1 and ROR2) are oncoembryonic antigens aberrantly expressed on hairy cell leukemia (HCL) B cells and various other cancers, but absent from healthy B cells and postpartum tissues. We generated novel, glycoengineered monoclonal antibodies—GE-zilovertamab (anti-ROR1) and GE-6E6 (anti-ROR2)—by removing core fucose residues from the Fc regions of their parental humanized IgG1 antibodies, zilovertamab (UC-961/cirmtuzumab) and 6E6, respectively. To assess their immunotherapeutic potential, we engineered the CD20+ B cell leukemia MEC1 cell line to stably express either ROR1 or ROR2, generating MEC1-ROR1 and MEC1-ROR2 cell lines. Co-culture of these targets with Jurkat-Lucia™ NFAT-CD16A (V158) reporter cells and antibody treatments revealed that glycoengineered antibodies (GE-zilovertamab and GE-6E6), but not their parental antibodies, robustly triggered luminescence activity in NFAT-CD16 cells in an antigen-specific manner. Chromium-51 (Cr51) release assays further demonstrated that GE-zilovertamab or GE-6E6, in the presence of human peripheral blood mononuclear cells (PBMCs), respectively induced potent and selective lysis of ROR1+ or ROR2+ MEC1 variants that was comparable to that of rituximab against CD20+ parental MEC1 cells, and consistently superior to the non-glycoengineered parental antibodies. These findings were extended to primary HCL cells, which express CD20, ROR1, and ROR2. GE-zilovertamab and GE-6E6 induced greater cytotoxicity against Cr51-labeled HCL cells than their parental antibodies and equaled or surpassed the efficacy of rituximab. Combination treatment with glycoengineered anti-ROR1 and anti-ROR2 antibodies mediated significantly higher killing of Cr51-labeled HCL cells than either agent alone, or rituximab, or the combination of the two parental antibodies. Importantly, in contrast to rituximab, GE-zilovertamab and GE-6E6 spared normal B cells, which lack expression of ROR1 and ROR2. In summary, our findings show that dual targeting with glycoengineered anti-ROR1 and anti-ROR2 antibodies achieves superior, selective ADCC against ROR1/2+ hairy cell leukemia, sparing normal B cells. This approach offers a promising and potentially safer immunotherapeutic strategy for patients with HCL or other ROR-expressing B-cell malignancy.

TPS7088 Background: Treatment options for patients with relapsed or refractory (R/R) chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) can be limited if patients do not respond to both Bruton tyrosine kinase inhibitors (BTKis) and B-cell lymphoma 2 inhibitors (BCL2is). Nemtabrutinib is a once-daily, potent, noncovalent, reversible BTKi with a distinct kinase profile that inhibits BTK and other B-cell receptor relevant kinases. The multicenter, open-label, single-arm, phase 2 BELLWAVE-003 study (NCT04728893) is designed to evaluate nemtabrutinib at the recommended phase 2 dose (RP2D) in participants with R/R CLL/SLL, Richter transformation, mantle cell lymphoma, marginal zone lymphoma, follicular lymphoma, and Waldenström macroglobulinemia. Cohort J will evaluate nemtabrutinib in participants with R/R CLL/SLL who are relapsed/refractory to both a BTKi and BCL2i. Methods: Key eligibility criteria for cohort J include participants aged ≥18 years with CLL/SLL whose disease is R/R to prior therapy with both a BTKi (covalent or irreversible) and a BCL2i, and an ECOG PS of 0 to 2. Additional use of noncovalent or reversible BTKis is permitted if disease is R/R to such therapy. Participants must have received and not responded to, been intolerant to, or determined by their treating physician to be a poor PI3Ki candidate or ineligible for PI3Ki per local (institution) guidelines. Exclusion criteria include prior exposure to nemtabrutinib, active CNS disease, and prior systemic therapy with a monoclonal antibody within 5 half-lives or 4 weeks before allocation. Overall, the BELLWAVE-003 study comprises a dose escalation and confirmation phase (part 1) to establish the RP2D, and a cohort expansion phase (part 2). Part 1 evaluated nemtabrutinib in ≥6 to ≤20 participants with R/R CLL/SLL after ≥2 prior lines of therapy. The RP2D has been established as nemtabrutinib 65 mg QD. In part 2, ~460 participants will be enrolled across 9 expansion cohorts. Approximately 40 participants will be enrolled in cohort J. Treatment will continue until unacceptable toxicity, disease progression, or withdrawal. Adverse events will be monitored throughout and graded using NCI CTCAE version 5.0. Hematologic toxicities in participants with CLL will be assessed using iwCLL 2018 criteria. CT/MRI and/or PET will be performed every 12 weeks unless needed more frequently. The primary end point for cohort J is ORR per iwCLL 2018 criteria by independent central review (ICR). Additional end points include DOR and PFS per iwCLL 2018 criteria by ICR, OS, and safety and tolerability. Recruitment is ongoing. This is the first clinical trial with a dedicated cohort to assess noncovalent BTKis in patients whose disease has failed to respond to both BTKi and BCL2i. Clinical trial information: NCT04728893 .

