BTK Mutations Research Articles

Characterization of BTK mutation pathologic status and association with known prognostic mutations (TP53, ATM, BIRC3, SF3B1, NOTCH1) in CLL: An AACR Project GENIE Analysis

Characterization of BTK mutation pathologic status and association with known prognostic mutations (TP53, ATM, BIRC3, SF3B1, NOTCH1) in CLL: An AACR Project GENIE Analysis

Strategies for overcoming resistance to Bruton's tyrosine kinase inhibitor zanubrutinib

AbstractBruton's tyrosine kinase (BTK) inhibitors have revolutionized the treatment of B‐cell malignancies. They target BTK, a key effector in the B‐cell receptor (BCR) signaling pathway, crucial for B‐cell survival and proliferation. The first‐in‐class irreversible BTK inhibitor, ibrutinib, was approved for various B‐cell malignancies but has limitations due to off‐target effects. Second‐generation inhibitors, such as acalabrutinib and zanubrutinib, offer improved selectivity and reduced side effects. However, resistance to BTK inhibitors, driven by BTK mutations, remains a challenge. Combinatorial therapies with PI3K inhibitors, immune checkpoint inhibitors, BH3 mimetics, and anti‐CD20 antibodies show promise in overcoming resistance. Noncovalent BTK inhibitors and proteolysis‐targeting chimeras (PROTACs) are emerging strategies with potential to combat resistance. Overall, advancements in BTK‐targeted therapies provide hope for improved outcomes in patients with B‐cell malignancies and a promising avenue to address drug resistance. Further research is needed to optimize combination therapies and identify optimal treatment regimens.

Real-world understanding of the unmet need for treatment of non-covalent BTKi in chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL).

e19028 Background: Non-covalent (nc) Bruton Tyrosine Kinase inhibitors (BTKi) for the treatment of Chronic Lymphocytic Leukemia and Small Lymphocytic Lymphoma (CLL/SLL) provide patients with a new treatment option after resistance to on-target BTK mutations or PLCγ2 mutations that bypass BTK inhibition. Given the December 1, 2023 accelerated FDA approval for pirtobrutinib, a nc-BTKi, in the treatment of CLL/SLL for treatment of patients who have received at least two prior lines of therapy with a covalent (c) BTKi and a B-Cell Lymphoma 2 Protein Inhibitor (BCL2i), we sought to understand the addressable population for treatment with nc-BTKI therapy in a real-world database of CLL/SLL patients. Methods: We used the Integra Connect PrecisionQ real-world de-identified database of over 3 million cancer pts across 500 sites of care to identify CLL/SLL pts for the analysis. The CLL patients included for the analysis were on a treatment in 2023 and must have had either >5 CLL/SLL diagnosed visits or more CLL/SLL visits than non-CLL/SLL visits if <5 CLL/SLL diagnosed visits. Descriptive analyses were used for this study. Results: Of the 6,992 patients treated for CLL/SLL in 2023, the median age at treatment start was 71, 62% were male, 65% identified as White, 6% as African American, and 29% as Other. Of the pts, 12% received a c-BTKi and BCL2i, 64% received a c-BTKi only, and 12% received a BCL2i only. Of the 12% of pts who received a c-BTKi and BCL2i, 191 pts started a subsequent line of therapy. Among pts who received a c-BTKi and BCL2i, 79% received a BTKi therapy first, 10% received a BCL2i therapy first, and 12% had a line of therapy with both a BTKi and BCL2i. We found 59 pts who received nc-BTKi; among those pts, 51% received a c-BTKi and BCL2i prior to initiation of nc-BTKi. The median age at first treatment for CLL/SLL was 71.7 for those who received a c-BTKi, 71.1 for those who received a BCL2i, and 70.8 for those who received nc-BTKi. The median age at first diagnosis for CLL/SLL was 68 for those who received a c-BTKi, 66 for those who received a BCL2i, and 63 for those who received nc-BTKi. The median time to first treatment was 470 days for pts treated with a c-BTKi, 507 days for pts treated with BCL2i, and 587 days for pts treated with nc-BTKi. Conclusions: We found that among nearly 7,000 treated patients in the database, 12% were treated with both a c-BTKi and BCL2i; of those, just 23% started a subsequent treatment. Among patients who were treated with a nc-BTKi we found their median age at treatment start and diagnosis was lower than those who received a c-BTKi or BCL2i respectively.

Abstract 5828: The genomic architecture of ibrutinib resistance in CLL: Oligoclonal progression with variable anatomical distribution

Abstract Progressive disease (PD) in patients with chronic lymphocytic leukemia (CLL) on ibrutinib often presents with acquired mutations in BTK and/or PLCG2. Reported variant allele frequencies (VAF) are often low, leading some to question the role of these mutations. Further, several co-existing mutations are often identified. Here we investigated the clonal composition of PD on single cell level, across time, and different anatomic compartments. 84 CLL patients (52 treatment-naïve; 32 relapsed/refractory) with either a TP53 aberration or age ≥65 years received single-agent ibrutinib 420 mg once daily until PD or limiting side effects on a phase 2 study (NCT01500733). The median progression-free survival was 86.6 months. 39 (46.4%) patients progressed, 31 with CLL (PD-CLL), and 8 with Richter’s transformation. Median time to PD-CLL was 57.2 months. In PD-CLL patients, peripheral blood (PB) was sent for genotyping of known hotspot mutations in BTK/PLCG2 (Neogenomics). We then designed digital droplet PCR probes to test for BTK C481S/R/Y and PLCG2 R665W/L845/S707F mutations in samples collected at baseline, during response to ibrutinib and at PD in 29 patients. 26 (89.7%) patients had detectable BTK/PLCG2 mutations (VAF range 0.04 - 0.96). 12 (41%) patients carried multiple BTK/PLCG2 mutations. In 11 evaluable cases single-cell DNA sequencing (Mission Bio) showed individual mutations present in separate cells consistent with branching evolution. Having confirmed oligoclonality, we calculated the cumulative cell fraction (cCF) of BTK/PLCG2 mutations in the 29 PD-CLL patients. 16 (55%) patients had cCF >0.5 (range 0.59-0.96), 10 patients (35%) had cCF < 0.5 (range 0.04-0.43) and 3 patients (10%) had no detectable mutations. BTK/PLCG2 mutations were detected in samples collected at median 21.6 months (range 0 - 75) prior to PD. In 11 cases, we tested lymph node (LN) biopsies at PD. In 6 cases with cCF <0.5 in PB, 4 corresponding LN samples had higher cCF (0.01/0.92; 0/0.85; 0.06/0.69; 0.46/0.99 for PB/LN, respectively). In 5 cases, PB contained 2-6 distinct subclones with cCF >0.5, in 3 cases the corresponding LN harbored fewer subclones than PB (6/3; 3/2; 3/0 for PB/LN, respectively). To characterize the genomic background of PD-CLL, we performed whole-exome sequencing of longitudinal samples from 26 patients. In addition to BTK/PLCG2, the most frequently mutated genes were TP53, SF3B1, NOTCH1, and POT1. Furthermore, copy number variations (CNA), such as del17p, del13q, del14q, del3p, and del8p, were common. In summary, in our cohort BTK/PLCG2 mutations were identified in 89.7% of PD-CLL patients. These mutations arise in oligoclonal populations with variable distribution across anatomical sites. In rare patients without BTK/PLCG2 mutations, the co-evolution of putative driver mutations and multiple CNAs generated genomic complexity reminiscent of lymphoma transformation. Citation Format: Chingiz Underbayev, Jonathan Chen, Hailey M. Harris, Lauren T. Vaughn, Andy Itsara, Adrian Wiestner. The genomic architecture of ibrutinib resistance in CLL: Oligoclonal progression with variable anatomical distribution [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 5828.

