Chronic Lymphocytic Leukemia Therapy Research Articles

Duvelisib Eliminates CLL B Cells, Impairs CLL-Supporting Cells, and Overcomes Ibrutinib Resistance in a Xenograft Model.

Inhibitors of Bruton's tyrosine kinase (BTKi) and PI3K (PI3Ki) have significantly improved therapy of chronic lymphocytic leukemia (CLL). However, the emergence of resistance to BTKi has introduced an unmet therapeutic need. Hence, we sought evidence for essential roles of PI3K-δi and PI3K-γi in treatment-naïve and BTKi-refractory CLL. Responses to PI3K-δi, PI3K-γi, and the dual-inhibitor duvelisib in each B, T, and myeloid cell compartments of CLL were studied in vitro, and in a xenograft mouse model using primary cells from treatment-naïve and ibrutinib-resistant patients, and finally, in a patient with ibrutinib-resistant CLL treated with duvelisib. We demonstrate the essential roles of PI3K-δ for CLL B-cell survival and migration, of PI3K-γ for T-cell migration and macrophage polarization, and of dual inhibition of PI3K-δ,γ for efficacious reduction of leukemia burden. We also show that samples from patients whose disease progressed on ibrutinib were responsive to duvelisib therapy in a xenograft model, irrespective of BTK mutations. In support of this, we report a patient with ibrutinib-resistant CLL, bearing a clone with BTK and PLCγ2 mutations, who responded immediately to single-agent duvelisib with redistribution lymphocytosis followed by a partial clinical remission associated with modulation of T and myeloid cells. Our data define the mechanism of action whereby dual inhibition of PI3K-δ,γ affects CLL B-cell numbers and T and myeloid cell pro-leukemia functions and support the use of duvelisib as a valuable approach for therapeutic interventions, including for patients refractory to BTKi.

Abstract 6396: BET inhibition alleviates T-cell dysfunction in chronic lymphocytic leukemia

Abstract Background: The chronic lymphocytic leukemia (CLL) tumor microenvironment (TME) is laden with hyporesponsive T-cells that permit disease persistence. Yet, redundant TME immunosuppressive mechanisms and epigenetic maintenance of T-cell exhaustion limit the efficacy of T-cell targeted therapies in CLL. Bromodomain and extra-terminal (BET) proteins regulate key pathways contributing to CLL pathogenesis and TME interactions, including T-cell function and differentiation. We hypothesize that blocking BET protein function can reverse T-cell exhaustion to yield durable tumor elimination in CLL. Methods: WT C57BL/6 mice were engrafted with Eμ-TCL1 spleen-derived lymphocytes, then treated daily with the novel pan-BET inhibitor, OPN-51107 (OPN5; 20mg/kg PO) for up to 4 weeks. Splenic gene expression was evaluated with the NanoString PanCancer iO360 panel. T-cell differentiation status, immune inhibitory receptor (IR) expression, proliferation (72 h ex vivo α-CD3/α-CD28 stimulation), and cytokine production (6 h ex vivo PMA/ionomycin stimulation) was measured via flow cytometry. CLL patient and healthy donor PBMCs were used for validation studies and to assess T-cell transcription factor (TF) expression via flow cytometry. Evaluation of BRD4 occupancy at select T-cell TFs via ChIP qPCR is ongoing. Results: OPN5 significantly increased cytotoxic cell signatures and reduced exhaustion-associated cell signatures in leukemic mice through inhibition of T-cell exhaustion signaling, as well as activation of Th1, natural killer cell, and IL-7 signaling pathways. Correspondingly, T-cells from OPN5-treated mice demonstrated greater ex vivo proliferative capacity and effector response to stimuli. A greater proportion of CD8+ T-cells from OPN5-treated mice were classified as naïve, and OPN5 significantly reduced KLRG1 expression on antigen-experienced CD8+ T-cells. Importantly, OPN5 curtailed IR co-expression (PD-1, PD-L1, VISTA, CD244, CD160, and LAG3) on splenic T-cells. These findings were confirmed with primary CLL cells ex vivo. OPN5 also impaired expression of terminal differentiation-associated TFs in CLL patient-derived T-cells, enriching for a TCF1+ progenitor T-cell population. While BTK inhibitors are known to similarly improve T-cell function in CLL, ibrutinib treatment was inadequate to revert CLL T-cell terminal differentiation. Future ATAC-sequencing analysis will inform how BET inhibition alleviates exhaustion-associated chromatin organization in CLL T-cells. Conclusion: BET inhibition dismantles immunosuppressive mechanisms in the CLL TME, alleviating CLL-induced T-cell dysfunction and terminal differentiation. These findings suggest that BET inhibition may be a useful component of combination strategies for the treatment of CLL to yield lasting anti-cancer immune memory and prevent relapsed/refractory disease. Citation Format: Audrey L. Smith, Alexandria P. Eiken, Sydney A. Skupa, Christopher R. D'Angelo, Avyakta Kallam, Matthew A. Lunning, Gregory Bociek, Julie M. Vose, Ben Powell, Gideon Bollag, Dalia El-Gamal. BET inhibition alleviates T-cell dysfunction in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6396.

Efficacy and Safety of Bruton Tyrosine Kinase Inhibitor Monotherapy Compared with Combination Therapy for Chronic Lymphocytic Leukemia and Small Lymphocytic Lymphoma: A Systematic Review and Meta-Analysis.