RNA splicing factor SF3B1 is one of the most recurrently mutated genes in chronic lymphocytic leukemia (CLL) and frequently co-occurs with chromosome 13q deletion [del(13q)]. This combination is associated with poor prognosis in CLL, suggesting these lesions increase CLL aggressiveness. While del(13q) in murine B cells (minimal deleted region of 13q14 includes DLEU1, DLEU2, and miR15a-16-1; Mdr mice), but not expression of Sf3b1-K700E, drives the initiation of CLL, we hypothesize that SF3B1 mutation accelerates CLL progression. In this study, we crossed mice with a B cell-specific Sf3b1-K700E allele with Mdr mice to determine the impact of Sf3b1 mutation on CLL progression. We found that the co-occurrence of these 2 lesions in murine B cells caused acceleration of CLL. We showed that Sf3b1-K700E impacted alternative RNA splicing of nuclear factor of activated T cells C1 (Nfatc1) and activated mTOR signaling and the MYC pathway, contributing to CLL acceleration. Moreover, concurrent inhibition of RNA splicing and the mTOR pathway led to cell death in vitro and in vivo in murine CLL cells with SF3B1 mutation and del(13q). Our results thus suggest that SF3B1 mutation contributes to the aggressiveness of CLL by activating the mTOR pathway through alternative splicing of Nfatc1, providing a rationale for targeting mTOR and RNA splicing in the subset of CLL patients with both SF3B1 mutations and del(13q).

7036 Background: First-line ibrutinib (Ibr) + venetoclax (Ven) treatment for CLL/SLL was tested in the phase 2 CAPTIVATE study, including minimal residual disease (MRD)–guided randomized discontinuation (MRD cohort) and Fixed Duration (FD) cohorts. We report final analysis results for patients (pts) treated with FD Ibr+Ven in the FD cohort and MRD cohort placebo arm. Methods: Pts ≤70 y with previously untreated CLL/SLL received 3 cycles of Ibr, then 12 cycles of Ibr+Ven (Ibr, 420 mg/d orally; Ven, 5-wk ramp up to 400 mg/d orally), up to 13 cycles in the MRD cohort placebo arm. On-study retreatment included single-agent Ibr; FD cohort pts with progressive disease (PD) >2 y after end of treatment (EOT) could be retreated with FD Ibr+Ven. Results: 202 pts completed FD Ibr+Ven (FD cohort, n=159; MRD cohort placebo arm, n=43). With median follow-up of 68.9 mo (range, 0.8–83.9), 5.5-y PFS and OS rates (95% CI) were 66% (58–72) and 97% (93–99), respectively. 5.5-y PFS rates (95% CI) in pts without and with del(17p)/mutated TP53 were 70% (62–76) and 36% (17–55), respectively. In pts with unmutated IGHV, 5.5-y PFS was 55% (45–64): 63% (49–74) in pts without, and 44% (28–60) in pts with, concomitant del(17p)/mutated TP53 /complex karyotype. The corresponding rates for pts with mutated IGHV were 79% (68–87), 85% (71–93), and 62% (34–81). Undetectable MRD (uMRD4; <10 –4 by flow cytometry) was achieved in peripheral blood (PB) in 54% of pts at C7 and 69% at EOT, and in bone marrow in 69% of pts at EOT. 5.5-y PFS rates (95% CI) were higher in pts with uMRD4 in PB at EOT (75% [67–82]) vs those with MRD (47% [33–59]). 64 pts had PD after completion of FD Ibr+Ven. 5.5-y freedom from next-line treatment was 73% (95% CI 66–79). Of 40 pts with available samples at PD to date, 1 had an acquired subclonal mutation in BCL2 of unclear significance (A113G, VAF 8.3%); none had acquired resistance-associated mutations in BTK or PLCG2 . 36 pts initiated retreatment with Ibr (n=25) or Ibr+Ven (n=11). With 28.4 mo median follow-up on Ibr retreatment (range, 3.7–59.1), ORR was 76% (best response: 1 CR; 1 nodular PR; 17 PR; 4 SD; 1 PD [Richter transformation]; 1 no assessment); 2-y PFS and OS rates from the start of retreatment were 91% and 96%, respectively. With 15.2 mo median follow-up on Ibr+Ven retreatment (range, 7.4–29.3), ORR was 82% (best response: 1 CR; 8 PR; 2 SD); 1-y PFS and OS rates from the start of retreatment were both 100%. Second malignancies occurred in 24 pts across the entire study period, including 12 initial treatment and 4 retreatment TEAEs. Conclusions: Ibr+Ven is an all-oral, once-daily, chemotherapy-free FD regimen for first-line treatment of CLL/SLL that continues to provide durable PFS and OS with long-term follow-up, including in pts with high-risk genomic features. Ibr-based retreatment provided durable responses in pts needing subsequent therapy after completion of FD Ibr+Ven. Clinical trial information: NCT02910583 .