Abstract 605: ABBV-101, a highly potent and selective clinical stage Bruton tyrosine kinase degrader, overcomes BTK mutation-induced resistance to BTK inhibitors

Abstract Bruton tyrosine kinase (BTK), a clinically validated target for various B-cell malignancies, plays a critical role in regulating cell growth, adhesion, and homing to lymphoid tissues that provide a microenvironment favorable to cancer cells. Despite the success of approved covalent BTK inhibitors in diseases such as CLL, most patients do not achieve complete response with monotherapy and eventually relapse, often via the emergence of the BTK-C481S mutation. Although second-generation reversible BTK inhibitors may temporarily overcome the C481S mutation, novel BTK mutations associated with resistance emerged in the clinic recently. Additionally, covalent BTK inhibitors have not shown sufficient differentiation from standard-of-care (SoC) in B-cell malignancies, such as diffuse large B cell lymphoma (DLBCL) to warrant approval to date. To address these unmet needs, a next-generation orally bioavailable BTK degrader ABBV-101 has been developed. Unlike BTK inhibitors that solely bind to and impede the catalytic domain of BTK, ABBV-101 eliminates the protein in a highly selective fashion. This degradation mechanism targets both the catalytic and scaffolding functions of BTK, and thus may enable deeper and more durable responses in patients with B-cell malignancies. Cellular activity of ABBV-101 was determined in a panel of DLBCL cell lines. We show that most non-germinal center B cell DLBCL (non-GCB-DLBCL) cell lines are sensitive to ABBV-101. To determine whether ABBV-101 could overcome BTK mutations-induced resistance, we engineered human DLBCL cell line TMD8 using CRISPR technology to express these BTK mutations and tested for sensitivity to ABBV-101 and BTK inhibitors. We show that ABBV-101 demonstrates similar potent activity against all BTK mutations associated with resistance to both covalent and reversible BTK inhibitors. The modulation of downstream pathway by ABBV-101 and how it differentiates from other BTK inhibitors will be presented. Further, ABBV-101 induces complete tumor regression in the BTK-C481S TMD8 DLBCL xenograft model. A spontaneous mouse CLL model carrying BTK-C481S mutation was developed through crossing BTK-C481S knock-in mice with the Emu-TCL1 transgenic mice in the C57BL/6 background. ABBV-101 completely inhibits BTK-C481S Emu-TCL1 CLL burden increase in the blood compartment and reduces CLL burden in the spleen and lymph nodes, whereas covalent BTK inhibitors show no activity. Finally, combination with a BCL-2 inhibitor enhances the efficacy of ABBV-101 in CLL and DLBCL models. Taken together, our results suggest that ABBV-101 has potent in vitro and in vivo efficacy. ABBV-101 is currently in phase 1 clinical trial for a variety of B-cell malignancies. ClinicalTrials.gov identifier: NCT05753501 Citation Format: Chin Pan, Jacob Riehm, Tarikere L. Gururaja, Haiyan Li, Henry Nguyen, Yongjiao Zhai, Xiaoping Xie, Rong-Xian Ding, Rebecca Matthew, Hana Y. Hoh, Zhaozhong J. Jia, Bo Liu, Cassandra P. Shu, Claudina A. Stevenson, Christine Will, Federico Innocenti, Wissam Assaily, Lloyd T. Lam, Alexey Rivkin. ABBV-101, a highly potent and selective clinical stage Bruton tyrosine kinase degrader, overcomes BTK mutation-induced resistance to BTK inhibitors [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 605.

Abstract 4669: Differential preclinical activity of Bruton tyrosine kinase inhibitors (BTKis) against BTK resistance-associated mutations

Abstract Background: Differences in profiles of acquired mutations of resistance to BTKis might have implications for treatment sequencing. We evaluated preclinical activity of BTKis (ibrutinib [Ibr], acalabrutinib [Acala], zanubrutinib [Zanu], and pirtobrutinib [Pirto]) against multiple clinically relevant BTK mutations and report on the clinical impact of BTK C481S in patients (pts) treated with Ibr. Methods: Inhibition of kinase activity by BTKis was evaluated in a cell-free kinase activity assay (Nanosyn). Cell-killing activity on CRISPR-generated BTK mutant knock-in TMD8 cells was assessed after 3 days of BTKi treatment. Time from first detection of C481S mutation to progressive disease (PD) was evaluated in pts treated with Ibr with previously untreated (n=247) or relapsed/refractory (R/R; n=172) CLL using pooled data from 5 clinical trials. Results: In vitro kinase activity was highest for Ibr compared with Acala or Zanu against C481S (Ibr IC50: 26.3 nM; Zanu: 84.8 nM; Acala: 338 nM) and T474I (Ibr: 1.2 nM; Zanu: 4.4 nM; Acala: 43.6 nM). Ibr maintained cell-killing activity against all tested BTK mutants, whereas limited activity was seen with Acala against C481S and A428D; with Zanu against C481S, L528W, and A428D; and with Pirto against T474I, L528W, V416L, and A428D (Table). In pts treated with Ibr, C481S was detected prior to clinical PD in 3 previously untreated pts and in 26 pts with R/R CLL; median (range) time from first detection of C481S to PD in these pts was 17.0 mo (11.2-34.3) and 10.0 mo (2.6-35.7), respectively. Conclusion: This study demonstrates activity of Ibr against all evaluated BTK variants, which are associated with resistance to covalent and noncovalent BTKis. Clinical data shows that pts who develop C481S mutations continue to derive benefit from Ibr for several months before PD. Together, these data suggest a role for Ibr in the treatment of pts with a broad spectrum of BTK variants. Table. Cell-killing activity of BTKis against wild type and mutant BTK EC50 (nM) BTK Wild type C481S T474I L528W V416L A428D Ibr 0.6 414.0 1.9 181.4 0.5 205.3 Acala 3.9 2973.8 43.3 3.5 106.3 3000.0 Zanu 0.7 2322.0 11.7 3000.0 0.6 1726.0 Pirto 8.2 12.6 2727.5 3000.0 1898.0 3000.0 Citation Format: Matthew S. Davids, Inhye E. Ahn, Jacob J. Riehm, Melih Acar, Vincent Girardi, Sima Patel, Tarikere Gururaja, Jennifer Woyach, Lloyd T. Lam, Edith Szafer-Glusman. Differential preclinical activity of Bruton tyrosine kinase inhibitors (BTKis) against BTK resistance-associated mutations [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 4669.