The effectiveness and safety of combination treatments such as chemoimmunotherapies in chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL/SLL) remain controversial. Bruton tyrosine kinase inhibitors (BTKis) are an effective therapy for CLL/SLL patients. This meta-analysis aimed to compare the efficacy and safety of BTKis versus combination therapy in CLL/SLL patients. We searched the PubMed, Cochrane, Medline, and Embase databases through February 2023 for relevant randomized controlled trials (RCTs). Four RCTs (including 1510 patients) were found and met the inclusion criteria. Progression-free survival (PFS) was significantly improved with BTKis when compared to the combination therapy (hazard ratio (HR), 0.30; 95% confidence interval (CI), 0.22-0.40), while a pooled analysis of overall survival did not favor single-agent BTKis over the combination therapy (HR, 0.87; 95% CI, 0.67-1.15). We observed consistent benefits for PFS among patients with high-risk disease characteristics. Although there was no difference in complete response between the two arms (risk ratio (RR), 0.54; 95% CI, 0.20-1.46), BTKi use was related to a better overall response rate (RR, 1.10; 95% CI, 1.04-1.16). The risk of grade ≥3 adverse events (AEs) was comparable between the two arms (RR, 0.82; 95% CI, 0.55-1.23). However, the risk of grade ≥3 AEs was significantly lower in the second-generation BTKi group than in the combination therapy group (RR, 0.73; 95% CI, 0.54-0.98). Overall, BTKis have superior efficacy compared to the combination regimens in patients with untreated or treated CLL/SLL without excess toxicity. Further studies are needed to confirm these results and determine the optimal therapy for managing patients with CLL/SLL.

Cellular Therapies in Chronic Lymphocytic Leukemia and Richter’s Transformation: Recent Developments in Chimeric Antigen Receptor T-Cells, Natural Killer Cells, and Allogeneic Stem Cell Transplant

Cellular therapies can be viewed as both the newest and oldest techniques for treating chronic lymphocytic leukemia (CLL) and Richter's transformation (RT). On one hand, allogeneic hematopoietic stem cell transplantation (alloHSCT) has been available for decades, though its use is diminishing with the increasing availability of effective novel targeted agents, especially in CLL. Among newer techniques, chimeric antigen receptor T-cells (CAR-T) have demonstrated astounding efficacy in several hematologic malignancies, leading to FDA approval and use in clinical practice. However, though CLL is the earliest disease type for which CAR-T were studied, development has been slower and has yet to lead to regulatory approval. Owing partially to its rarity but also due to the aggressive behavior of RT, CAR-T in RT have only been minimally explored. Here, we will focus on the applications of cellular therapies in CLL and RT, specifically reviewing more recent data related to alloHSCT in the novel-agent era and CAR-T cell development in CLL/RT, focusing on safety and efficacy successes and limitations. We will review strategies to improve upon CAR-T efficacy and discuss ongoing trials utilizing CAR-T in CLL/RT, as well as emerging technologies, such as allogeneic CAR-T and natural killer CAR (CAR NK) cells.

Ocular toxoplasmosis during frontline venetoclax therapy for chronic lymphocytic leukaemia - more than meets the eye.

A 67-year-old woman presented with a right central scotoma and reduced Snellen visual acuity (<6/60), 4 months after commencing frontline obinutuzumab–venetoclax (Ven–O) therapy for 13q-deleted, IGHV-mutated, chronic lymphocytic leukaemia (CLL). At the time of ocular symptoms, her full blood count comprised haemoglobin 131 g/L, lymphocytes 0.7 × 109/L, neutrophils 0.8 × 109/L, platelets 40 × 109/L and total IgG < 3.2 g/L. Repeat bone marrow trephine revealed minor residual CLL only (5%–10%). She received valaciclovir only as supportive therapy. Serial ophthalmic examinations demonstrated an enlarging macular lesion with mild vitritis consistent with retinochoroiditis (retinal photograph, Figure 2 without satellite lesions or evidence of pre-existing retinal scarring. Spectral-domain optical coherence tomography Figure 1 revealed full-thickness retinal necrosis with hyper-reflectivity and disorganisation of retinal layers. Polymerase chain reaction (PCR) of aqueous humour demonstrated the presence of Toxoplasma gondii, consistent with active ocular toxoplasmosis. T. gondii IgG but no IgM antibodies were detected both pre-treatment and 4 weeks from the onset of ocular symptoms. Computed tomography imaging of the brain was normal, and serum T. gondii PCR was negative. T. gondii is a common infectious cause of retinochoroiditis. Both acquired ocular toxoplasmosis and reactivation of previous infection may be observed in immune-competent individuals. Ocular toxoplasmosis has also been described in relapsed/refractory CLL, including those treated with continuous venetoclax. Prior fludarabine exposure has been cited as a risk factor for opportunistic infection. However, reports of toxoplasmosis during frontline CLL therapy are limited and, to our knowledge, have not included the Ven–O combination. The patient completed systemic and intra-vitreal clindamycin with topical prednisolone drops, noting gradual improvement of her visual acuity to 6/36 and slow healing of the macular lesion on serial examination. Given the quantitative immune dysfunction observed and evidence of past T. gondii infection, opportunistic reactivation of toxoplasmosis was considered. However, the absence of pre-existing toxoplasmosis retinal scar or bilateral ocular involvement argued against infection reactivation. Absence of infection dissemination, including no central nervous system involvement as seen with acquired immune deficiency syndromes such as HIV, also argue against infection occurring as a function of immune compromise. Nonetheless, our patient remains on suppressive antibiotic therapy during Ven–O therapy and 4-weekly prophylactic intravenous immunoglobulin. This case highlights the complexities of infection diagnoses and management in the context of frontline novel therapies in CLL. Appropriate supportive treatments remain uncertain. Rory Bennett, Lyndell L. Lim, Constantine S. Tam interpreted the case information and wrote the manuscript. CST: Honoraria for AbbVie, AstraZeneca, Janssen, BeiGene and LOXO. Research funding from AbbVie, Janssen and BeiGene Documented verbal consent obtained from the patient prior to preparation of manuscript.

