Landscape Of Chronic Lymphocytic Leukemia Research Articles

Current Therapeutic Sequencing in Chronic Lymphocytic Leukemia

The treatment landscape of chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults, is constantly changing. CLL patients can be divided into three risk categories, based on their IGHV mutational status and the occurrence of TP53 disruption and/or complex karyotype. For the first-line treatment of low- and intermediate-risk CLL, both the BCL2 inhibitor venetoclax plus obinutuzumab and the second generation BTK inhibitors (BTKi), namely acalabrutinib and zanubrutinib, are valuable and effective options. Conversely, venetoclax-based fixed duration therapies have not shown remarkable results in high-risk CLL patients, while continuous treatment with acalabrutinib and zanubrutinib displayed favorable outcomes, similar to those obtained in TP53 wild-type patients. The development of acquired resistance to pathway inhibitors is still a clinical challenge, and the optimal treatment sequencing of relapsed/refractory CLL is not completely established. Covalent BTKi-refractory patients should be treated with venetoclax plus rituximab, whereas venetoclax-refractory CLL may be treated with second generation BTKi in the case of early relapse, while venetoclax plus rituximab might be used if late relapse has occurred. On these grounds, here we provide an overview of the current state-of-the-art therapeutic algorithms for treatment-naïve patients, as well as for relapsed/refractory disease.

Dual-targeted regimens for the frontline treatment of CLL.

The treatment landscape of chronic lymphocytic leukemia (CLL) has evolved considerably over the past decade due to the development of effective novel agents with varying mechanisms of action, including Bruton tyrosine kinase (BTK) and B-cell lymphoma 2 (BCL2) inhibitors. Extrapolating upon the success of anti-CD20-directed chemoimmunotherapy, a dual-targeted approach has been explored in treatment-naive patients with CLL. Anti-CD20 monoclonal antibody combinations with BTK inhibitors as well as BCL2 inhibitors have demonstrated superiority over traditional cytotoxic chemoimmunotherapy regimens such as fludarabine, cyclophosphamide, and rituximab; bendamustine-rituximab; and obinutuzumab-chlorambucil. Impressive clinical benefit is seen in both younger and older patients, those with comorbidities, and, most importantly, those with poor prognostic features. Given this success, combinations of BTK inhibitors and venetoclax have been explored in clinical trials. These dual-targeted regimens provide remarkable efficacy while allowing for an all-oral approach and fixed duration of treatment. Current investigations under way are evaluating the utility of a triplet approach with the addition of obinutuzumab in comparison to a doublet approach.

Impact of Real-World Treatment Sequencing Patterns on Time to Next Treatment among Patients with Chronic Lymphocytic Leukemia in the United States

Background: The treatment landscape for chronic lymphocytic leukemia (CLL) is evolving at a rapid pace, with development of several targeted therapies. The real-world applicability of clinical trial effectiveness is markedly influenced by treatment choices, the specific characteristics of patients' diseases, their overall health, and individual preferences. However, there is limited evidence to understand the impact of multiple treatment options on real-world effectiveness in CLL in routine clinical practice. This study aimed to characterize recent real-world treatment sequencing patterns and associated time to next treatment among patients with CLL in the United States. Methods: This retrospective observational study included patients diagnosed with CLL (≥18 years at diagnosis) from January 1, 2018, through January 1, 2023, who received ≥1 treatment after initial diagnosis. Patients were sourced from the ConcertAI RWD360 dataset, a real-world de-identified dataset >6 million patients from across the United States derived from various electronic health record systems and integrated with a large administrative open claims dataset. Patients were followed through the end of the record or death, whichever occurred first. Patients were classified into the following treatment categories: chemotherapy (including bendamustine-based and other chemotherapy), anti-CD20-based, Bruton tyrosine kinase inhibitor (BTKi)-based (ibrutinib, acalabrutinib, or zanubrutinib), and venetoclax-based therapy. Demographic and clinical characteristics, treatment sequencing pattern, and time to next treatment were examined across lines of therapy for each treatment category. Kaplan-Meier analysis was used to estimate median time to next treatment. Results: The study included 3,431 patients with CLL (median age: 69 years; male: 63.8%). There was a significant variation in average age among the 4 treatment groups ( P<0.0001). Compared with the overall population, patients who received anti-CD20-based therapy were slightly older (median age: 71 years), while patients who received venetoclax-based therapy were relatively younger (median age: 67 years). Patients were primarily treated in community rather than academic settings (76.4% vs 21.3%). First-line patients were most commonly treated with BTKi (60.1%), venetoclax (16.0%), and anti-CD20 (15.4%) regimens. Among patients who initiated BTKi therapies, zanubrutinib use (2.6%) was limited; this may be due to the recent approval date (January 2023). Among CLL patients, 35.0% received second-line treatment and BTKi stayed dominant (35.7%), followed by anti-CD20 (29.6%) and venetoclax (25.8%) therapies. From first- to second-line therapy, the prevailing sequencing pattern was from either one BTKi to another BTKi (23.5%), venetoclax to anti-CD20 (13.2%), or BTKi to an anti-CD20 (9.7%). From second- to third-line therapy, patients most frequently switched from anti-CD20 to venetoclax (24.6%), anti-CD20 to BTKi (13.3%), or one BTKi to another BTKi (12.6%). From third- to fourth-line therapy, the predominant sequencing pattern was from venetoclax to anti-CD20 (21.3%), anti-CD20 back to venetoclax (20.6%), or one BTKi to another BTKi (11.6%). Median time to next treatment in first line was significantly longest for patients receiving BTKi (53.9 months) and shortest for patients treated with venetoclax (7.7 months) ( P<0.0001) (Figure 1). Median time to next treatment in second line was not reached among patients using BTKi, while it was significantly shorter for those on venetoclax and anti-CD20 (10.4 months and 1.5 months, respectively; P<0.0001) (Figure 2). Conclusions: This studydemonstrated unique treatment sequencing patterns among patients with CLL in the real-world setting.As the most commonly used regimen, BTKi therapy was shown to have significantly improved time to next treatment compared with other treatment regimens in first and second lines of therapies. Future studies with extended follow-up are needed to allow for assessment of newer treatments and enable evaluation of long-term clinical outcomes.