Abstract 1960: CFON-026 is a potent non-covalent BTK inhibitor suitable for combination therapy with covalent BTK inhibitors for early eradication of resistance mutations

Abstract Bruton’s Tyrosine Kinase (BTK) inhibitor drugs are a mainstay in the treatment of CLL, WM and MCL. They are not curative because of the emergence of BTK inhibitor resistance mutations in as much as 67% of relapsed patients (in CLL) [1]. Frequently observed mutations include BTKC481S, which abrogates covalent binding of irreversible drugs such as acalabrutinib, ibrutinib and zanubrutinib, and BTKT474I and BTKV416L which both abrogate binding of the non-covalent inhibitor drug pirtobrutinib [1, 2]. To combat resistance, third generation BTK inhibitors are needed, which must 1. bind to unique regions in the tumor driving kinases that are not marred by resistance mutations, 2. have sufficient potency for sustained inhibition of BTK signaling, and 3. be sufficiently selective to afford efficacious doses and suitable for a combination regimen. We have developed a platform called Energetically Privileged Ligands (EPriLs), based on a macrocycle scaffold that binds non-covalently in the kinase ATP pocket. Their unique binding mode involves connecting the N-terminal and C-terminal lobes of the general kinase fold in a continuous β-sheet, in this way avoiding hotspots for kinase inhibitor resistance mutations. The potent binding of the macrocycle ensures a very long residence time. Using the EPriL platform, we have developed CFON-026, a next generation non-covalent BTK inhibitor with a covalent-like residence time as demonstrated by surface plasmon resonance (SPR) and cellular washout experiments. The biochemical binding KD to BTKWT, determined by SPR, is 0.14 nM. CFON-026 binds mutants with KDs of 0.4 nM (BTKC481S) and and 1.8 nM (BTKT474I). X-ray crystal structures of CFON-026 bound to BTKWT and BTKT474I confirm the unique binding mode and water network around the active site. CFON-026 inhibits the growth of the BTK dependent cell lines TMD8 and Rec-1 with IC50s of 0.45 nM and 1.7 nM, respectively. CFON-026 is very active on TMD8 variants containing endogenous BTK inhibitor resistance mutations (BTKC481S, BTKV416L, BTKT474I and BTKL528W, IC50 range 1-100 nM). CFON-026 has favorable metabolic stability, pharmacokinetic properties, and safety characteristics, and induces complete tumor regression in a TMD8 xenograft model. Since BTK resistance mutations can be detected diagnostically 12-18 months before clinical relapse, CFON-026 could be applied as combination partner to prolong progression free survival on first line BTK inhibitor therapy. Experiments with mixed populations of BTK mutant cell lines, to model a heterogenous tumor, show that CFON-026 can deepen the efficacy of ibrutinib, acalabrutinib and zanubrutinib. In conclusion, CFON-026 is a novel EPriL-based non-covalent BTK inhibitor that could enhance and prolong responses of existing covalent BTK inhibitor therapy. [1] Woyach et al., J Clin Oncol (2017). [2] Wang et al., N. Eng J Med (2022). Citation Format: Jos de Man, Michelle Muller, Joost C.M. Uitdehaag, Freek van Cauter, Sander van Gemert, Milan Hoffmann, Yvonne G.T.H. van Mil, Winfried R. Mulder, Martine B.W. Prinsen, Jan Gerard Sterrenburg, Diep Vu, Joeri de Wit, Erik Ensing, Rogier C. Buijsman. CFON-026 is a potent non-covalent BTK inhibitor suitable for combination therapy with covalent BTK inhibitors for early eradication of resistance mutations [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 1960.

Five-year follow-up of a phase 2 study of ibrutinib plus fludarabine, cyclophosphamide, and rituximab as initial therapy in CLL

AbstractWe previously reported high rates of undetectable minimal residual disease <10−4 (uMRD4) with ibrutinib plus fludarabine, cyclophosphamide, and rituximab (iFCR) followed by 2-year ibrutinib maintenance (I-M) in treatment-naïve chronic lymphocytic leukemia (CLL). Here, we report updated data from this phase 2 study with a median follow-up of 63 months. Of 85 patients enrolled, including 5 (6%) with deletion 17p or TP53 mutation, 91% completed iFCR and 2-year I-M. Five-year progression-free survival (PFS) and overall survival were 94% (95% confidence interval [CI], 89%-100%) and 99% (95% CI, 96%-100%), respectively. No additional deaths have occurred with this extended follow-up. No difference in PFS was observed by immunoglobulin heavy-chain variable region gene status or duration of I-M. High rates of peripheral blood (PB) uMRD4 were maintained (72% at the end of iFCR, 66% at the end of 2-year I-M, and 44% at 4.5 years from treatment initiation). Thirteen patients developed MRD conversion without clinical progression, mostly (77%) after stopping ibrutinib. None had Bruton tyrosine kinase (BTK) mutations. One patient had PLCG2 mutation. Six of these patients underwent ibrutinib retreatment per protocol. Median time on ibrutinib retreatment was 34 months. The cumulative incidence of atrial fibrillation was 8%. Second malignancy or nonmalignant hematologic disease occurred in 13%, mostly nonmelanoma skin cancer. Overall, iFCR with 2-year I-M achieved durably deep responses in patients with diverse CLL genetic markers. Re-emergent clones lacked BTK mutation and retained sensitivity to ibrutinib upon retreatment. This trial is registered at www.clinicaltrials.gov as #NCT02251548.

Dysregulation of Toll-Like Receptor Signaling-Associated Gene Expression in X-Linked Agammaglobulinemia: Implications for Correlations Genotype-Phenotype and Disease Expression.

In X-linked agammaglobulinemia (XLA), the diversity of BTK variants complicates the study of genotype-phenotype correlations. Since BTK negatively regulates toll-like receptors (TLRs), we investigated if distinct BTK mutation types selectively modulate TLR pathways, affecting disease expression. Using reverse transcription-quantitative polymerase chain reaction, we quantified ten TLR signaling-related genes in XLA patients with missense (n = 3) and nonsense (n = 5) BTK mutations and healthy controls (n = 17). BTK, IRAK2, PIK3R4, REL, TFRC, and UBE2N were predominantly downregulated, while RIPK2, TLR3, TLR10, and TLR6 showed variable regulation. The missense XLA group exhibited significant downregulation of IRAK2, PIK3R4, REL, and TFRC and upregulation of TLR3 and/or TLR6. Hypo-expression of TLR3, TLR6, and TLR10 may increase susceptibility to infections, while hyper-expression might contribute to chronic inflammatory conditions like arthritis or inflammatory bowel disease. Our findings shed light on the important inflammatory component characteristic of some XLA patients, even under optimal therapeutic conditions.

Overcoming Resistance in Chronic Lymphocytic Leukemia-Maybe Less Is More?

Acquired mutations in BTK, PLCG2, and BCL2 are associated with resistance to continuous targeted agent therapy in chronic lymphocytic leukemia (CLL). Here, we discuss new evidence that limiting the duration of CLL therapy may prevent the evolution of such resistance mutations, potentially facilitating effective retreatment strategies. See related article by Jain et al., p. 498.

Clinical Characteristics, Treatment Strategies, and Outcomes for CLL Patients with BTK Mutation; A Single Center Study