Prolonged SARS-CoV-2 Infection and Organizing Pneumonia in a Patient with Follicular Lymphoma, Treated with Obinutuzumab—Challenging Recognition and Treatment

A Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) led to a pandemic outbreak in 2019. COVID-19's course and its treatment in immunocompromised patients are uncertain. Furthermore, there is a possibility of protracted SARS-CoV-2 infection and the need for repeated antiviral treatment. Monoclonal antibodies against CD20, which are used, among other things, in the therapy of chronic lymphocytic leukaemia and follicular lymphoma, can induct immunosuppression. We present a case report of a patient with follicular lymphoma, treated with obinutuzumab, who was diagnosed with prolonged, ongoing SARS-CoV-2 infection and related organizing pneumonia. The recognition and the treatment were challenging which makes this case noteworthy. Antiviral therapy with several medications was administrated to our patient and their temporary, positive effect was observed. Moreover, high-dose intravenous immunoglobulin was applied, because slowly decreasing IgM and IgG levels were observed. The patient also received standard treatment of organizing pneumonia. We believe that such a complex approach can create an opportunity for recovery. Physicians should be conscious of the course and treatment possibilities facing similar cases.

The evolving use of measurable residual disease in chronic lymphocytic leukemia clinical trials.

Measurable residual disease (MRD) status in chronic lymphocytic leukemia (CLL), assessed on and after treatment, correlates with increased progression-free and overall survival benefit. More recently, MRD assessment has been included in large clinical trials as a primary outcome and is increasingly used in routine practice as a prognostic tool, a therapeutic goal, and potentially a trigger for early intervention. Modern therapy for CLL delivers prolonged remissions, causing readout of traditional trial outcomes such as progression-free and overall survival to be inherently delayed. This represents a barrier for the rapid incorporation of novel drugs to the overall therapeutic armamentarium. MRD offers a dynamic and robust platform for the assessment of treatment efficacy in CLL, complementing traditional outcome measures and accelerating access to novel drugs. Here, we provide a comprehensive review of recent major clinical trials of CLL therapy, focusing on small-molecule inhibitors and monoclonal antibody combinations that have recently emerged as the standard frontline and relapse treatment options. We explore the assessment and reporting of MRD (including novel techniques) and the challenges of standardization and provide a comprehensive review of the relevance and adequacy of MRD as a clinical trial endpoint. We further discuss the impact that MRD data have on clinical decision-making and how it can influence a patient's experience. Finally, we evaluate how upcoming trial design and clinical practice are evolving in the face of MRD-driven outcomes.

Sialylation regulates migration in chronic lymphocytic leukemia.

Sialylation is the terminal addition of sialic acid to underlying glycans. It plays a prominent role in cell adhesion and immune regulation. Sialylated structures found on adhesion molecules, such as CD49d, mediate the interactions between cancer cells and the microenvironment, facilitating metastatic seeding in target organs. Chronic lymphocytic leukemia (CLL) is a clonal B-cell malignancy characterized by the accumulation of CD5-positive B cells in the peripheral blood, bone marrow and lymph nodes. CLL cells proliferate mainly in the lymph node "proliferation centers", where the microenvironment provides pro-survival signals. Thus, migration and homing into these protective niches play a crucial role in CLL biology. In recent years, therapeutic strategies aimed at inducing the egress of CLL cells from the lymph nodes and bone marrow into the circulation have been highly successful. In this study, the sialylation status of 79 untreated and 24 ibrutinib-treated CLL patients was characterized by flow cytometry. Moreover, the effect of sialic acid removal on migration was tested by a transwell assay. Finally, we examined the sialylation status of CD49d by Western blot analysis. We found that CLL cells are highly sialylated, particularly those characterized by an "activated" immune phenotype. Notably, sialylation regulates CLL migration through the post-translational modification of CD49d. Finally, we showed that therapeutic agents that induce CLL mobilization from their protective niches, such as ibrutinib, modulate sialic acid levels. We propose that sialylation is an important regulator of CLL trafficking and may represent a novel target to further improve CLL therapy.