Patients with chronic lymphocytic leukemia (CLL) experience variable clinical course and duration of therapeutic response. While prior studies have shown that specific genetic drivers can shape leukemia growth kinetics and influence the natural course of CLL, the longitudinal patterns and genetic determinants of clonal evolution underlying response and resistance to time-limited therapies are not fully defined. To address this, we performed longitudinal analyses of 107 patients treated with time-limited frontline CLL therapies, either chemotherapy or chemoimmunotherapy (CIT, n=62), or fixed-duration venetoclax-obinutuzumab (VO, n=30) or ibrutinib-venetoclax (IV, n=15). We combined quarterly monitoring of measurable residual disease (MRD) levels with genetic characterization of CLL from blood samples collected serially before and during therapy, at MRD sampling time points after therapy, and at clinical relapse. A median of 6 samples were genetically characterized per patient (range 4 to 15), including a median of 4 post-therapy MRD samples (range 2-11). Genetic analysis of MRD samples was carried out using ultra-deep, patient-specific targeted sequencing focusing on mutations representing distinct subclones of each CLL. Subclones were defined based on branches of the phylogenetic tree inferred using PhylogicNDT from paired pretreatment and relapse whole-exome sequencing data. Altogether, 3058 baits capturing subclone-specific mutations (median 6 baits/subclone) were deployed to track 561 subclones (median 5 subclones/CLL) across intervening MRD time points. We used duplex sequencing to reduce sequencing errors at MRD time points and obtained an average duplex depth of 2870x (range 156-5181x) (~110,000x raw depth) per sample, with individual variants reaching duplex depths >10,000x allowing detection of subclones with cancer cell fractions (CCF) of ≥10% at MRD ≥4 x 10-3. We integrated serial white blood cell counts, MRD and clone-specific CCF data and applied the Markov Chain Monte Carlo method to model decay and repopulation rates of each subclone during and after therapy, respectively. Overall, we discerned 4 archetypes of clonal dynamics present across the CIT and VO cohorts. Archetypes 1-3 were eachmarkedby the outgrowth of a particular subclone while on therapy, consistent with their relative insensitivity to treatment, but differed with respect to the post-therapy repopulation kinetics of the therapy-insensitive subclone relative to other subclones. For Archetype 1 (26% CIT; 33% VO), the therapy-insensitive subclone remained stably dominant after therapy and was the sole basis of CLL relapse. While chemoresistant subclones typically harbored mutations in canonical CLL driver genes (primarily TP53, ATM, SF3B1, POT1, CHEK2, SAMHD1, IKZF3 and DIS3), therapy-insensitive subclones in VO-treated cases exhibited greater genetic heterogeneity, encompassing mutations in CLL drivers such as SPEN and ARID1A,mutations in cancer drivers not recurrently seen in CLL, and copy-number events. Archetype 2 (35% CIT; 48% VO) was marked by the continued expansion of the therapy-insensitive subclone, reflecting its accelerated regrowth kinetics relative to other subclones. After CIT, these subclones typically harbored chemoresistance-conferring mutations, while after VO, repopulating subclones were genetically diverse and carried mutations in such genes as NFKBIE, SPEN, NOTCH1, BIRC3, MGA, DYRK1A and RFX7. In contrast, Archetype 3 (21% CIT; 14% VO) was typified by the initial therapy-insensitive subclone being outcompeted by more therapy-sensitive ones that rapidly regrew, driving relapse. For example, in 3 of 5 patients where TP53-, ATM-, or SF3B1-mutant subclones were relatively sensitive to VO, CLL relapse coincided with preferential post-therapy expansion of these subclones, in line with their fitness advantage during disease progression. Finally, Archetype 4, observed predominantly in CIT-treated patients (18% CIT; 5% VO), featured emergence and expansion of novel subclones post-therapy, presumably induced by new mutation events, that were undetectable at the time of treatment initiation. Analysis of the IV cohort is in progress. Collectively, these archetypes delineate the diverse evolutionary trajectories that CLL subclones can follow post-therapy, providing a framework for understanding CLL relapse and guide strategies to anticipate clonal evolution, counteract subclonal selection, and ultimately prevent relapse.