Background: The population of patients with chronic lymphocytic leukemia (CLL) with disease refractory to Bruton tyrosine kinase inhibitors (BTKi) is growing given the widespread use of these agents. We conducted a single center study to evaluate the disease characteristics, treatment strategies, and outcomes for patients with BTK mutation. Methods: The archives of personalized genetic medicine at the H Lee Moffitt Cancer Center and Research Institute were accessed to identify cases of BTK mutation confirmed by next generation sequencing that were reviewed between 2016-2023. Individual records of patients identified were examined to gather data regarding clinical history, genetic testing, and treatment following detection of BTK mutation according to an Institutional Review Board approved protocol. Each patient record was followed for 2 years post detection of BTK mutation or till July 2023, whichever came first, to determine progression free survival (PFS) on next line of therapy. Results: A total of 30 patients were identified. The median age was 67.5 (interquartile range (IQR) 61-73.5) years and 15 (50.0%) of the patients were female. Twenty-three (76.7%) of the patients were on ibrutinib, while 7 (23.3%) were on acalabrutinib. Only 4 (13.3%) patients received BTKi as the first line of therapy. The majority of patients received BTKi as the second line of treatment (46.7%), while 30.3% received it as 3rd line, and 10.0% received it as 4th line. The median time to progression on BTKi 52.5 months. The most common presentation of progression was worsening lymphocytosis alone (36.7%), followed by lymphadenopathy (LAD) and lymphocytosis (33.3%), and LAD alone (20.0%). The most common BTK mutation variant seen was C481S that occurred in 21 (70.0%) cases. C481S mutation occurred independently in the absence of another BTK variant mutation in 18 of these patients. C481R mutation was seen in 20.0% of the cases and was always seen concurrently with another variant mutation (either C481F, C481Y, or C841S). The median number of other mutations present concurrently with BTK mutation was 5 (IQR 3-6). Half (50.0%) of the cases had concurrent TP53 mutation while 30.0% had concurrent PLCG2 mutation. Other commonly seen mutations included NOTCH1 (26.7%), SF3B1 (26.7%), ATM (23.3%), MED12 (16.7%), ASXL1 (16.7%), BIRC3 (13.3%), and TET2 (13.3%) (Figure 1). Following confirmation of BTKi mutation, most patients (63.3%) were started on a venetoclax based regimen. Six patients (20.0%) continued on the same BTKi with addition of venetoclax, 10 (33.3%) patients were switched to venetoclax and immunotherapy (either obinutuzumab or rituximab), and 3 (10.0%) patients were started on single agent venetoclax. Two patients (6.7%) were switched to a non-covalent BTKi while another 2 (6.7%) patients were kept on the same BTKi with the addition of immunotherapy. Three (10.0%) patients were taken to chimeric T cell receptor (CAR-T) cell therapy of which one patient had no response and was started on salvage treatment with duvelisib. Another two patients (6.7%) had Richter's transformation and were treated with chemoimmunotherapy. The overall median PFS for next line of therapy was 18 months with no statistical difference between venetoclax and non-venetoclax regimens (not reached vs 8 months, p=0.25). Conclusion: Our results reveal that durable responses can be achieved by switching to venetoclax based regimens in patients with BTKi. Though the early results of the use of noncovalent BTKi in this setting aree encouraging, the durability of response is limited as new BTK mutations are selected and future therapeutic alternatives are needed for these subset of patients.

Understanding Resistance Mechanisms and Growth Kinetics of CLL Treated with Covalent and Non-Covalent BTK Inhibitors

Resistance mechanisms to covalent BTK inhibitors (cBTKi) are well studied, with mutations in BTK at the 481 target binding site most commonly identified, and, less commonly, activating mutations in PLCϒ 2. Second site non-481 BTK mutations have been identified among patients on non-covalent BTK inhibitors (ncBTKi). The present study was designed to further evaluate these resistance mechanisms using dense serial sampling during therapy with both classes of BTKi and using mathematical modeling to analyze the rates of growth and decline of specific mutated clones. We performed whole exome sequencing on matched tumor and normal samples from 19 CLL patients treated with BTK inhibitors (Ibrutinib, Ibr, 42% (8/19); Acalabrutinib, Acala, 21% (4/19) and Pirtobrutinib, Pirto, 37% (7/19)). The median number of longitudinal samples per patient per cohort was 6 for ibr (N=51 total samples), 11 for acala (N=46) and 15 for pirto (N=105). The median number of prior therapies in both the ibr and acala cohorts was 2 and high-risk genomic features del(17p) and del (11q) were both at 50% in acala cohort and 50% and 38% in the ibr group respectively. The ibr cohort had no prior BTKi therapy whereas 1 patient in the acala cohort had received spebrutinib, another cBTKi. The average time to progression (TTP) in the ibr and acala groups was comparable at 44.1 and 44.4 months respectively. The pirto cohort was more heavily pretreated with a median of 3 prior therapies and del(17p) in 28.6% and del(11q) in 42.8%. All patients in this cohort had prior cBTKi (4 patients had 1 and 3 patients had ≥2 cBTKi) and average TTP was shorter at 15.1 months. At progression (PD), mutations in BTK C481S were found in 5 /8 patients (55%) in the ibr cohort with cancer cell fraction (CCF) &amp;gt; 80% in 3 patients. Two of these patients also have mutations in PLCϒ 2 (CCF &amp;gt; 30%). One patient in the cohort has an acquired PLCϒ 2 mutation (D1140N) with CCF&amp;gt;70%, without BTK C481S. Investigation of this patient's resistant clone growth kinetics (using WBC counts) demonstrates that the PLCϒ2 clone which was not detected at baseline takes over the population at ibr progression. Two patients in this cohort did not have either BTK or PLCϒ 2 mutations; however, one patient had 2 expanding subclones with known driver mutations ( TP53, BIRC3, del 8p). All 4 patients in the acala cohort have resistant clones harboring BTK C481S (CCF 16-75%) mutations, with T474I mutations present in 2 patients (CCF 4% and 26%) at progression. Growth kinetics analysis of the C481S clones for 3 patients shows steady increase during acala therapy, suggesting CLL progression in these two patients is driven by the C481S mutations. All but one patient in the pirto cohort had developed PD on cBTKi and 71% (5/7) had BTK C481S mutations prior to starting pirto (CCF 26 - 99%). In all 5 cases, the C481S clone declines on pirto and at PD is replaced by the BTK gatekeeper mutation T474I (CCF 76 - 99%) in 3 patients, the kinase-inactive mutation BTK L528W (CCF 30%) in 1 patient, and mutations in other driver genes ( XPO1, MED12) in 1 patient. Growth kinetics analysis for 1 patient shows exponential decay of the C481S clone under pirto treatment, followed by slow steady growth of the T474I clone, with a sharp increase right before PD. In another patient, exponential growth of the T474I clone occurs during pirto treatment while the C481S clone steadily declines. Interestingly this patient also had an existing PLCϒ 2 mutation (L845F, CCF 42%) at baseline, acquired during ibr treatment, which also steadily decreased on pirto. In another patient previously treated with ibr, with baseline BTK mutations in C481R (CCF 29%), C481S (CCF 18%), and L528W (CCF 2%), the L528W clone steadily increases, while both C481S and C481R clones decrease during PD. This patient exhibits extreme multiclonal diversity of BTK mutations, with 8 different BTK mutations at the DNA level at the C481 site present both at baseline and at PD, with 56% reduction in total wild type reads at PD. Of the remaining two patients without baseline C481S mutations, one patient acquired T474I (CCF 99%) and the other patient acquired 1q gain (MCL1) and t(8;14) at progression. Growth kinetics of different BTKi can vary based on their specific mechanism of action. The approach of combining WES data with growth pattern modeling can help unravel the complexities of tumor evolution and drug resistance for the different classes of BTKi. Data on comparative clone growth rates will be presented at the meeting.