INTEGRATED BIOINFORMATIC ANALYSIS TO EVALUATE TARGET GENES AND PATHWAYS IN CHRONIC LYMPHOCYTIC LEUKEMIA

Objective: The most common type of leukemia, chronic lymphocytic leukemia (CLL), is characterized by progressive accumulation of monoclonal B cells with a specific immunophenotype in the blood, bone marrow, and lymphoid organ. The goal of this research was to use bioinformatic analysis to comprehend the molecular mechanisms causing CLL and to investigate potential targets for the diagnosis and therapy of CLL.&#x0D; Material and Method: Expression data from CLL patients with accession numbers GSE22529 and GSE26725 were downloaded from the GEO database for bioinformatic analysis. GSE22529 data was studied with samples from 41 CLL patients and 11 healthy groups, while GSE26725 data was studied with blood samples from 12 CLL patients and 5 healthy groups. GEO2R was used to find differentially expressed genes (DEGs) in CLL patient samples and healthy control samples. The DAVID program was used to perform GO and KEGG enrichment analyses on DEGs. Using the Cytoscape software, a protein-protein interaction (PPI) network was created, and hub genes associated with CLL were identified.&#x0D; Result and Discussion: DEGs with p 0.05 and log2FC 0, log2FC&gt;0 were chosen after analysis with GEO2R. In the GSE22529 dataset, 942 genes had higher expression levels in CLL patients compared with controls, while the expression of 1007 genes decreased. In the GSE26725 dataset, CLL patients had lower expression levels for 916 genes compared with controls, while 939 genes showed an increase in expression. 229 DEGs with higher expression levels and 308 DEGs with lower expression levels were found in both sets of data. It has been observed that these common genes, whose expression has changed, are enriched in protein processing in the ER, Chemokine, B-cell receptor, T-cell receptor, protein export pathways. Additionally, DDOST, RPL18, RPL18A, RPL19, RPL31, GNB3, GNB4, GNG11, GNGT1, NEDD8, UBE2M RBX1, FBXO21, SKP1, KLHL9 and CAND1 were identified as the most important genes. Our study's findings demonstrated that newly discovered genes and pathways may be candidates for CLL biomarkers that can be used for both the diagnosis and drug treatment of the disease.

How we approach the perioperative management of patients with chronic lymphocytic leukaemia receiving continuous cancer-directed therapies.

Historically, invasive procedures and surgeries were deferred in patients with haematological malignancies including advanced stage chronic lymphocytic leukaemia (CLL) because of limited life expectancy. However, novel, and often continuous, treatments have markedly improved outcomes in CLL. Some patients may expect years of treatment response and disease control, overcoming the short life expectancy that deters interventionalists. Such patients now often undergo various invasive procedures including major surgery. To inform peri-operative management, we summarize the relevant side effects and drug interactions of continuous CLL therapies, highlight potential surgical risks, and provide recommendations on withholding specific CLL drugs around invasive procedures.

An Optimized Single Nucleotide Polymorphism-Based Detection Method Suggests That Allelic Variants in the 3' Untranslated Region of RRAS2 Correlate with Treatment Response in Chronic Lymphocytic Leukemia Patients.

Unlike classical RAS genes, oncogenic mutations on RRAS2 are seldomly found in human cancer. By contrast, RRAS2 is frequently found overexpressed in a number of human tumors, including B and T cell lymphomas, breast, gastric, head and neck cancers. In this regard, we have recently shown that overexpression of wild-type RRAS2 drives the formation of both chronic lymphocytic leukemia (CLL) and breast cancer in mice. In support for the relevance of overexpression of wild type RRAS2 in human cancer, we have found that RRAS2 expression is influenced by the presence of a specific single nucleotide polymorphism (SNP) located in the 3'-untranslated region (UTR) of the RRAS2 mRNA. Perhaps more importantly, the presence of the alternate C, rather than the G allele, at the RRAS2 SNP designated as rs8570 is also associated with worse patient prognosis in CLL. This indicates that the detection of this SNP allelic variants can be informative to predict RRAS2 expression levels and disease long-term evolution in patients. Here, we describe a polymerase chain reaction (PCR)-based method that facilitates the rapid and easy determination of G and C allelic variants of the SNP. Using this approach, we confirm that the C allelic variant is associated with higher expression levels of RRAS2 transcripts and poor patient prognosis. However, we have also found that expression of the C allelic variants correlates with better response to ibrutinib, a Bruton kinase inhibitor commonly used in CLL treatments. This suggests that this method for detecting the RRAS2 rs8570 SNP might be a useful as a tool to predict both patient prognosis and response to targeted therapy in CLL.

Bcl-2 Inhibition Induces a Synergistic Effect in Combination with Doxorubicin in Chronic Lymphocytic Leukemia

Background: Chronic lymphocytic leukemia (CLL), a common neoplastic disease, is associated with the accumulation of B-lymphocytes in the hematopoietic organs. A main characteristic of CLL cells is the failure to undergo apoptosis, thus resulting in resistance to several chemotherapeutics. Doxorubicin (DOX), an anthracycline widely used for treating various neoplasms including CLL, induces apoptosis via several mechanisms. Despite this, CLL cells become resistant to DOX. A major protein known as B-cell lymphoma-2 (Bcl-2), known to exert direct anti-apoptotic effects on the cell, is reported to be overexpressed in CLL cells. Methods: We aimed to silence the Bcl-2 gene by siRNA. Mononuclear cells were isolated from the peripheral blood and bone marrow of eleven untreated CLL patients by Ficoll-Paque. To transfect cells, we used Lipofectamine. Bcl-2 expression was investigated using qRT-PCR. Next, we studied the effect of Bcl-2 silencing in combination with DOX treatment on the viability of cells by an MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Results: Bcl-2 expression was significantly suppressed in CLL cells following siRNA transfection via lipofectamine. Based on the results of MTT, the inhibition of Bcl-2 sensitized CLL cells to DOX treatment. Also, the effect of the treatment was time-dependent. Conclusion: These findings imply that combination therapy of CLL using anti-Bcl-2 siRNA and DOX can be considered a practical therapeutic approach that should be further evaluated in future studies.

Selecting initial therapy in CLL.

Targeted therapy is a powerful treatment option in chronic lymphocytic leukemia (CLL) that has outperformed conventional chemoimmunotherapy in most clinical settings. Except for selected young, fit patients with a mutated immunoglobulin heavy chain variable region gene, most patients benefit from targeted therapy with either a continuous BTK inhibitor or 1-year fixed-duration venetoclax-obinutuzumab as first-line treatment of CLL. Treatment selection is driven by patient-, treatment-, and disease-related factors, encompassing patient preference, concomitant medications, comorbidities, safety profile of the regimen, and TP53 aberration. Clinical trials are actively investigating the simultaneous inhibition of Bruton's tyrosine kinase (BTK) and B-cell lymphoma 2 (BCL-2) proteins with or without a CD20 monoclonal antibody, which can achieve deep response in most patients (52%-89% undetectable minimal residual disease in bone marrow).