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. 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. 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). 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.

Introduction:Bruton tyrosine kinase inhibitors (BTKi) are highly effective therapy for chronic lymphocytic leukemia (CLL) but can lead to long-term side effects. Methods:We performed a retrospective analysis of venetoclax consolidation of 20 patients with CLL after initial BTKi discontinuation. Patients were administered either single-agent venetoclax or in combination with obinutuzumab. Results:The best peripheral blood minimal residual disease (MRD) response was undetectable MRD, occurring in 89% of patients. With a median follow-up of 47.4 months, both the median progression-free survival and time-to-next treatment were not reached. Conclusion:Venetoclax consolidation after BTKi is a feasible treatment strategy for patients with CLL, with encouraging efficacy deserving further study. Clinical Trial Registration: N/A.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis and limited therapeutic options. Leukemic stem cells (LSCs), which drive disease progression and confer resistance to therapy, pose a significant challenge to conventional treatment strategies. In this study, we identified and characterized the inhibitory mechanisms of TH37, a small molecule derived from traditional Chinese medicine, which selectively targets AML blasts and LSCs. Our analyses identified peroxiredoxin 1 (PRDX1), an enzyme that catalyzes the breakdown of hydrogen peroxide (a reactive oxygen species), as the primary molecular target of TH37. We demonstrated that TH37 directly interacts with PRDX1, inhibiting its enzymatic activity and thereby elevating intracellular reactive oxygen species levels in AML cells. PRDX1 was found to be overexpressed in AML, and its expression correlated with poor prognosis and the activation of AML- and cancer-associated pathways. Targeting PRDX1, either through lentiviral short-hairpin RNA-mediated silencing or TH37 treatment, induced apoptosis, reduced colony formation, and impaired the engraftment and growth of AML cells in immunodeficient mouse models. Furthermore, TH37 synergized with conventional chemotherapeutic agent to significantly reduce the viability and colony-forming capacity of AML cells. These findings demonstrate the critical role of PRDX1 in AML pathogenesis and highlight its potential as a key therapeutic target to improve clinical outcomes for AML patients.