Extended Follow-up and Resistance Mutations in CLL Patients Treated with Acalabrutinib

Background: Covalent Bruton tyrosine kinase inhibitor (cBTKi) is a standard treatment for chronic lymphocytic leukemia (CLL). Understanding of acquired resistance to cBTKi therapy has largely come from data on patients (pts) treated with the first-in-class cBTKi ibrutinib. Mutations in BTK C481 and to a lesser extent PLCG2 are frequently detected in ibrutinib-treated pts at the time of progressive disease (PD). In contrast, much less is known about genetic mechanisms of drug resistance in pts treated with next generation cBTKi acalabrutinib and zanubrutinib. A recent study identified 3 pts with BTK C481 mutations and no pts with PLCG2 mutations among 8 acalabrutinib-treated pts with progressive disease (PD) (Black, et al. Blood 2022). A second study found a kinase-dead BTK L528W mutation in 7/13 zanubrutinib-treated pts (Blombery, et al. Blood Adv 2022). Although cBTKi share the same mechanism of action by binding to BTK C481, the latter report discloses differences in clonal selection and underscores the need for further investigation in pts treated with next generation cBTKi. In a phase 2 study of acalabrutinib in pts with treatment naïve (TN) CLL or small lymphocytic lymphoma (SLL) with 17p deletion, or TP53 or NOTCH1 mutation, or relapsed/refractory (R/R) CLL/SLL regardless of cytogenetics or somatic mutations (Sun, et al. Blood 2020), we sought to understand clonal evolution during treatment. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from 48 pts at baseline, after 6 months, then annually on treatment until PD. Current analysis includes 14 PD pts [excluding 3 Richter transformation (RT) pts] comprising 10 R/R and 4 TN CLL pts. Additionally, paired baseline and PD bone marrow (BM) from 6 pts and lymph node (LN) samples from 2 pts were included. Targeted next-generation sequencing of ~700 recurrently mutated genes in hematological malignancies (Collins, et al. Leuk Lymphoma. 2021) was performed using DNA isolated from CD19 + enriched cryopreserved PBMCs and BM aspirates, as well as fresh-frozen core LN biopsies. Filtered variants in putative CLL driver genes (Knisbacher, et al. Nat Genet. 2022) with variant allele frequencies (VAF) ≥1% for BTK and PLCG2, ≥5% for variants with known oncological significance, and ≥7.5% for all other variants were included in downstream analysis. Results: After a median follow-up of 6.5 years (IQR 2.9-6.3), 23/48 (48%) pts developed PD (20 CLL, 3 RT). PD occurred in 17/32 (53%) R/R pts and 6/16 (38%) TN pts. Median progression-free survival was reached at 6.0 years. Best response was complete remission in 2/20 (10%) pts and partial remission in 18/20 (90%) pts with CLL PD. IGHV was unmutated in all but one PD patient. At baseline, 34 CLL driver genes were mutated in 41/45 pts (91%; excluding 3 RT pts) with most mutations in NOTCH1, TP53, ATM, SF3B1, BIRC3, and NFKBIE. During acalabrutinib treatment but before PD, 14/45 pts (31%; 8 R/R and 6 TN) acquired mutations in 11 driver genes, most commonly in BRAF (n=4), CREBBP (n=4), and KMT2D (n=2). At the time of PD, 11/14 pts (79%) had newly acquired mutations in 4 genes, predominantly BTK (n=6) and PLCG2 (n=4). BTK C481 mutations (VAF 2-42%) were detected in 6/14 (43%; 4 R/R and 2 TN) pts and T474I mutation (VAF 4-46%) in 3/14 (21%; 1 R/R and 2 TN) pts. Paired BM samples in 2 pts and LN samples in 2 pts showed the same BTK mutations as PBMCs with similar VAFs. PLCG2 mutations (VAF 1-8%) were found in 4/14 (29%; 2 R/R and 2 TN) pts. BTK C481 co-occurred with BTK T474I in 3 pts, but on different sequencing reads suggesting independent subclones, and with a PLCG2 mutation in 1 pt. There have been no pts with BTK L528 mutations. In conclusion, mutations in BTK and PLCG2 are the predominant resistance mechanism to acalabrutinib. Among BTK mutations, C481 was the most common. All T474I mutations co-occurred with C481.

MS-553 Therapy Inhibits BCR Signaling and Increases Survival Dependence on BCL-2 in BTK-Inhibitor Resistant CLL

B-cell receptor (BCR) signaling pathway inhibitors and B-cell lymphoma-2 (BCL-2) antagonist venetoclax (Ven) have transformed the treatment of chronic lymphocytic leukemia (CLL), including in high-risk patients with 17p deletion and/or TP53 mutations and complex karyotype. Despite the durable remissions observed with inhibitors of Bruton's Tyrosine Kinase (BTKi), a significant proportion of patients develop resistance with acquired mutations in BTK and its downstream target phospholipase Cγ2 ( PLCG2). Patients who progress on BTKi including ibrutinib (ibr) often receive Ven but eventually relapse, underscoring an urgent need for new treatment strategies. Protein kinase C-β (PKC-ß) is a protein downstream of BCR signaling (BTK-PLCG2) and is essential for CLL cell survival and proliferation in vivo. Here we investigate MS-553, an inhibitor of PKC-ß, with a focus of combination strategies in CLL patient samples including relapsed/refractory CLL. We show that CLL cells in contact with stroma upregulate PKC-ßII, its immediate downstream effectors, pGSK and pERK as well as ß-catenin in CLL patients including those with mutant TP53 and 17p (n=8). Correspondingly, MS-553 (5µM, 48h) exposure reduced levels of p-PKC-ßII and its downstream effectors (p-ERK, p-GSK) in a dose dependent manner regardless of TP53 mutation in both CLL cells and those supported by NK.Tert (stromal cell line derived from bone marrow, BM, and mimics BM microenvironment). Pre-treatment (48h, 5µM MS-553) of stromal NK.Tert cells with MS-553 prevented the upregulation of PKC-ßII within adjacent CLL cells implicating the stroma as a major source of PKC-ß activation in CLL cells. We also tested whether MS-553 could inhibit PKC-ß signaling in CLL cells exposed to phorbol myristate acetate (PMA-400nM, 90 min), which simulates BCR activation. MS-553 (5µM, 48h) exposure caused downregulation of BTK effectors (pPKC-ßII, pERK, pGSK), BCL-2 family member proteins (MCL-1 and BCLxl), and ß-catenin (n=10). In parallel experiments, we used BH3 profiling to show that CLL cells' (n=8) exposure to MS-553 in vitro developed increased dependence on BCL-2 and BCLxl for survival (Bim, Bad, XXA1_y4ek, 5-fold± 2-fold increase) in both wild type (WT) and mutant (Mut) CLL cells, suggesting a mechanistic rationale for synergy with Ven. To confirm this, CLL cells were exposed to MS-553, Ven or a combination of MS-553 and Ven. ~3-fold and 9-fold synergistic increase in the mitochondrial dysfunction (measured by cytochrome C release, 12h, 5µM MS-553, 1nM Ven) was observed in stromal supported WT and Mut CLL respectively in MS-553 versus Ven+MS-553 treated group. Similarly, ~3-fold increase was observed in WT and Mut CLL alone group and more than 10-fold increase was found in stromal supported WT and Mut group in cell death determined as percent apoptotic index (measured by annexin V-PI staining, 18h, 5µM MS-553, 1nM Ven) in MS-553 versus Ven+MS-553 group respectively. Finally, we validated our preclinical findings in CLL samples obtained from four patients on MS-553 therapy (NCT03492125, Range = cycles 0-19, MS-553 28 days/cycle). All patients showed an increase in survival dependence towards BCL-2 and BCLxl by Cycle 3-6 of MS-553 therapy. This was accompanied by downregulation of ß-catenin, pPKC-ßII, pGSK, pERK along with BCLxl and MCL-1 after Cycle 10 or later of MS-553 therapy (data summary table1). Overall, our data provides clear evidence that MS-553 inhibits BCR signaling in vitro and in patients on MS-553 therapy. It also provides a rationale for combinations with Ven as a treatment partner based on its ability to increase survival dependence on BCL-2.