Accuracy of predicting IgHV mutation status in chronic lymphocytic leukemia using RNA expression profiling and machine learning

Background: Immunoglobulin heavy chain (IgHV) mutation status is a unique prognostic indicator for predicting clinical course and response to targeted therapy in chronic lymphocytic leukemia (CLL). Most of the tests for the IgHV mutation status require extensive and complicated genomic evaluation. We explored the potential of using RNA expression data generated from routine targeted RNA sequencing by Next Generation Sequencing (NGS) along with machine learning for the prediction of the IgHV mutation status.

Patterns of use of biological and genetic markers for chronic lymphocytic leukemia and acute myeloid leukemia in Puerto Rico.

The use of markers has stimulated the development of more appropriate targeted therapies for chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). We assessed the use and prevalence of biological and genetic markers of CLL and AML in the homogeneous Hispanic population of Puerto Rico. We used the Puerto Rico CLL/AML Population-Based Registry, which combines information from linked databases. Logistic regression models were used to examine factors associated with biological and genetic testing. A total of 926 patients 18 years or older diagnosed with CLL (n=518) and AML (n=408) during 2011-2015 were included in this analysis. Cytogenetic testing (FISH) was reported for 441 (85.1%) of the CLL patients; of those, 24.0% had the presence of trisomy 12, 9.5% carried deletion 11q, 50.3% carried deletion 13q, and 6.3% carried deletion 17p. Regarding AML, patients with cytogenetics and molecular tests were considered to determine the risk category (254 patients), of which 39.8% showed poor or adverse risk. Older age and having more comorbidities among patients with CLL were associated with a lower likelihood of receiving a FISH test. Although prognostic genetic testing is required for treatment decisions, the amount of testing in this Hispanic cohort is far from ideal. Furthermore, some tests were not homogeneously distributed in the population, which requires further exploration and monitoring. This study contributes to the field by informing the medical community about the use and prevalence of biological and genetic markers of CLL and AML. Similarly, it has the potential to improve the management of CLL and AML through benchmarking.

NRX-0492 degrades wild-type and C481 mutant BTK and demonstrates in vivo activity in CLL patient-derived xenografts

AbstractBruton tyrosine kinase (BTK) is essential for B-cell receptor (BCR) signaling, a driver of chronic lymphocytic leukemia (CLL). Covalent inhibitors bind C481 in the active site of BTK and have become a preferred CLL therapy. Disease progression on covalent BTK inhibitors is commonly associated with C481 mutations. Here, we investigated a targeted protein degrader, NRX-0492, that links a noncovalent BTK-binding domain to cereblon, an adaptor protein of the E3 ubiquitin ligase complex. NRX-0492 selectively catalyzes ubiquitylation and proteasomal degradation of BTK. In primary CLL cells, NRX-0492 induced rapid and sustained degradation of both wild-type and C481 mutant BTK at half maximal degradation concentration (DC50) of ≤0.2 nM and DC90 of ≤0.5 nM, respectively. Sustained degrader activity was maintained for at least 24 hours after washout and was equally observed in high-risk (deletion 17p) and standard-risk (deletion 13q only) CLL subtypes. In in vitro testing against treatment-naïve CLL samples, NRX-0492 was as effective as ibrutinib at inhibiting BCR-mediated signaling, transcriptional programs, and chemokine secretion. In patient-derived xenografts, orally administered NRX-0492 induced BTK degradation and inhibited activation and proliferation of CLL cells in blood and spleen and remained efficacious against primary C481S mutant CLL cells collected from a patient progressing on ibrutinib. Oral bioavailability, >90% degradation of BTK at subnanomolar concentrations, and sustained pharmacodynamic effects after drug clearance make this class of targeted protein degraders uniquely suitable for clinical translation, in particular as a strategy to overcome BTK inhibitor resistance. Clinical studies testing this approach have been initiated (NCT04830137, NCT05131022).

Real-World Comparison of Time to Next Treatment for Patients with CLL Initiated on First-Line Treatment with Ibrutinib Versus Acalabrutinib