Background: Chronic lymphocytic leukemia (CLL)-associated hypogammaglobulinemia is well-established. A degree of humoral immune reconstitution has been reported after ibrutinib and after venetoclax plus rituximab (Ven + R). We assessed improvement in immunoglobulin levels after different Ven-based treatments, including Ven monotherapy, combinations with rituximab or obinutuzumab (Ven + O), and combination with ibrutinib (Ven + I). Methods: Patients with CLL who received treatment with Ven at our single center during the years 2012-2024 were evaluated in this retrospective chart review analysis. Recurrent courses of Ven were each considered discretely. Patients were excluded from analysis if they did not have at least 1 quantitative immunoglobulin measurement both within 1 year prior to Ven initiation and post-treatment. Patients with a paraprotein were excluded from analysis of that Ig class. Patients who received IVIG were excluded from IgG analysis. Data were extracted from time of Ven treatment start until subsequent treatment for CLL. Results: 91 cases of Ven treatment were identified for chart review, including 86 unique patients and 5 instances of Ven retreatment. 5 patients were excluded from IgM analysis due to IgM paraprotein. 51 patients were excluded from IgG analysis due to being on IVIG. Patient population had a median age of 69 at Ven initiation. 79% (72 of 91 patients) had received prior CLL treatment, with a median number of 3 prior unique treatment regimens. Ven treatments were: Ven monotherapy- 35% (32 patients); Ven + R- 18% (16 patients); Ven + O- 31% (28 patients); and Ven + I- 16% (15 patients). Median time on Ven was 15.1 months. Overall, 74% (67 patients) achieved uMRD (< 0.01% CLL cells in peripheral blood). 50.5% (46 patients) had eventual CLL progression either on or after Ven treatment, with median 17.3 months from the end of Ven to next treatment (range 0 – 59.5 months). For the overall population, median IgA levels decreased from pre-treatment to end of treatment (38 to 24.5 mg/dL, p=0.01; paired t-test). This was followed by a gradual increase starting 12 months after end of treatment, reaching statistical significance at month 36 (median IgA 52 mg/dL, p = 0.02). IgM levels trended similarly, with a significant increase from a median of 20 mg/dL at baseline to 37 mg/dL at month 36 (p = 0.02). IgG levels did not change significantly, though the exclusion of patients on IVIG limited this analysis. The different treatment regimens had distinct effects on IgA levels. Patients treated with Ven + CD20 mAb had a significant reduction in the IgA at the end of treatment (baseline median 41 mg/dL to 20.5 mg/dL, p=0.024). Levels then rose gradually during treatment-free remissions, and were significantly higher than baseline by month 36 (median 53 mg/dL, p=0.03). The IgA trajectories were similar for patients treated with Ven + R or Ven + G. In contrast, patients treated with Ven monotherapy did not have a statistically significant decrease of IgA levels (baseline median 34 mg/dL to 26.5 mg/dL, p = 0.1) and there was no increase from baseline after treatment. Notably, patients treated with Ven + I did not have a statistically significant reduction after treatment, but did have a significant increase in IgA from baseline at later time points (baseline median 41 mg/dL to 84.5 mg/dL at month 48, p = 0.05). We assessed for factors associated with superior IgA increase, defined as either a 50% improvement or improvement to the reference range (>80 mg/dL for our institution) at any time after Ven treatment. 21% (19 of 91 patients) had a superior IgA increase. Treatment regimen (Ven mono versus Ven + CD20 versus Ven + I) was also not associated with superior IgA increase. 84% of patients with superior IgA increase had a uMRD response to treatment (16 of 19 patients), compared to a 71% uMRD rate for patients who did not (51 of 72 patients), but this difference was also not statistically significant (p= 0.3, Chi-squared test). Conclusions: Our findings support that humoral immune reconstitution can be achieved after Ven-based treatment, based on recovery of IgA levels. IgA levels improved after Ven + CD20 therapy, as per prior reports, though Ven + CD20 mAb resulted in more significant initial reduction in IgA levels than Ven monotherapy or Ven + I. Interestingly, we did not find an association between superior IgA increases and depth of response.

Deletion of 17p (del(17p)) and/or TP53 mutated (p53MU) and/or complex karyotype (CK) are adverse prognostic factors with chemotherapy or targeted therapy that define high-risk chronic lymphocytic leukemia (CLL). Del(17p) causes loss of one allele of TP53. Somatic mutations occur in the other allele of TP53 in about 80% of del(17p) CLL. ROR1 is an embryonic receptor for Wnt5a, which can induce ROR1-signaling leading to activation of ERK1/2, NF-κB and NRF2 target genes in CLL cells. Anti-ROR1 antibody (zilovertamab) therapy, which blocks ROR1-signaling, inhibited expression of such target genes in CLL cells of patients (pts) treated in a phase I study. Prior work also found that pts with CLL cells with high-level ROR1 (ROR1Hi) had an inferior progression-free survival and overall survival compared to pts with CLL cells with low-levels of ROR1 (ROR1Lo). Our gene set enrichment analysis revealed that NRF2 target genes and cell-cycle control genes were significantly enriched in ROR1Hi versus ROR1Lo CLL cells or in pre-treatment CLL cells (D0) versus CLL cells of the same pts treated with zilovertamab (D28). Most pronounced was CCNA1, which encodes cyclin A1 and is transcriptionally activated by wild-type p53 or most p53MU. NRF2 activation can enhance cyclin A1 activation by inducing NRF2 target genes such as NQO1, which can bind p53MU and mitigate its proteasomal degradation. We assessed 493 untreated CLL for ROR1 expression, FISH, and cytogenetics. We found that cases with ROR1Hi CLL had a significantly higher proportion of del(17p) and/or CK than cases with ROR1Lo CLL. This suggests that high-level ROR1-signaling may be conducive to the survival of CLL cells with del(17p) and genomic instability associated with high-level oxidative stress. We examined for this using a CLL-cell line, MEC1, which lacks ROR1 but makes high levels of Wnt5a and has p53MU (Q317fs*29) with del(17p). We also used MEC1Wnt5a-/- cells, in which we deleted WNT5A. MEC1 or MEC1Wnt5a-/- cells were transduced with a lentivirus vector encoding ROR1 to generate MEC1-ROR1 or MEC1-ROR1Wnt5a-/- cells. Relative to MEC1 or MEC1-ROR1Wnt5a-/-, MEC1-ROR1 cells had significantly increased expression of NRF2 target genes and cell-cycle control genes, NQO1, p53MU, and cyclin A1, and increased resistance to drug induced oxidative stress. Similarly, we found that culture of ROR1+ del(17p) CLL cells with Wnt5a increased expression of NQO1, cyclin A1, and p53MU, and enhanced their resistance to drug induced oxidative stress; this effect of Wnt5a could be blocked by concomitant treatment with zilovertamab. As such, inhibiting ROR1-signaling may reduce resistance to oxidative stress and prove selectively deleterious to CLL cells with del(17p) and CK, thereby mitigating their adverse prognostic implications for pts with CLL. Emanuela M. Ghia, Can Huang, Michael Y. Choi, George F. Widhopf, Thomas J. Kipps. ROR1-dependent mutant TP53 and cyclin A1 increases, via NRF2 activation, potentially potentiate genomic instability in chronic lymphocytic leukemia cells with del(17p) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7176.

Supplementary Figure S1. Kaplan-Meier curves of progression-free survival (A) and overall survival (B) in the total pooled population of patients treated with fixed-duration ibrutinib plus venetoclax in the FD cohort and the MRD cohort placebo arm. Tick marks indicate censored patients.

Matrix metalloproteinase-9 (MMP-9) facilitates the extravasation and lymphoid-tissue infiltration of chronic lymphocytic leukemia (CLL) cells. Prior studies found that high level expression of MMP-9 in CLL associates more aggressive disease. We find that circulating CLL cells that express high levels the onco-embryonic protein ROR1 express significantly higher levels of MMP-9. Stimulation of CLL cells with Wnt5a could enhance expression and the release of MMP-9 into the culture media and increase the capacity of CLL cells to invade Matrigel in a Boyden-Chamber Assay. Such effects of Wnt5a could not be inhibited by BTK inhibitors such as ibrutinib or zanubrutinib, but could be blocked by zilovertamab, a humanized mAb specific for ROR1. We found that siRNA silencing of NF-κB-p65 or use of an NF-κB inhibitor (CAS 545380-34-5) blocked the capacity of Wnt5a to induce MMP-9 or enhance the invasive capacity of treated CLL cells. Moreover, siRNA directed silencing of MMP9 or treatment with an MMP-9 inhibitor (CAS 1177749-58-4) also blocked the invasive capability of CLL cells induced by Wnt5a. We conclude that Wnt5a-induced ROR1-signaling can induce expression of MMP-9 on CLL cells through activation of NF-κB, thereby enhancing the extravasation and lymphoid-tissue infiltration required for CLL cell trafficking.

Supplementary Table S1. Patient demographics and disease characteristics at baseline by cohort.

Fixed-duration venetoclax-rituximab (VenR) in patients with relapsed/refractory chronic lymphocytic leukemia (CLL) in the phase 3 MURANO trial (NCT02005471) resulted in superior progression-free survival (PFS) and overall survival (OS) vs bendamustine-rituximab (BR). We report the final analyses of MURANO (median 7 years follow-up). Patients were randomized to VenR (venetoclax 400 mg daily for 2 years plus monthly rituximab for 6 months; n = 194) or BR (6 months; n = 195). In a substudy, patients with progressive disease (PD) received VenR as retreatment or crossover from BR. At the final data cut (3 August 2022), median PFS with VenR was 54.7 vs 17.0 months with BR. Seven-year PFS with VenR was 23.0%. Seven-year OS was 69.6% and 51.0%, respectively. Among VenR-treated patients with undetectable (u) minimal residual disease (MRD) and no PD at end of treatment (EOT) (n = 83), median PFS from EOT was 52.5 vs 18.0 months in patients with MRD at EOT (n = 35; P < 0.0001). Fourteen patients had enduring uMRD. Three distinct mutations in BCL2 in four patients were identified. In the substudy, 25 patients were retreated with VenR and nine patients crossed over to VenR; median PFS was 23 and 27 months, and best overall response rate was 72% and 89%, respectively. At the end of combination treatment, following retreatment or crossover, eight and six patients achieved uMRD, respectively. No new safety findings were observed. Overall, these final MURANO analyses support consideration of fixed-duration VenR therapy for patients with relapsed/refractory CLL.

The mutational landscape of phylogenetically ultraconserved elements (UCEs), especially those in noncoding DNAs (ncUCEs), and their functional relevance in cancers remain poorly characterized. Here, we perform a systematic analysis of whole-genome and in-house targeted UCE sequencing datasets from more than 3000 patients with cancer of 13,736 UCEs and demonstrate that ncUCE somatic alterations are common. Using a multiplexed CRISPR knockout screen in colorectal cancer cells, we show that the loss of several altered ncUCEs significantly affects cell proliferation. In-depth functional studies in vitro and in vivo further reveal that specific ncUCEs can be enhancers of tumor suppressors (such as ARID1B) and silencers of oncogenic proteins (such as RPS13). Moreover, several miRNAs located in ncUCEs are recurrently mutated. Mutations in miR-142 locus can affect the Drosha-mediated processing of precursor miRNAs, resulting in the down-regulation of the mature transcript. These results provide systematic evidence that specific ncUCEs play diverse regulatory roles in cancer.

Breast cancer stem-like cells (CSCs) are enriched following treatment with chemotherapy, and posited as having a high level of plasticity and enhanced tumor-initiation capacity, which can enable cancer relapse. Here, we show that such features are shared by breast cancer (BCA) cells that express receptor tyrosine kinase-like orphan receptor (ROR2), which is expressed primarily during embryogenesis and by various cancers. We find that Wnt5a can induce ROR2 homooligomerization to activate noncanonical Wnt signaling and enhance tumor-initiation capacity of BCA cells. Molecular analysis reveals that the cysteine-rich domain and transmembrane domain are required for ROR2 homooligomerization to activate ROR2. Treatment with a newly generated monoclonal antibody (mAb) specific for ROR2 can block Wnt5a-induced ROR2 homooligomerization, ROR2-dependent noncanonical Wnt signaling, and impair the capacity of BCA patient-derived xenografts to initiate tumor in immune-deficient mice. Collectively, these results indicate that targeting ROR2 (e.g., using mAb) suppresses BCA stemness and, thereby, may prevent BCA relapse.

BACKGROUND:Chronic lymphocytic leukemia (CLL) is considerably more common in Americans compared with Asians. The basis for these differences and implications for therapy outcomes are controversial and mostly unknown. METHODS:We compared baseline co-variates, therapies, and outcomes from 2 databases, Flatiron Health database in the United States (N = 15 786) and Tianjin CAMS database from China (N = 2996). RESULTS:Chinese subjects had younger age at diagnosis, more advanced Rai stage and an increased prevalence of lymphadenoma, thrombocytopenia, and increased β2-microglobulin. Americans had higher rates of unmutated IGHV, TP53 deletion, and cytogenetic abnormalities. These differences persisted after adjusting for age, Rai stage, and IGHV mutation state. There were also substantial differences in therapy patterns between the cohorts. Median survival in Chinese was 9.7 vs 7.5 years in Americans (P < .001). In sub-group analyses, Chinese CLL had better 5-year survivals with chemotherapy (69% [95% CI, 66, 72%] vs 49% [47, 52%]; P < .001), immune therapies (67% [63, 72%] vs 65% [64, 66%]; P = .041), and targeted therapies (85% [81, 88%] vs 65% [64, 67%]; P < .001). These advantages were pronounced among older patients and those with early-stage, mutated IGHV and without TP53 deletion. CONCLUSION:This cross-sectional study identifies significant clinical and treatment outcome disparities in CLL between Eastern and Western populations, attributed to distinct genetic and molecular profiles.