Characterization and Preclinical Evaluation of AS-1763, an Oral, Potent and Selective Noncovalent BTK Inhibitor, in Chronic Lymphocytic Leukemia

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors (cBTKi) have transformed treatment landscape of chronic lymphocytic leukemia (CLL). These inhibitors bind to C481 residue in the kinase domain of BTK. This is also the site for the most common mutations rendering cells resistant to cBTKi. To circumvent this limitation of the cBTKi, non-covalent BTKi (ncBTKi) such as pirtobrutinib have been developed. Recently, non-C481 BTK mutations have been reported in patients with CLL at the time of disease progression during pirtobrutinib treatment. These observations underscore the need for ncBTKi that can target C481 as well as non-C481 mutations of BTK. AS-1763 is a potent, highly selective, orally available, and ncBTKi, equipotent against both wild-type and C481S-mutated BTK when tested in biochemical assays. In vivo, AS-1763 demonstrated significant antitumor effects in OCI-LY10 tumor xenograft models harboring wild-type or C481S mutant BTK (Kawahata et al. J Med Chem 64:14129, 2021). Study Design and Methods: In the present project, first we used cell free assay systems to evaluate selectivity and potency of AS-1763 by a kinome-wide profiling and inhibitory effect of AS-1763 in enzyme assays using recombinant mutant BTK. Second, we examined dose- and time-dependent inhibition of mutant BTK that is expressed in HEK293 cell line. Third, we tested biological, biochemical, and molecular impact of AS-1763 in primary CLL cells. Fourth, we determined sensitivity of CLL cells to AS-1763 when combined with other targeted agents. Results: AS-1763 showed a highly selective profile for BTK in a panel of 291 kinase assays with >260-fold selectivity except 3 Tec family kinases (BMX, ITK, and TEC). We have generated a total of 17 recombinant BTK mutant proteins (C481, T474, L528 variants and other BTK mutants; Table 1) reported in the literature or predicted by single nucleotide change in the codon, and established assay methods to measure inhibitory potency of BTKi for those BTK mutants (Table 1). AS-1763 showed potent inhibitory activities for those BTK mutants while inhibitory potencies of other cBTKi and ncBTKi were diminished against some BTK mutants such as T474 and/or L528 mutations. AS-1763 exhibited dose-dependent and slow-off rate inhibitions of BTK autophosphorylation (pY223) in HEK293 cells transfected with various BTK mutants. Furthermore, the observed inhibitory effects of AS-1763 on the BTK autophosphorylation (pY223) in HEK293 cells were continued up to 24 h after washing out of AS-1763. In primary CLL samples, AS-1763, pirtobrutinib or ibrutinib induced a modest apoptosis. AS-1763 effectively inhibited the BCR signaling pathway in a dose dependent manner as evidenced by downregulation of pY223-BTK expression which was also observed in cBTKi and ncBTKi relapsed/refractory CLL samples. In vitro incubations with AS-1763 inhibited CLL cell spontaneous migration, decreased CCL3/CCL4 levels in culture supernatant and was accompanied with the inhibition of intracellular calcium release and B-cell activation, as measured by surface CD86 expression. Besides, AS-1763 incubation for 24 hours was shown to modulate the expression of BCL-2 family proteins with the downregulation of MCL-1 and BCL-xl. Interestingly, in vitro treatment of CLL patient samples with AS-1763 demonstrated a notable elevation in cellular ROS and mitochondrial superoxide levels starting at 1 µM, concurrently impacting SOD1 expression in CLL patient samples. Evaluation of drug interaction models utilizing Compusyn and Synergy Finder applications predominantly indicated additive effects between AS-1763 with BCL-2 inhibitor, venetoclax as well as p53 activator APR-246. Consistent with this data, AS-1763 and venetoclax combinations showed high-rates of apoptosis in samples that were relapsed/refractory to cBTKi and ncBTKi. Conclusions: AS-1763 is a selective ncBTKi that inhibits both wild-type and mutant BTKs listed in Table 1. In CLL cells, AS-1763 was effective in inhibiting BCR pathway signaling and sensitized cells to other agents such as venetoclax. Based on these encouraging data we have initiated a clinical trial to test AS-1763 in patients with CLL and other B cell malignancies who have failed or are intolerant to at least two prior lines of systemic therapy, including cBTKi (NCT05602363 Clinical Trials.gov).