Introduction: Treatment guidelines recommend Bruton's tyrosine kinase inhibitors (BTKi) ibrutinib (once-daily) and acalabrutinib (twice-daily) as preferred regimens for chronic lymphocytic leukemia (CLL); however, there are no studies comparing the effectiveness of ibrutinib and acalabrutinib as first-line (1L) therapy in CLL. This study investigated time to next treatment (TTNT) for patients with CLL initiated on 1L ibrutinib or acalabrutinib in real-world (RW) clinical practice in the US. Methods: Specialty pharmacy electronic medical records from academic integrated care networks (11/21/2018-4/30/2022) were used to identify adults with CLL who initiated ibrutinib or acalabrutinib in 1L (index date) on or after the date of acalabrutinib approval for CLL (11/21/2019). A washout period of ≥12 months without antineoplastic agent use pre-index was required to confirm use as 1L therapy. Patients were excluded if there was concomitant use of another antineoplastic agent during the first 28 days post-index. Characteristics evaluated in the 12-month baseline period were compared between ibrutinib and acalabrutinib cohorts using t-tests for continuous variables and chi-square tests for categorical variables. TTNT was defined as the time from the index date to the initiation a next or additional treatment. Patients without a next/additional treatment were censored at death or end of data (4/30/2022). Patients with a within-class BTKi switch at any time post-index were censored at time of switch, as this may have indicated a switch due to tolerability rather than progression. Patients who added an anti-CD20 antibody or venetoclax to the BTKi within 180 days post-index were censored at time of add-on, as these may not have indicated overt disease progression but rather late initiation of a second anti-cancer agent as a 1L combination treatment strategy. Beyond 180 days, anti-CD20 or venetoclax add-ons were considered as a next/additional treatment. TTNT was described using Kaplan Meier curves and compared between ibrutinib and acalabrutinib using a Cox proportional hazards model adjusted for the following baseline variables: age, gender, region, race, year of index date, Quan-Charlson Comorbidity Index (Quan-CCI), chronic pulmonary disease (CPD), peripheral vascular disease, hypertension, atrial fibrillation (AF), metastatic cancer, use of corticosteroids, and use of antiplatelets. Results: Among 710 and 373 patients in the ibrutinib and acalabrutinib cohorts, respectively, mean age was 71.5 and 72.4 years (P=0.159) and 38.5% and 38.3% were female (P=0.971; Table). Mean baseline Quan-CCI score was similar for ibrutinib and acalabrutinib (3.1 vs 3.0; P=0.597); a higher proportion of patients in the ibrutinib cohort had CPD (13.6% vs 8.8%; P=0.024), peripheral vascular disease (7.8% vs 4.1%; P=0.022), and hypertension (41.4% vs 32.2%; P=0.003) at baseline, while AF was similar (7.0% vs 9.9%, P=0.098). Baseline corticosteroid use was lower in the ibrutinib cohort (14.5% vs 20.1%; P=0.018), but antiplatelet use was higher (7.0% vs 3.5%; P=0.017). Mean (median [range]) follow-up was 17.1 (18.1 [1.0-29.3]) months for the ibrutinib cohort and 12.5 (11.9 [1.0-29.1]) months for the acalabrutinib cohort. Four/710 (0.6%) patients in the ibrutinib cohort and 16/373 (4.3%) in the acalabrutinib cohort added an anti-CD20 antibody to the BTKi within 180 days and were censored at that point. Ten/710 (1.4%) and 8/373 (2.1%) patients added venetoclax to ibrutinib or acalabrutinib within 180 days and were censored at that point. The proportion of patients in the ibrutinib cohort who initiated a next or additional treatment was 5.9% relative to 7.5% for acalabrutinib. After adjusting for baseline characteristics, patients treated with acalabrutinib were 89% more likely to start a next or additional treatment than those treated with ibrutinib (hazard ratio = 1.89 [95% confidence interval] = [1.12, 3.13]; P=0.016; Figure). Results were similar when censoring anti-CD20 add-ons at any time (hazard ratio = 1.82 [95% confidence interval] = [1.08, 3.03]; P=0.025). Conclusions: In this large US RW study, patients with CLL treated with 1L acalabrutinib were significantly more likely to start a next treatment or add/intensify therapy than those treated with 1L ibrutinib. RW treatment patterns also suggest a higher propensity to add an anti-CD20 antibody to acalabrutinib versus ibrutinib in 1L CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An Ex Vivo Monocyte Stimulation Test Predicts Severity of COVID-19 Infection in Patients with Chronic Lymphocytic Leukemia (CLL)