Prevalence of Resistance-Associated Bruton Tyrosine Kinase (BTK) C481 Mutations By Prior Treatment Status Among Patients with Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL): A Real-World Observational Study

Introduction: BTK mutations, particularly at C481, are an important cause of acquired resistance to covalent BTK inhibitors (BTKi) among pts with CLL/SLL. Almost two-thirds of pts whose disease progresses on a covalent BTKi have a BTK C481 mutation detectable at progression. Furthermore, BTK C481 mutations have been detected up to 28 mo before progression among pts with CLL/SLL treated with a covalent BTKi. The early identification of pts who are at risk for developing covalent BTKi resistance is critical to optimize clinical treatment of pts with CLL/SLL. In this cross-sectional study, the prevalence of BTK C481 mutations was evaluated in a real-world population of patients with CLL/SLL. Methods: Patients with CLL/SLL undergoing standard-of-care treatment at Aarhus University Hospital and Gødstrup Hospital in Denmark were recruited. Eligible patients were ≥18 y old, had a confirmed diagnosis of CLL/SLL, provided a whole blood sample during their clinical visit, and had CLL cells detectable by flow cytometry. DNA was assayed from newly collected blood samples for 6 BTK C481 mutations known to confer resistance to covalent BTKi (c.1442G&amp;gt;C, c.1441T&amp;gt;A, c.1442_1443delinsCT, c.1442G&amp;gt;T, c.1441T&amp;gt;C, c.1442G&amp;gt;A), using droplet digital polymerase chain reaction with assay sensitivities of 0.1% variant allele frequency or less depending on the variant. A patient was considered to have a BTK C481 mutation “detected” if any of the variants were present above the limit of detection. Demographic, clinical, and treatment characteristics were extracted from medical records. Patients were grouped by treatment status at the time of blood collection: treatment naive (group 1), treated with therapies other than a BTKi (BTKi naive; group 2), and previously treated with or currently receiving a covalent BTKi (BTKi experienced; group 3). The primary objectives were to determine the presence of BTK C481 mutations in pts who had not received prior BTKi and the natural history of BTK C481 mutations in BTKi-experienced pts with CLL/SLL. Demographic and clinical variables were summarized descriptively. The prevalence of BTK C481 mutations was calculated and reported as a percentage with estimated 95% CIs. Results: Between October 2021 and December 2022, 130 patients with clinical and BTK C481 mutation data available were enrolled (group 1, n = 53; group 2, n = 29; group 3, n = 48). The median (range) age at diagnosis was 66 y (48-85) in group 1, 66 y (37-86) in group 2, and 62 y (43-78) in group 3. The median (range) age at first therapy was 72 y (39-86) in group 2 and 65 y (44-80) in group 3. In group 1, 38 pts (72%) had Rai stage 0 disease at diagnosis compared with 8 pts (28%) in group 2 and 13 (27%) in group 3. IGHV was unmutated in 8 pts (15%) in group 1, 13 (45%) in group 2, and 33 (69%) in group 3. In group 2, 18 pts (62%) had received 1 prior line of therapy, 4 (14%) had received 2 prior lines, and 4 (14%) had received ≥3 prior lines (not including therapy received at the time of blood collection). In group 3, 17 pts (35%) had received 1 prior line of therapy, 12 (25%) had received 2, and 11 (23%) had received ≥3. BTK C481 mutations were detected in 0 patients (0%) in group 1, 0 pts (0%) in group 2, and in 10 pts (21%; 95% CI, 10-35) in group 3. Of the 10 pts with detectable BTK C481 mutations, 5 had received a BTKi as prior therapy and 5 were receiving a BTKi at the time of blood collection (Table). The median cumulative exposure to a covalent BTKi in group 3 was 27 mo (range, 1-67), with 32 pts having a cumulative exposure of ≤36 mo. BTK C481 mutations were only detected among the 16 pts with cumulative exposure to a covalent BTKi of &amp;gt;36 mo. Of 37 pts who were receiving BTKi therapy at the time of blood collection, 5 (14%) had a detectable BTK C481 mutation. Conclusions: In this real-world cross-sectional study, BTK C481 mutations were observed in 21% of BTKi-experienced pts with CLL/SLL, all of whom had a cumulative exposure to BTKis of &amp;gt;36 mo. Because two-thirds of BTKi-experienced pts had &amp;lt;36 mo of BTKi exposure, the prevalence of BTK C481 mutations may be higher in populations with longer BTKi exposure. Further analyses of the natural history of BTK C481 mutations will help identify patients who are at risk of covalent BTKi resistance. Recruitment for this study is ongoing.

Investigation into Intraclonal Heterogeneity of CXCR4 DimCD5 Bright Chronic Lymphocytic Leukemia Cells Identifies Distinct Activation Signatures

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease associated with diverse clinical and molecular profiles, suggesting long-term clonal evolution driven by genetic diversification from a small, often undetected, subset of leukemic cells endowed with superior growth capacities. More importantly, this diversification process enhances the opportunity of CLL subclones to survive selective pressure, such as BTK or PLCG2 mutations that can abolish the effect of BTK inhibitors. While this evolutionary process is inevitable in leukemic cells, therapeutic intervention targeting the cells that have just replicated their DNA, and hence could have accumulated new genomic mutation, would be an effective strategy to significantly delay or abort the emergence of deleterious mutations. In CLL, a series of studies involving consumption of 2H 2O determined that CXCR4 DimCD5 Bright cells (proliferative fraction, PF) of CLL clones contain most of the recently divided cells, identified by the presence of 2H-DNA. This conclusion was supported by finding elevated numbers of Ki67 + and MCM6 + cells in the same fraction, when compared to other CLL cells. Furthermore, PF is superior in: lymphoid tissue homing in xenograft models; activating T cells; the capacity to alter tumor microenvironment (TME) by Th2 skewing; and expression of AICDA which can increase the likelihood of off-target genomic mutations. To permit a focused analysis of PF that would allow us to intercept this evolutionary loop, we characterized the single cell transcriptomes of PF from 4 U-CLL and 4 M-CLL patients immediately prior to ibrutinib treatment. Consistent with previous reports, the majority of PF (herein APC-PF) cells express certain antigen presentation molecules with a gene signature suggestive of T cell regulation and cell-cell interactions. Additionally, we discovered a small subpopulation with an upregulation of MIR155HG, HSP90AB, CCL3, CCL4, MYC and a concomitant MYC activation signature (herein MYC-PF). Since miR-155 expression can be induced by CD40L or B-cell-activating factors, this PF subset may reflect prior BCR ligation and/or T cell co-stimulation. However, the strongest BCR activation signature was found in another PF subpopulation (herein BCR-PF) with an enrichment of germinal center-related genes (e.g., BACH2, PAX5, FOXP1) and CLL-associated molecules ( BCL2, ROR1, MSI2, PLCG2). Furthermore, the BCR-PF cells are least likely to be resting (in G1/G0) compared to others. Collectively, these findings suggest effective BCR engagement, either from the TME or cell-autonomously. Corroborating these findings with our previous studies documenting the CXCR4/CD5 fractions and the expression of protein proliferation markers, our transcriptomic analyses suggest that the BCR-PF may be the most immediate emigrant from the proliferation centers (PC) within CLL lymph nodes (LN). To probe this possibility, spatial transcriptomic analysis was performed on FFPE LN tissues from surgical biopsies from 3 U-CLL and 1 M-CLL patients. Single cell deconvolution was used to determine the relative abundance of cells within the anatomically defined PCs that resemble the three PF subpopulations transcriptomically. Our results suggest that PCs are more enriched in cells resembling BCR-PF, followed by cells bearing the APC-PF signature. To extend on these observations, we have documented that the unique surface markers expressed in these PF subfractions positively correlate with our transcriptomic results. Taken together, these results not only highlighted the molecular heterogeneity of CLL, but also lead to the identification of two PF subpopulations that may provide novel actionable therapeutic targets to significantly halt CLL clonal evolution.