Patient Preferences for Fixed Versus Treat-to-Progression Therapies in Chronic Lymphocytic Leukemia

The treatment landscape for chronic lymphocytic leukemia (CLL) has evolved in the last 8 years to include novel targeted therapies as alternatives to chemoimmunotherapy. These treatments vary in their benefit-risk profiles, mode and frequency of administration, and duration of treatment. Previous preference studies in CLL have evaluated preferences for treatment attributes such as efficacy, tolerability, and mode and frequency of administration. However, few have assessed preferences for treatment duration independently of mode of administration. This study sought to further understand the factors that influence patient preferences for treatment duration and to quantify the tradeoffs that patients were willing to accept to have a fixed duration therapy versus a treat-to-progression therapy. The study was conducted in 2 phases: (1) a qualitative phase of in-depth individual interviews with patients to identify the factors that influence patient preferences for treating until progression versus a fixed duration as well as the CLL treatment attributes that patients consider most important, and (2) a quantitative phase to estimate the tradeoffs that patients would accept among CLL treatment attributes. In the qualitative phase, the semistructured interview guide included open-ended questions and probes to understand perceptions of fixed-duration treatments compared with treat-to-progression regimens and to elicit a list of treatment attributes that influence CLL treatment preference. The results of the interviews informed the development of a web-based discrete-choice experiment (DCE) survey. Respondents to the online survey answered 12 DCE questions, each offering a choice between 2 hypothetical treatment profiles defined by 7 attributes with varying levels, including treatment duration, which included levels for treat to progression and fixed duration for either 6 or 12 months. Data were analyzed using a random-parameters logit model, and estimated preference weights were used to calculate the maximum acceptable risk (MAR) of treatment-related adverse events the average respondent would accept in exchange for a move from a treat-to-progression to a fixed-duration therapy. The MAR is estimated as the ratio of the relative importance of an improvement in an attribute to the relative importance of a unit change in the level of risk (i.e., tumor lysis syndrome (TLS), atrial fibrillation, or fatigue). Interviews were conducted with 20 adults with a self-reported diagnosis of CLL. The mean age was 59 years, and 55% of participants (n = 11) identified as female. The mean time since diagnosis of CLL was 3 years, and 70% of participants (n = 14) had received treatment for CLL. Overall, 93 treatment attributes were spontaneously reported, including treatment duration. When probed, treatment duration was reported by 50% of participants as “very important” in their treatment decision. Almost all participants (n = 17) preferred a treatment with a fixed duration compared with treat-to-progression, assuming each had the same efficacy. Table 1 reports patients' perceived benefits and drawbacks of fixed-duration versus treat-to-progression therapies. The DCE survey was completed by 229 adults with a self-reported diagnosis of CLL for at least 3 months. The mean age was 66 years, and nearly 60% of the sample identified as female (n = 136). About 60% of the sample (n = 138) was diagnosed 5 or more years ago, and 152 respondents (66%) had experience with treatment for CLL. The results of the preference analysis showed that respondents preferred a fixed duration of either 6 months or 12 months versus treat-to-progression and, based on the MAR estimates, were willing to accept the following levels of risks to have a fixed-duration treatment versus treat to progression: more than a 3% increased risk of TLS, 6%-7% increased risk of atrial fibrillation, and 21%-26% increased risk of fatigue (Table 2). Past research has shown efficacy as the most important factor, yet qualitative interview participants also identified treatment duration as an important factor in their decision when choosing a CLL therapy. This finding was confirmed by the quantitative preference study, which revealed a preference for fixed-duration therapies over treat-to-progression regardless of the timeframe (6 or 12 months). Results from this study can help inform shared decision-making when considering alternative therapies for CLL.

Real-world adherence and discontinuation among Medicare beneficiaries initiating venetoclax vs. BTKis in relapsed/refractory chronic lymphocytic leukemia

The treatment landscape for chronic lymphocytic leukemia (CLL) has been transformed by the availability of Bruton’s tyrosine kinase inhibitors (BTKis) and the B-cell lymphoma 2 (BCL-2) inhibitor venetoclax. Despite clinical trial data supporting these novel oral agents, evidence evaluating real-world adherence is limited. This study used 2015–2019 Medicare claims data for elderly patients with relapsed/refractory CLL to assess differences in real-world adherence and discontinuation in the 12 months after treatment initiation. In the final sample of 711 venetoclax patients and 1,566 BTKi patients, we found that those initiating venetoclax tended to be younger (mean age 75.6 [SD 6.0] vs 77.6 [SD 6.9] years, p < .001) but had poorer clinical characteristics. After risk-adjustment, the venetoclax group had higher adherence (61.9% vs. 45.4%, p < .0001) and lower discontinuation when compared to the BTKi group (28.5% vs. 47.4%, p < .001). These favorable real-world findings underscore the importance of developing well-tolerated novel combinations for older adults.

Ultra-deep mutational landscape in chronic lymphocytic leukemia uncovers dynamics of resistance to targeted therapies.

BTK inhibitors, Bcl-2 inhibitors, and other targeted therapies have significantly improved the outcomes of patients with chronic lymphocytic leukemia (CLL). With increased survivorship, monitoring disease and deciphering potential mechanisms of resistance to these agents are critical for devising effective treatment strategies. We used duplex sequencing, a technology that enables detection of mutations at ultra-low allelic frequencies, to identify mutations in five genes associated with drug resistance in CLL and followed their evolution in two patients who received multiple targeted therapies and ultimately developed disease progression on pirtobrutinib. In both patients we detected variants that expanded and reached significant cancer cell fractions (CCF). In patient R001, multiple known resistance mutations in both BTK and PLCG2 appeared following progression on zanubrutinib (BTK p.L528W, p.C481S; PLCG2 S707F, L845F, R665W, and D993H). In contrast, patient R002 developed multiple BTK mutations following acalabrutinib treatment, including known resistance mutations p.C481R, p.T474I and p.C481S. We found that pirtobrutinib was able to suppress, but not completely eradicate, BTK p.C481S mutations in both patients, but other resistance mutations such as mutations in PLCG2 and new BTK mutations increased while the patients were receiving pirtobrutinib. For example, BTK p.L528W in patient R001 increased in frequency more than 1,000-fold (from a CCF of 0.02% to 35%), and the CCF in p.T474I in patient R002 increased from 0.03% to 4.2% (more than 100-fold). Our data illuminate the evolutionary dynamics of resistant clones over the patients' disease course and under selective pressure from different targeted treatments.

How I Manage Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL), is a hematologic malignancy characterized by the uncontrolled proliferation of mature B lymphocytes. CLL is the most prevalent leukemia in Western countries. Its presentation can range from asymptomatic with the incidental finding of absolute lymphocytosis on a routine blood test, to symptomatic disease requiring immediate intervention. Prognosis of the disease is defined by the presence or absence of specific mutations such as TP53, chromosomal abnormalities such as del(17p), a type of IGHV mutational status, and elevation of B2M and LDH. Treatment of CLL in the United States and Europe has evolved over the recent years thanks to the development of targeted therapies. The standard of care has shifted from traditional chemoimmunotherapy approaches to targeted therapies including Bruton tyrosine kinase inhibitors (BTKis) and BCL2 inhibitors, administered either as monotherapy or in combination with CD20 monoclonal antibodies. Several clinical trials have also recently evaluated combinations of BTKi and venetoclax and showed the combination to be well tolerated and able to induce deep remissions. Targeted therapies have a good safety profile overall; however, they also have unique toxicities that are important to recognize. Diarrhea, fatigue, arthralgia, infections, cytopenias, bleeding, and cardiovascular toxicities (including atrial fibrillation, ventricular arrhythmias, and hypertension) are the adverse events (AEs) commonly associated with BTKis. Initiation of therapy with venetoclax requires close monitoring because of the risk for tumor lysis syndrome associated with this agent, particularly in patients with a high disease burden. Development of newer target therapies is ongoing and the therapeutic landscape in CLL is expanding rapidly.

Population-level impact of ibrutinib for chronic lymphocytic leukemia in British Columbia, Canada

Ibrutinib has dramatically changed the treatment landscape for chronic lymphocytic leukemia (CLL) since its availability in British Columbia (BC), Canada in 2014. We analyzed patterns of use and real-world survival outcomes in 370 patients who received ibrutinib for first-line (1 L, n = 35) and relapsed/refractory (R/R, n = 335) CLL between 2014–2018 in BC. Dose reductions and interruptions were frequent in 32% and 27%, respectively. With a median follow-up of 27.6 months, 35% of patients discontinued ibrutinib, primarily for adverse events (AEs) rather than progressive disease. Over the course of treatment, 87% of patients experienced at least one adverse event. The 2-year overall survival (OS) and event-free survival (EFS) were excellent at 83.9% and 76.1%, respectively, with medians not reached. However, patients who discontinued ibrutinib had a median OS of 32.5 months and median EFS of only 3.8 months from time of discontinuation, highlighting the need to minimize toxicity in the real-world.

Chromatin activation profiling of stereotyped chronic lymphocytic leukemias reveals a subset 8–specific signature

AbstractThe chromatin activation landscape of chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptor immunoglobulin is currently unknown. In this study, we report the results of a whole-genome chromatin profiling of histone 3 lysine 27 acetylation of 22 CLLs from major subsets, which were compared against nonstereotyped CLLs and normal B-cell subpopulations. Although subsets 1, 2, and 4 did not differ much from their nonstereotyped CLL counterparts, subset 8 displayed a remarkably distinct chromatin activation profile. In particular, we identified 209 de novo active regulatory elements in this subset, which showed similar patterns with U-CLLs undergoing Richter transformation. These regions were enriched for binding sites of 9 overexpressed transcription factors. In 78 of 209 regions, we identified 113 candidate overexpressed target genes, 11 regions being associated with more than 2 adjacent genes. These included blocks of up to 7 genes, suggesting local coupregulation within the same genome compartment. Our findings further underscore the uniqueness of subset 8 CLL, notable for the highest risk of Richter’s transformation among all CLLs and provide additional clues to decipher the molecular basis of its clinical behavior.

Landscape of BCL2 Resistance Mutations in a Real-World Cohort of Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia Treated with Venetoclax

The oral, highly selective Bcl2 inhibitor venetoclax has substantially improved the therapeutic landscape of chronic lymphocytic leukemia (CLL). Despite the remarkable response rates in patients with relapsed/refractory (R/R) disease, acquired resistance is the leading cause of treatment failure, with somatic BCL2 mutations being the predominant genetic drivers underpinning venetoclax resistance. To assess the correlation between disease progression and the most common BCL2 mutations G101V and D103Y, sensitive (10-4) screening for the most common BCL2 mutations G101V and D103Y was performed in 67 R/R CLL patients during venetoclax single-agent or venetoclax-rituximab combination therapy. With a median follow-up time of 23 months, BCL2 G101V and D103Y were detected in 10.4% (7/67) and 11.9% (8/67) of the cases, respectively, with four patients harboring both resistance mutations. Ten out of eleven patients carrying BCL2 G101V and/or D103Y experienced relapse during the follow-up period, representing 43.5% of the cases (10/23) showing clinical signs of disease progression. All BCL2 G101V or D103Y variants were detected in patients receiving venetoclax as a continuous single-agent treatment while these mutations were not observed during or after fixed-duration venetoclax therapy. Targeted ultra-deep sequencing of BCL2 uncovered three additional variants in four patient samples obtained at relapse, suggesting convergent evolution and implying a cooperating role of BCL2 mutations in driving venetoclax resistance. This cohort is the largest R/R CLL patient population reported to date in which BCL2 resistance mutations were investigated. Our study demonstrates the feasibility and clinical value of sensitive screening for BCL2 resistance mutations in R/R CLL.

Molecular Landscape of Chronic Lymphocytic Leukemia Using Targeted Gene Panel Sequencing

Introduction Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease with various genetic abnormalities that can affect the treatment and survival of patients. Recently, by the advent of next-generation sequencing (NGS) techniques recurrently mutated genes have been described in CLL. Driver gene mutations such as NOTCH1, SF3B1, TP53, and MYD88 have been considered to have prognostic impact in CLL. The aim of this study was to detect pathogenic variants of commonly mutated genes in CLL patients and to explore their associations with IGHV mutational status and cytogenetic abnormalities. Patients and Methods The samples were collected from bone marrow or peripheral blood of 124 CLL patients. Genomic DNA was isolated using QIAamp Blood Mini kit. NGS analysis was performed using Twist Custom Panel and bidirectional sequencing with a minimum coverage of 1000x on Illumina NextSeq according to the manufacturers’ instructions. Data were analyzed by NextGene software. The following 27 recurrently mutated CLL genes were studied: PIK3CD, PIK3CA, SF3B1, MYD88, FBXW7, BRAF, NOTCH1, PTEN, BIRC3, PTPN6, MAP2K1, TP53, STAT5B, STAT3, CD79B, TCF3, MAPK1, DDX3X,ATM, BCL2, BTK, KRAS, NRAS, CARD11, CXCR4, PLCG2, POT1. Variants below 5% variant allele frequency (VAF) were filtered out from the analysis. Variants were considered subclonal with 5-10% VAF and clonal when VAF was >10%. Classification of variants were performed according to the American College of Medical Genetics and Genomics (ACMG) criteria. Results Our NGS study revealed a total of 96 variants in 124 CLL patients. Overall, 63/124 patients (50.8%) harbored at least one mutation, the majority of them (60/96, 62.5%) was clonal. In 36 patients only one pathogenic variant was detected, while 88 patients carried two or more (2-4) genetic alterations. The most frequently mutated gene was the NOTCH1, followed by TP53, SF3B1, FBXW7, and BRIC3. Subclonal variants were also detected in 36/124 patients (29%), particularly in NOTCH1,FBXW7, BIRC3, and PIK3CA genes. TP53 and NOTCH1 variants occured mostly in unmutated IGHV CLL cases. Patients with mutated IGHV harbored mainly FBXW7 and BIRC3 mutationss. FBXW7 variants were detected in 9/125 (7%) patients, that is a higher frequency compared to other studies. Fluorescence in situ hybridization (FISH) was performed in 113 cases. The majority of these patients (84 cases, 74%) had one or more cytogenetic abnormality. In this cohort, 13q deletion was the most common aberration (65%, 55/84 patients), followed by 11q deletion (26%, 22/84 patients) and 17q deletion (14%, 12/84 patients). Deletion of 17p co-occured with TP53 single nucleotide variants in 10 of 12 patients. SF3B1, NOTCH1, BIRC3 and TP53 variants occured with 11q deletion in 10 cases. Trisomy 12 was detected with NOTCH1, BIRC3 and FBXW7 variants, and showed negative association with unfavorable SF3B1 mutations. Conclusions Our results confirmed the presence of high number of clonal and subclonal variants in the most frequently mutated genes (NOTCH1, TP53, SF3B1, FBXW7, BIRC3). TP53, NOTCH1 and SF3B1 mutations were enriched with unfavorable biologic factors, as they were found to be associated with unmutated IGHV and cytogenetic aberrations, such as del17p, del11q and trisomy 12. Cases with NOTCH1 variants harbored all types of the investigated cytogenetic alterations (del13q, del11q, de17p, trisomy 12) and were associated exclusively with unmutated IGHV. TP53 variants occured mostly in unmutated IGHV CLL cases together with del17p. Patients with SF3B1 variants showed del11q, del13q and mostly unmutated IGHV. FBXW7 variants enriched in both of unmutated and mutated IGHV cases and harbored del13q, de17p and trisomy 12. FBXW7 and NOTCH1 pathogenic variants have the same biological consequences in CLL, therefore presence of FBXW7 mutations may have clinical relevance regarding anti-CD20 therapy. Taken together, these NGS results complemented with the study of IGHV mutational status and cytogenetic data can contribute to better prognostic workup and management of our CLL patients.

PLX51107 Enhances the Anti-Tumor Activity of Targeted Therapy in Chronic Lymphocytic Leukemia When Combined with Venetoclax

Background: The development of drugs targeting BTK and BCL2 have dramatically improved the therapeutic landscape in chronic lymphocytic leukemia (CLL). However, resistance to these agents have been reported due to non-recurrent changes in oncogenic pathways and gene expression signatures. The bromodomain and extra terminal (BET) family proteins (BRD2, BRD3, BRD4 and BRDT) are epigenetic reader proteins that recognize acetylated lysine residues in histones. They play a critical role in mediating gene transcription and have been considered as highly promising targets in several diseases including cancer. Of these, the BRD4 protein is highly enriched in super enhancers and regulate gene transcription of relevant oncogenes such as MYC, CDK genes, cyclin-D1, BCL2 and MCL-1. BRD4 inhibitors (BRD4i) block the transcription of these key oncogenes through the displacement of BRDs and other epigenetic modifiers from chromatin. In this study, we investigated the preclinical activity of BRD4i PLX51107, and their cooperative role with BCL2 inhibitor Venetoclax in CLL. Methods: 56 primary CLL cells were isolated via histopaque-1077 (Sigma-Aldrich, St Louis MO) from peripheral blood apheresis samples from consenting adults using Roswell Park Comprehensive Cancer Center protocol CIC-01-16. Peripheral blood mononuclear cells (PBMC) were seeded at 3 x106/mL in a 384 or 6 well tissue culture plates and exposed to different doses of PLX51107 (1-20uM) as a single agent or in combination with Venetoclax (1-100nM) for 48 and 72hrs (Selleck Chemicals, Houston TX). Cell viability was assessed using Cell Titer-glo (Promega, Madison WI) with all data being normalized to untreated controls. Synergy was calculated using Calcusyn (Cambridge, UK) software to determine Coefficient of Synergy (CI) values. Anti-tumor effect was also analyzed by induction of apoptosis using Annexin-V/PI staining by flow cytometry at 72hrs. Cells pellets were saved for western blotting to determine expression of BRD4 members and different members of the B-cell receptor, NF-kB pathway as also the BCL-2 family of proteins. Bcl-2 family profiling was performed via Milliplex Bcl-2 family protein panels 1 and 2 to look at overall changes in Bcl-2 family expression. Results: Single agent exposure to PLX51107 displayed time and dose dependent induction of apoptosis of CLL cells (Figure 1a). Next, we examined the interaction between the PLX51107 and Venetoclax. In 72hr exposure the combination of PLX51107 with Venetoclax with an ATM deletion was synergistic in 4 of 8 samples and 4 of 7 samples with p53 deletion. Annexin/PI staining yielded apoptotic cell death in the single agent and combination exposure with Venetoclax. Western blotting showed a modulation of both BRD4 proteins as well as Bcl-2 family member proteins (Figure 1b). Bcl-2 family profiling via Milliplex panels demonstrated the ratio of Bcl-2/Bcl x/L was much higher in samples with a high Venetoclax IC50 (136/1) compared to Venetoclax low IC50 (54/1). (Figure 1) -A) Samples with p53 deletion and corresponding synergy. (B) BCL2 family protein expression of CLL sample 214c. Conclusions: Our data indicates that a) PLX51107 has an antitumor effect in CLL cells; b) the combinational treatment of Venetoclax with PLX51107 highly improved the apoptotic effect of single agent treatments. A better understanding of the key signaling pathways for CLL cells' proliferation and survival impacted by BRD4i in combination with Venetoclax would provide a proof-of-concept rationale for studies validating BRD4i as epigenetic approach to target BCR signaling in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Management of relapsed/refractory Chronic Lymphocytic Leukemia.

The therapeutic landscape for chronic lymphocytic leukemia (CLL) has changed dramatically over the past decade as our understanding of the biology of CLL has advanced, allowing the development of oral therapies targeting key drivers of CLL. Currently, inhibitors of Bruton's Tyrosine Kinase and the BH3 mimetic venetoclax are standards of care for both frontline and relapsed/refractory CLL. Sequencing of available therapies, therefore, has become a major challenge of therapy. In this review, we will focus on the current landscape for the treatment of relapsed/refractory CLL. We will also discuss important considerations when sequencing these available treatments. The recent advances in this disease are significant steps forward, and raise new questions of how these available drugs should be given as well as how we can continue to improve the treatment of CLL.

Abstract IA26: Understanding CLL and Richter’s syndrome biology through mouse models of human genetics

Abstract Large-scale human next generation sequencing studies have progressively defined the complex genetic landscape of chronic lymphocytic leukemia (CLL) and Richter’s syndrome (RS), yet the difficulties in rapid manipulation of primary CLL cells and the limited availability of cell lines have challenged studies aimed at interrogating the function of the so-called disease “drivers”, defined in human studies through statistical inference. Mouse models represent powerful tools to study mechanisms of normal and malignant B cell biology. Conditional knock-in/knock-out systems have allowed B-cell restricted modeling of CLL genetic lesions, with MDR mice being the first example of a genetically-faithful CLL model, recapitulating the most common genomic aberration of human disease, del(13q). Since then, a series of models generated by our group and others have allowed to uncover key functional changes underlying disease onset and transformation, including in the context of combined mutation in the splicing factor Sf3b1 and the DNA damage response gene Atm, sole mutation in the B cell developmental factor Ikzf3, the nuclear export receptor Xpo1, the ribosomal protein Rps15, and the DNA methyltransferase Dnmt3a (the latter not a CLL lesion per se, but recapitulating disordered methylation patterns typical of CLL). The CRISPR-Cas9 gene editing technology has further enabled B-cell restricted introduction of multiple combinatorial loss-of-function lesions, allowing the generation of faithful models not only of CLL but, importantly, of transformation into RS. In this presentation, I will describe the application of mouse models to studies of CLL and RS pathogenesis and treatment, with a focus on key aspects uncovered through the characterization of genetically- inspired mouse lines of recurrent disease drivers. I will provide a perspective on how these novel models combined with new technology allowed the dissection of mechanisms of disease evolution and response to therapy with greater depth than previously possible, representing valuable platforms for functional genomic analyses. Citation Format: Elisa ten Hacken. Understanding CLL and Richter’s syndrome biology through mouse models of human genetics [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr IA26.

Resistance to Bruton tyrosine kinase inhibition in chronic lymphocytic leukaemia and non-Hodgkin lymphoma.

Bruton tyrosine kinase inhibitors (BTKi) have transformed the therapeutic landscape of chronic lymphocytic leukaemia (CLL) and non-Hodgkin lymphoma. However, primary and acquired resistance to BTKi can be seen due to a variety of mechanisms including tumour intrinsic and extrinsic mechanisms such as gene mutations, activation of bypass signalling pathways and tumour microenvironment. Herein, we provide an updated review of the key clinical data of BTKi treatment in CLL, mantle cell lymphoma, and diffuse large B-cell lymphoma (DLBCL). We incorporate the most recent findings regarding mechanisms of resistance to covalent and non-covalent inhibitors, including ibrutinib, acalabrutinib, zanubrutinib and pirtobrutinib. We also cover the clinical sensitivity of certain molecular subtypes of DLBCL to an ibrutinib-containing regimen. Lastly, we summarise ongoing clinical investigations aimed at overcoming resistance via use of BTKi-containing combined therapies or the novel non-covalent BTKi. The review article targets an audience of clinical practitioners, clinical investigators and translational researchers.

Beyond ibrutinib: novel BTK inhibitors for the treatment of chronic lymphocytic leukemia.

Ibrutinib was the first Bruton tyrosine kinase inhibitor (BTKi) approved for clinical use, contributing to a dramatic change in the treatment landscape of chronic lymphocytic leukemia (CLL). This review provides an overview of next-generation BTKi that have been recently approved or are being investigated for the treatment of CLL, specifically highlighting differences and similarities compared to ibrutinib. Acalabrutinib presented comparable response rates to ibrutinib with lower rates of adverse events and is currently approved for the treatment of CLL. Zanubrutinib displayed excellent response rates with a lower incidence of BTKi-related adverse events, but major rates of neutropenia, and its approval is awaited. With the aim of overcoming drug resistance, noncovalent BTKi have been developed. Of all the explored agents to date, pirtobrutinib has shown promising results with manageable toxicities. For the treatment of CLL, several effective therapeutic strategies to target BTK are or will soon be available: these drugs present different safety profiles, thus making it possible to tailor the treatment choice according to patient's characteristics. Importantly, noncovalent BTKi will provide a therapeutic chance also for those relapsed/refractory CLL patients who are BTKi-resistant and are considered an unmet clinical need.

Zanubrutinib joins the CLL treatment buffet.

The advent of targeted therapies has transformed the treatment landscape of chronic lymphocytic leukaemia (CLL). Although chemoimmunotherapy constituted the standard of care in earlier decades, Bruton tyrosine kinase (BTK) inhibitors (ibrutinib and acalabrutinib), BCL2 inhibitors (venetoclax), and combinations thereof are currently the cornerstone of CLL management. With the intention of minimising off-target adverse effects, new BTK inhibitors, such as zanubrutinib and acalabrutinib, have been developed and approved for the treatment of several B-cell malignancies.

Time-limited, chemotherapy-free treatment comes of age in chronic lymphocytic leukaemia.

The therapeutic landscape of chronic lymphocytic leukaemia has been transformed by the advent of drugs targeting Bruton tyrosine kinase and BCL2. 1 Kay NE Hampel PJ Van Dyke DL Parikh SA CLL update 2022: a continuing evolution in care. Blood Rev. 2022; 100930100930 Google Scholar Pivotal studies showed the superiority of such inhibitors for chronic lymphocytic leukaemia treatment compared with chemoimmunotherapy. 2 Byrd JC Brown JR O'Brien S et al. Ibrutinib versus ofatumumab in previously treated chronic lymphoid leukemia. N Engl J Med. 2014; 371: 213-223 Crossref PubMed Scopus (1189) Google Scholar , 3 Sharman JP Egyed M Jurczak W et al. Acalabrutinib with or without obinutuzumab versus chlorambucil and obinutuzmab for treatment-naive chronic lymphocytic leukaemia (ELEVATE TN): a randomised, controlled, phase 3 trial. Lancet. 2020; 395: 1278-1291 Summary Full Text Full Text PDF PubMed Scopus (216) Google Scholar , 4 Seymour JF Kipps TJ Eichhorst B et al. Venetoclax–rituximab in relapsed or refractory chronic lymphocytic leukemia. N Engl J Med. 2018; 378: 1107-1120 Crossref PubMed Scopus (475) Google Scholar , 5 Al-Sawaf O Zhang C Tandon M et al. Venetoclax plus obinutuzumab versus chlorambucil plus obinutuzumab for previously untreated chronic lymphocytic leukaemia (CLL14): follow-up results from a multicentre, open-label, randomised, phase 3 trial. Lancet Oncol. 2020; 21: 1188-1200 Summary Full Text Full Text PDF PubMed Scopus (112) Google Scholar Bruton tyrosine-kinase inhibitors such as ibrutinib and acalabrutinib are administered until progression or toxicity and, although highly effective, cardiovascular toxicities and mutations associated with resistance are known to ensue for some patients. 2 Byrd JC Brown JR O'Brien S et al. Ibrutinib versus ofatumumab in previously treated chronic lymphoid leukemia. N Engl J Med. 2014; 371: 213-223 Crossref PubMed Scopus (1189) Google Scholar , 3 Sharman JP Egyed M Jurczak W et al. Acalabrutinib with or without obinutuzumab versus chlorambucil and obinutuzmab for treatment-naive chronic lymphocytic leukaemia (ELEVATE TN): a randomised, controlled, phase 3 trial. Lancet. 2020; 395: 1278-1291 Summary Full Text Full Text PDF PubMed Scopus (216) Google Scholar , 4 Seymour JF Kipps TJ Eichhorst B et al. Venetoclax–rituximab in relapsed or refractory chronic lymphocytic leukemia. N Engl J Med. 2018; 378: 1107-1120 Crossref PubMed Scopus (475) Google Scholar , 5 Al-Sawaf O Zhang C Tandon M et al. Venetoclax plus obinutuzumab versus chlorambucil plus obinutuzumab for previously untreated chronic lymphocytic leukaemia (CLL14): follow-up results from a multicentre, open-label, randomised, phase 3 trial. Lancet Oncol. 2020; 21: 1188-1200 Summary Full Text Full Text PDF PubMed Scopus (112) Google Scholar Minimal residual disease-guided stop and start of venetoclax plus ibrutinib for patients with relapsed or refractory chronic lymphocytic leukaemia (HOVON141/VISION): primary analysis of an open-label, randomised, phase 2 trialThese data point to a favourable benefit–risk profile of MRD-guided, time-limited treatment with ibrutinib plus venetoclax for patients with relapsed or refractory chronic lymphocytic leukaemia, suggesting that MRD-guided cessation and reinitiation is feasible in this patient population. Full-Text PDF

Ibrutinib Does Not Impact CCR7-Mediated Homeostatic Migration in T-Cells from Chronic Lymphocytic Leukemia Patients.

Simple SummaryBruton’s tyrosine kinase inhibitor ibrutinib restores T-cell immunity and increases circulating T-cell numbers in chronic lymphocytic leukemia (CLL) patients. Recent evidence suggests T-cell enhanced expansion, rather than increased egress from secondary lymphoid organs (SLO), as a root cause for ibrutinib-induced lymphocytosis. However, this physiological change might reflect, as well, a forced retention in PB derived of impaired migration to SLO. With the aim to investigate the impact of ibrutinib on CCR7-mediated homeostasis in CLL T-cells, we have documented receptor expression in a large cohort of ibrutinib-treated patients, and performed different in vivo and in vitro migration models. We show that ibrutinib has no effect on CCR7 expression or receptor-mediated T-cell chemotaxis, homing to SLO, and interstitial migration. Together, these results indicate that ibrutinib T-lymphocytosis is not caused by accumulation in the blood stream, and therefore back the T-cell expansion as the most plausible cause.Bruton’s tyrosine kinase inhibitor ibrutinib has significantly changed treatment landscape in chronic lymphocytic leukemia (CLL). Growing evidence supports ibrutinib to work beyond the effect on tumor cells by means of, for example, restoring functionality of the T-cell compartment and increasing circulating T-cell numbers. Recent evidence suggests T-cell enhanced expansion, rather than increased egress from secondary lymphoid organs (SLO), as a root cause for ibrutinib-induced lymphocytosis. However, whether the latter physiological change is also a consequence of a forced retention in blood remains undisclosed. Since CCR7 is the main chemokine receptor taking over the homing of T-cells from peripheral compartments to lymph nodes and other SLO, we aimed to investigate the impact of ibrutinib on CCR7 functionality in T-cells. To this end, we documented receptor expression in T-cells from a large cohort of ibrutinib-treated CLL patients, and performed different in vivo and in vitro migration models. Overall, our data confirm that CCR7 expression or receptor-mediated migration in CLL T-cells is not affected by ibrutinib. Furthermore, it does not modulate CCR7-driven homing nor nodal interstitial migration. Together, our results support that ibrutinib-induced CLL T-cell accumulation in the blood stream is not derived from an impairment of CCR7-driven recirculation between the SLO and bloodstream, and therefore T-cell expansion is the most plausible cause.