Introduction The severity of coronavirus disease 2019 (COVID-19) is associated with the development of a cytokine storm driven by classical monocytes. Activation of the NLRP3 inflammasome mediates the production of IL-1β - a key pro-inflammatory cytokine that in excessive quantities causes tissue injury. Most CLL patients have an impaired humoral response to vaccination with subsequent higher risk of complications from COVID-19 infection. Currently, no pre-exposure test exists to assess the risk of a dysregulated immune response to COVID-19. We previously reported that open reading frame 8 (ORF8), a SARS-CoV-2 protein, when glycosylated and secreted into the blood, stimulates CD14+ monocytes and activates the NLRP3 pathway to produce IL-1β (Ruan et al, AAI 2022). We hypothesized that functional characteristics of CLL patients’ monocytes may predispose patients to develop severe COVID-19. First, we developed the ORF8 monocyte stimulation test (OMST), which measures monocyte reactivity via flow cytometry after stimulation of patient monocytes ex vivo with ORF8. Secondly, we applied the OMST to CLL patients to learn about its association with severe COVID-19 infection. Methods We identified patients from the Mayo Clinic CLL Database who were diagnosed with COVID-19 infection via a positive SARS-CoV-2 RT-PCR nasal swab between July 2020 to February 2022 and had a cryopreserved pre-COVID-19 peripheral blood mononuclear sample (PBMC) available. Our study was approved by the IRB, and samples were stored in the Mayo Clinic Predolin Biobank. For the OMST, samples were thawed and either stimulated with ORF8 or unstimulated (control) for 24 hours. The samples were harvested and intracellular cytokine/chemokine levels (IL-1β, IL-8, and CCL-2) in monocytes were analyzed by flow cytometry using the depicted gating strategy and formulas to calculate delta MFI (dMFI) and adjusted MFI (Figure 1). Clinical outcomes were abstracted by chart review. Severe infection was defined as hospitalization due to COVID-19 infection. Fisher's exact test, Wilcoxon rank sum test, and matched paired analysis were used to compare categorical, continuous, and paired variables, respectively. Receiver-operating curves (ROC) were used to calculate the area under the curve (AUC), sensitivity, specificity, and optimal cutoff levels for a reactive OMST. The optimal cutoff point was chosen as the point with the highest Youden index. Time to severe COVID-19 infection was calculated using the Kaplan-Meier method and associations of factors with severe COVID-19 were assessed using Cox proportional hazard model (CPHM). Results 60 patients were identified. The median age at COVID-19 diagnosis was 74 years (63-80). The median number of COVID-19 vaccinations before infection was 0 (range 0-3). The median time interval between vaccination and infection was 106 days (2-322). At diagnosis, 25 (42%) patients were receiving CLL-directed therapy and 22 (37%) were on a BTK-inhibitor. 22 patients had severe COVID-19 and 6 patients died. The OMST demonstrated that CD14+ monocytes had a significantly higher dMFI for IL-1β, IL-8, and CCL-2 after ORF8 stimulation compared to controls (495 vs. 158, p<0.0001), (298 vs. 121, p<0.001), and (385 vs. 171, p <0.0001) respectively. By ROC analysis, the AUC for IL-1β adjusted MFI was 0.68, 0.67 for IL-8 adjusted MFI, and 0.37 for CCL-2 adjusted MFI. A reactive OMST was defined by ROC as an IL-1β adjusted MFI >= 0.18 (sensitivity 67%, specificity 75%). The median time to hospitalization after COVID-19 infection for patients with a reactive OMST was 12 days (95% CI: 2 - not reached) versus not reached (NR) for patients with a non-reactive OMST [95% CI: NR-NR] with a hazard ratio of 7.7 (95% CI: 2.4-132, p=0.005) [Figure 2]. The following variables were not significant on univariate CPHM: age, gender, IGHV mutation, CLL-IPI risk, Charlson Comorbidity score, smoking status, number of mRNA COVID-19 vaccinations, receipt of monoclonal antibodies, or on therapy for CLL. Conclusions We have developed a novel assay, the OMST, that measures the reactivity of human monocytes to ORF8 and has significant value in determining the severity of COVID-19 infection in CLL patients. These findings are independent of patient age, sex, comorbidity score, prognostic models, and vaccination status. The OMST has the potential to assess monocyte reactivity and its implications in a broad range of clinical situations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anti-CD70 Monoclonal Antibodies Show Efficacy in Preclinical Models for Chronic Lymphoblastic Leukemia

Although therapy for chronic lymphocytic leukemia (CLL) has improved significantly with combination therapies that include ibrutinib and/or anti-B cell mAbs, resistance can develop, and patients can discontinue therapy for various reasons. Therefore, effective, alternative therapies are needed. CD70, a TNF family member, and its receptor, CD27, are mainly expressed on B and T lymphocytes. Different from normal B cells that only transiently express CD70 upon activation, CLL B cells stably express the molecule at relatively high levels, suggesting a fundamental biologic abnormality and adding another example of the dysregulation of the immune system in CLL. Here we investigated the role of CD70 in CLL using blocking anti-CD70 mAbs in preclinical mouse models of the disease. We first confirmed stable expression of CD27 and CD70 in patient samples as well as on murine TCL1 CLL B cells, including TCL1-192 cells, a set of primary murine leukemic cells within which > 95% cells react to autoantigen stimulation via the B-cell receptor (BCR). Comparing with healthy BM, we found that primary CLL patient B cells express significantly higher levels of the CD27 - CD70 receptor-ligand pair at both mRNA and protein levels, as do TCL-1 mice. We next investigated the effects of an anti-mouse CD70 effector function silenced/blocking mAb, FcD-FR70, on TCL1-192 CLL cells in vitro and in vivo in preclinical models. In vitro, treating TCL1 CLL cells with FcD-FR70 significantly inhibited CD40L-induced CLL cell growth. In vivo, twice weekly 10 mg/kg treatments with FcD-FR70 antibody significantly reduced the number of murine CLL B cells in spleens, lymph nodes and blood. The levels of reduction were comparable to those observed in anti-CD19 mAb or ibrutinib treated mice. There was a significantly better treatment outcome when therapy was initiated earlier. Moreover, the level of soluble mouse CD27, the product of CD27 cleavage after interaction with CD70, was significantly reduced in sera of animals treated with FcD-FR70. Notably, FcD-FR70, but not anti-CD19 mAb or ibrutinib, significantly reduced CD70 membrane levels. CD27 expression levels were not altered by all treatments. As CD27 and CD70 are inversely expressed in B cells, due to tight control at the transcriptional and posttranslational levels, this result strongly suggests preferential depletion of malignant cells with the high CD70 expression, which also affects levels of soluble CD27. We then performed bulk RNAseq analysis in CD19+CD5+ splenic cells from mice treated with control or FcD-FR70 antibody. Genes differing by >1.5 fold (P < 0.01) by FcD-FR70 treatment between the two groups included upregulated cell cycle regulator cyren and interferon gamma inducible protein IfI211, as well as downregulated IL10, CCR7, PIM3, PIM1, TGFb3, TNFaip3, JunB and CD69. Importantly, IPA analysis of the altered genes suggested inhibition of TLR7, TLR9, TNF, NFKB, IL4, BCR, BTK, and CD40L signaling pathways. Thus, FcD-FR70 treatment blocked key survival pathways, inducing death of CLL B cells. Finally, we studied the effect of anti-CD70 antibodies on CLL B-T cell interactions. Adding the FcD-FR70 mAb to an allo-MLR significantly blocked T cell proliferation and survival induced by TCL1-192 leukemic B cells (P < 0.01). We then tested the effects of 41D12-FcD, a blocking mAb in human IgG1 format that binds with picomolar affinity to human CD70, in a primary CLL patient derived xenograft model. This revealed a significant reduction of malignant cells in the spleen, BM, and peritoneum (P < 0.001). Interestingly, the efficacy of the blocking 41D12-FcD antibody was associated with a significant decrease of human T cells, suggesting that the antibody directly blocked the proliferation of malignant B cells and also inhibited T cell-CLL cell interactions. Notably, no significant changes were seen in the differentiation of T cells, expression of CTLA4, PD1, PD-L1, and levels of Th1/Th2 cytokines after anti-CD70 treatment. Overall, data from our preclinical models suggest targeting CD70 in CLL alters BCR, CD40L, IL4, and TNF receptor signaling and delays CLL disease progression. Although the involvement of CD70 and the relationships between CLL B cells and non-malignant cells needs further study, the availability of clinical stage therapeutic anti-CD70 antibodies supports the use anti-CD70 immunotherapy in combination therapies in CLL.