CD19-Directed CAR T-Cells (CD19-CAR) Combined with Acalabrutinib Achieves Durable Remissions in Patients with Relapsed or Refractory (r/r) Mantle Cell Lymphoma (MCL)

Background: MCL is an aggressive non-Hodgkin lymphoma (NHL) subtype which remains incurable despite improvements in progression free survival with consolidative autologous transplant and the introduction of novel targeted therapies [Dreyling et al. Blood 2012]. CD19-directed chimeric antigen receptor (CAR) T-cell therapy (CD19-CAR) has shown durable disease control in r/r MCL, but current standard of care products are limited by high rates of grade 3+ immune-related toxicities [Wang et al. NEJM 2020]. We hypothesized that combining a naïve/stem-memory (T N/SCM) phenotype-enriched CD19-CAR T-cell product with the selective BTK inhibitor (BTKi) acalabrutinib would attenuate the rates of severe toxicities, improve durable remission rates, and obviate the need for indefinite use of acalabrutinib in r/r MCL. Methods: We conducted a phase 1 single center, prospective clinical trial (NCT04484012) evaluating a single infusion of T N/SCM-enriched CD19.28.z.EGFRt-CAR at a target dose of either 2x10 8 (DL1) or 5x10 8 (DL2) CAR+ cells (≤20% lower dose allowed) in combination with ongoing acalabrutinib 100 mg twice daily for patients with r/r MCL after at least 1 prior line of therapy. Patients were required to undergo at least 3 months but less than 7 months of treatment with a BTKi prior to enrollment without evidence of progression and with measurable disease per Lugano criteria. All patients were switched to acalabrutinib for protocol therapy prior to lymphodepletion, and planned to continue until day 180 post CD19-CAR infusion at which time all therapy would be discontinued. We report outcomes from the dose finding portion of the study. Descriptive statistics were utilized for baseline characteristics and survival analyses were calculated utilizing the Kaplan-Meier method. Investigator-assessed response rates were based on Lugano criteria. Minimal residual disease (MRD) status was determined using either multiparameter flow cytometry (FCM) or next-generation sequencing (NGS). Results: To date, 8 patients enrolled and have received CD19-CAR (n=4 at DL1; n=4 at DL2). Manufacturing was successful in 100% of patients. Median time from leukapheresis to product infusion was 53 days (range, 41-114). Treated patients were 75% male; with a median age of 63 years (range, 38-70); and median number of prior therapies of 2 (range, 2-3). All patients had bone marrow involvement, and had high risk features including progression of disease within 24 months (n=6), relapse after autologous HCT (n=2), TP53 mutations (n=6), Ki-67 ≥30% (n=4), and/or complex karyotype (n=1). Prior to enrollment, all patients had received BTKi (n=8 acalabrutinib) for a median of 6.1 months (range, 4.5-7.3). No BTK and PLC-γ mutations were detected in patients with available samples. The combination of acalabrutinib and CD19-CAR was well tolerated; 5 patients (63%; n=1 grade 2; n=4 grade 1) had cytokine release syndrome (CRS), with no severe cases. No patients (0%) had immune effector cell associated neurotoxicity syndrome (ICANS) [Figure 1A]. No patient required corticosteroids for toxicity management, and only 2 patients (n=2/5, 40%) required tocilizumab for CRS. There were no serious adverse events (SAEs), and no treatment discontinuations due to AEs observed. The best ORR was 88% [n=6 CR (75%); n=1 PR (13%)], with median time to best response of 28 days. In those 6 patients who achieved CR, 2 patients were FCM-MRD negative (&amp;lt;10 -4) and 2 were NGS-MRD negative (&amp;lt;10 -6) (n=4/6, 67%) [Figure 1B]. After a median follow-up of 18.5 months (range, 8.3-28.9), 5 of 6 patients remain in CR, while1 patient who achieved an initial CR relapsed at 9.2 months. At 1-year post-infusion, the cumulative incidence of relapse and NRM were 30% (95% CI, 3%-67%) and 0%, respectively. The 1-year PFS and OS were 70% (95% CI: 22%-92%) and 100%, respectively. Conclusion: The combination of CD19-CAR with acalabrutinib was well tolerated and effective, yielding an ORR 88% and MRD- rate 50% in a cohort of patients with high-risk MCL. Notably, CRS events were manageable and low grade, and no ICANS was observed. Enrollment is ongoing in the expansion cohort (DL2), and additional clinical and correlative analyses will be presented at the meeting.

Single-Cell Long-Read Sequencing Enables the Dissection of BTK Subclonal Dynamics in CLL Treatment

Introduction: BTK inhibition is an effective frontline therapy for patients with CLL. However, patients who develop BTK C481S mutations relapse quickly. We recently published a longitudinal study investigating subclonal evolution in 38 patients receiving BTKi treatment for CLL. Of the 38 patients, 6 developed a subclone containing a BTK C481S mutation identified in the whole-exome sequencing (WES) data at the time of relapse. To understand whether a BTK C481S mutation alone can drive relapse or if an additional CLL-relevant mutation is required, we investigate these subclones on a single-cell level using PacBio Multiplexed Arrays Sequencing (MAS-seq). MAS-seq generates single-cell, long-read RNA sequencing data, which is highly accurate and provides coverage across the entire mRNA transcript. Methods: For each patient, B cells were isolated from a pre-BTKi treatment sample and a sample taken at the time of relapse. Single-cell cDNA library preps were generated using the 10X Genomics Chromium platform. The cDNA was then sequenced using MAS-Seq. After processing the MAS-seq data using PacBio's Isoseq pipeline, Seurat was used to perform a single-cell analysis for each sample. We used scBayes, a Bayesian-statistical approach developed in our lab (Qiao et al., Genome Research, in review), to assign subclone identity to individual cells based on the expression of previously identified somatic mutations. The transcript-wide coverage of MAS-seq increases the likelihood of coverage at the somatic mutation sites compared to traditional single-cell RNA sequencing methods. Results: We have sequenced and analyzed samples from 4 patients in this study (pt 1-4). In the pretreatment sample of pt 1, two subclones containing different TP53 mutations were present. At the time of relapse, a BTK C481S subclone evolved from one of these TP53 subclones, becoming the dominant clonal population. No other CLL-relevant mutations were detected in this patient. Pts 2-4 had an additional CLL-relevant mutation evolve with the BTK C481S mutation in the dominant subclone at the time of relapse. In pt 2, a subclone with an NFKBIE and the BTK mutation evolved from a cell population where BRAF, BCOR, and EGFR2 mutations were present. The BTK C481S subclone in Pt 3 also had new KMT2D and CREBBP mutations. This subclone evolved from a cell population with an ASXL1 and SF3B1 mutation. In pts 1-3, the BTK subclone was only seen at the time of relapse. Pt 4 did not have any CLL-relevant mutations identified before treatment. In a sample taken 6 months before relapse, a BTK C481S subclone is visible at a low frequency with no other CLL-relevant mutations. At the time of relapse, a DICER1 variant, a gene recently implicated in CLL (Knisbacher et al., Nature Genetics, 2022), evolved from the BTK subclone and is seen at the time of relapse as the dominant subclone. Furthermore, the full-transcript sequencing at the single-cell level enabled us to identify a second BTK C481S subclone present at the time of relapse and determine that it was independent of the subclone containing both the BTK C481S and DICER1 mutations. This second BTK subclone had no other CLL-relevant mutations and remained at a very low frequency. Conclusion: Single-cell long-read RNA sequencing via MAS-seq provides transcript-wide coverage, which enables genotyping at somatic mutation sites. Using this method, we found that the dominant relapse clones all contained a BTK C481S mutation, which either co-evolved with additional CLL-relevant mutations (3/4 patients) or occurred on the background of existing TP53 mutations (1/4 patients).

Absence of BTK, BCL2, and PLCG2 Mutations in Chronic Lymphocytic Leukemia Relapsing after First-Line Treatment with Fixed-Duration Ibrutinib plus Venetoclax.

Mutations in BTK, PLCG2, and BCL2 have been reported in patients with progressive disease (PD) on continuous single-agent BTK or BCL2 inhibitor treatment. We tested for these mutations in samples from patients with PD after completion of first-line treatment with fixed-duration ibrutinib plus venetoclax for chronic lymphocytic leukemia (CLL) in the phase II CAPTIVATE study. A total of 191 patients completed fixed-duration ibrutinib plus venetoclax (three cycles of ibrutinib then 12-13 cycles of ibrutinib plus venetoclax). Genomic risk features [del(11q), del(13q), del(17p), trisomy 12, complex karyotype, unmutated IGHV, TP53 mutated] and mutations in genes recurrently mutated in CLL (ATM, BIRC3, BRAF, CHD2, EZH2, FBXW7, MYD88, NOTCH1, POT1, RPS15, SF3B1, XPO1) were assessed at baseline in patients with and without PD at data cutoff; gene variants and resistance-associated mutations in BTK, PLCG2, or BCL2 were evaluated at PD. Of 191 patients completing fixed-duration ibrutinib plus venetoclax, with median follow-up of 38.9 months, 29 (15%) developed PD. No baseline risk feature or gene mutation was significantly associated with development of PD. No previously reported resistance-associated mutations in BTK, PLCG2, or BCL2 were detected at PD in 25 patients with available samples. Of the 29 patients with PD, 19 have required retreatment (single-agent ibrutinib, n = 16, or ibrutinib plus venetoclax, n = 3); 17 achieved partial response or better, 1 achieved stable disease, and 1 is pending response assessment. First-line fixed-duration combination treatment with ibrutinib plus venetoclax may mitigate development of resistance mechanisms associated with continuous single-agent targeted therapies, allowing for effective retreatment. See related commentary by Al-Sawaf and Davids, p. 471.

Acquired Mutations in Patients (Pts) with Relapsed/Refractory (R/R) Chronic Lymphocytic Leukemia (CLL) That Progressed in the ALPINE Study

Introduction:Pts who receive covalent Bruton tyrosine kinase inhibitors (cBTKis) for CLL can develop drug resistance, leading to disease progression. Often, cBTKi resistance results from the emergence of subclones with mutations at the cBTKi binding site (C481). Less frequent, non-C481 mutations, including gatekeeper residue T474 and kinase-impaired L528 mutations, have been reported in pts with progression on cBTKis. Most characterizations of cBTKi resistance mutations are derived from small or retrospective pt populations. To gain further insight into the genetic mechanisms of cBTKi resistance in a randomized population of pts with CLL, we performed next-generation sequencing (NGS) on samples from pts who had progression on zanubrutinib (zanu) or ibrutinib (ibr) in the phase 3 ALPINE study (NCT03734016; Brown et al. NEJM. 2023). Methods:Progressive disease (PD) in ALPINE pts was assessed by independent review committee (IRC; n=139; zanu, n=54; ibr, n=85) and/or by investigator (INV; n=132; zanu, n=54; ibr, n=78) using Hallek et al ( Blood. 2008) criteria. Peripheral blood samples were collected at or after PD from 57 pts (zanu, n=26; ibr, n=31); 94.7% (54/57) had paired baseline samples. In 57 pts with PD, the median study follow-up was 25.7 mo; the median number of prior lines of therapy was 1 (range: zanu, 1-3; ibr, 1-7). Four pts (zanu, n=2; ibr, n=2) had Richter transformation (RT) at PD. A high-sensitivity NGS panel (PredicineHEME) with full exon coverage of 106 genes, including BTK, PLCG2 and 27 putative CLL driver genes (Knisbacher et al. Nat Gen. 2022), was used for mutation analysis. All acquired mutations in BTK and PLCG2were reported at a variant allele frequency (VAF) of ≥0.25%. For all other genes, pathogenic mutations (assessed using VarSome) with a VAF of ≥1% are reported. Del(17p), del(11q), trisomy 12, del(13q), IGHV mutation status, and complex karyotype (CKT) status were assessed in baseline samples. Results: No BTK mutations were detected at baseline, but 2 pts had PLCG2 mutations (zanu, R589C; ibr, E914STOP). To determine rates of BTK/PLCG2 or CLL driver gene mutations, only pts with paired baseline and PD samples and without RT at PD (n=52; zanu, n=24; ibr, n=28) were included (Table 1). Nine pts acquired BTK/ PLCG2 mutations: 8 (zanu, n=5 [20.8%]; ibr, n=3 [10.7%]) had BTK mutations, and 2 (both in ibr; 7.1%) had PLCG2 mutations; 1 pt had both BTK and PLCG2 mutations. Of 18 single nucleotide variants (SNV) in BTK, 77.8% (n=14; zanu, n=11; ibr, n=3) were at C481. Non-C481 mutations were detected in 12.5% of pts (3/24) with progression on zanu (Table 2). All 9 pts with acquired BTK/ PLCG2 mutations had PD assessed by IRC and INV during treatment, except 1 ibr pt who had PD assessed by INV after treatment. Median treatment duration was shorter in pts with PD without BTK mutations (zanu: n=19; 16.8 mo; range, 4.4-33.3 mo; ibr: n=25; 15.9 mo; range, 5.9-29.4 mo) than in pts with PD with acquired BTK mutations (zanu: n=5; 29.7 mo; range, 18.4-34.6 mo; ibr: n=3; 30.8 mo; range, 11.8-34.5 mo). At baseline, SNV or indels in 18/27 driver genes were observed in 48/52 pts. Most frequent: NOTCH1 (n=21), TP53 (n=19), and ATM (n=8). Copy number aberrations (CNA) in 9/27 driver genes were observed in 23 pts. Most frequent: CCND2 (n=10, amplification [amp]), ATM (n=8, deletion [del]), TP53 (n=6, del), and KMT2D (n=6, amp). At PD, acquired SNV were observed in 3 pts ( SETD2, ASXL1, and SF3B1 n=1 each; all ibr). Acquired CNA in driver genes were observed in 10 pts ( KRAS amp: zanu, n=3; NRAS amp: ibr, n=2; CDKN1B amp: zanu, n=2, ibr, n=1; BIRC3 del: ibr, n=2). Acquisition of driver gene mutations was not associated with del(17p), IGHV mutation, or CKT status. Conclusion: Of the 52 pts, most (82.6%) did not have acquired BTK or PLCG2 mutations. Among the zanu pts, 3/24 (12.5%) developed non-C481 BTK mutations. This rate was lower than that reported by Woyach et al (ICML 2023); shorter follow-up and fewer prior therapies in the ALPINE study may explain this discrepancy. Additionally, these data suggest that BTK and/or PLCG2 mutations are not the sole factors driving PD in this pt population; the presence or acquisition of mutations in driver genes with known negative prognostic value in CLL may also contribute to PD in pts without BTK or PLCG2 mutations. Given the low incidence of non-C481 mutations in ALPINE pts with PD, pts with CLL who were treated with cBTKis are likely to remain sensitive to other BTK-targeting therapies.