Systematic Review and Network Meta-Analysis of Frontline Targeted Therapy in Chronic Lymphocytic Leukemia Shows Comparable Efficacy but Distinct Toxicity

Introduction: Targeted therapies inhibiting Bruton Tyrosine Kinase (BTK) (ibrutinib, acalabrutinib, and zanubrutinib) and Bcl-2 (venetoclax) have improved survival when given first line (1L) to patients with chronic lymphocytic leukemia (CLL). However, no head-to-head trials of 1L targeted therapies exist to guide clinicians on selection of optimal therapy for their patients. We performed a systematic review and network meta-analysis (NMA) to understand the relative efficacy and toxicity of 1L CLL regimens, including newer regimens of ibrutinib/venetoclax (IV) and zanubrutinib not previously assessed in NMA. Methods: We performed a systematic literature search from 2009-2022 for randomized trials of treatment-naïve adults with CLL who were deemed unfit due to age or comorbidities. Endpoints were progression free survival (PFS) at 24 and 48 months, overall response rate (ORR), percentage of patients with grade 3 or higher adverse event (AE), discontinuation due to AE, and rates of AEs of clinical interest. We used a Bayesian hierarchical random-effects models for mixed multiple treatment comparisons to estimate the relative treatment effect of each intervention compared with a common comparator. Results: Our search yielded 12 clinical trials including 15 unique 1L regimens (Fig 1). PFS and ORR of common comparators were consistent across trials except 24-month PFS for chlorambucil/obinutuzumab (CO) which ranged from 31%-64% likely due to variable duration of chlorambucil and variable inclusion of patients with del(17p). Compared to CO, only acalabrutinib/obinutuzumab (AO) showed statistically significant improvement in 24-month PFS with hazard ratio (HR) 0.13 and 95% confidence interval (CI) 0.017-0.97 (Fig 1). In general, targeted therapies trended towards longer PFS compared to chemotherapy-based regimens, though there is insufficient power to demonstrate statistical significance. We found no statistically significant differences in 24-month PFS, 48-month PFS, or ORR between targeted therapies. Regarding toxicity, no statistically significant differences in proportion of patients experiencing grade 3 or higher AE were found across regimens, though single agent acalabrutinib and zanubrutinib trended toward less toxicity than CO with risk ratio (RR) 0.71, 95% CI 0.45-1.1 and RR 0.66, 95% CI 0.30-1.5, respectively. No statistically significant differences in discontinuation rates were found, though IV trended towards higher discontinuation rate than CO (RR 6.1, 95% CI 0.71-74). There were no statistically significant differences in rates of atrial fibrillation, infection, anemia, or neutropenia. However, grade 3 or higher thrombocytopenia was significantly lower with zanubrutinib than CO (RR 0.069, 95% CI 0.0048-0.92). Conclusions: Using NMA, we demonstrate that PFS is improved from the pre-targeted therapy standard of care by AO, but, amongst targeted therapies, no significant differences in efficacy were identified. Our results differ from previously published NMAs which have shown superior PFS with AO compared to other targeted therapies, likely due to inclusion of additional trials of common comparators (Foa et al, CLL5) as well as newer trials assessing IV and zanubrutinib (GLOW, SEQUOIA cohort 1) in this analysis. All four trials add heterogeneity to common comparator outcomes, resulting in widened confidence intervals and loss of statistical significance. This is the first assessment, to our knowledge, of relative rates of specific AEs, and we found that zanubrutinib showed lower rates of thrombocytopenia than pre-targeted therapy standard of care. Establishing the absence of significant difference in efficacy of frontline targeted therapies in CLL allows the clinician and patient to select therapy based on other factors, namely planned duration, toxicity profile, medication interactions, and cost. Figure 1. 24-month PFS Network and Forest Plot 1Chl = Chlorambucil; Acal = Acalabrutinib; Acal/Obin = Acalabrutinib/Obinutuzumab; BR = Bendamustine/Rituximab; Chl/Ofa = Chlorambucil/Ofatumumab; Chl/Ritux = Chlorambucil/Rituximab; Chl/Ritux + Mtn = Chlorambucil/Rituximab + maintenance Rituximab; Flu = Fludarabine; Ibr = Ibrutinib; Ibr/Obin = Ibrutinib/Obinutuzumab; Ibr/Ritux = Ibrutinib/Rituximab; Ibr/Ven = Ibrutinib/Venetoclax; Ven/Obin = Venetoclax/Obinutuzumab; Zan = Zanubrutinib Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal