B-cell Chronic Lymphocytic Leukemia Research Articles

Palladium and Platinum Complexes of the Antimetabolite Fludarabine with Vastly Enhanced Selectivity for Tumour over Non-Malignant Cells.

The purine derivative fludarabine is part of frontline therapy for chronic lymphocytic leukaemia (CLL). It has shown positive effects on solid tumours such as melanoma, breast, and colon carcinoma in clinical phase I studies. As the treatment of CLL cells with combinations of fludarabine and metal complexes of antitumoural natural products, e.g., illudin M ferrocene, has led to synergistically enhanced apoptosis, in this research study different complexes of fludarabine itself. Four complexes bearing a trans-[Br(PPh3)2]Pt/Pd fragment attached to atom C-8 via formal η1-sigma or η2-carbene bonds were synthesised in two or three steps without protecting polar groups on the arabinose or adenine. The platinum complexes were more cytotoxic than their palladium analogues, with low single-digit micromolar IC50 values against cells of various solid tumour entities, including cisplatin-resistant ones and certain B-cell lymphoma and CLL, presumably due to the ten-fold higher cellular uptake of the platinum complexes. However, the palladium complexes interacted more readily with isolated Calf thymus DNA. Interestingly, the platinum complexes showed vastly greater selectivity for cancer over non-malignant cells when compared with fludarabine.

Cell Surface Lipoprotein Lipase Enzyme as A Prognostic Indicator in B-Cell Chronic Lymphocytic Leukemia

Lipoprotein lipase (LPL) has emerged as a distinct prognostic marker for chronic lymphocytic leukemia (CLL), a heterogeneous disease with a similar heterogeneity in life expectancy. While many prior studies have focused on tumor expression of LPL mRNA, LPL surface protein expression has been less robustly studied as a prognostic marker. A novel antibody developed at Geisel School of Medicine at Dartmouth has been previously utilized in immunohistochemistry assays of breast and prostate cancer. With this study we aimed to use this antibody as a flow cytometry marker of surface LPL expression correlated with overall survival and time to first treatment. Of the 19 patients studied, our data show that LPL surface protein expression as measured by flow cytometry trended toward a protective effect on overall survival and overall better prognosis.

SGR‐1505‐101: A PHASE 1, OPEN‐LABEL, MULTICENTER, DOSE‐ESCALATION STUDY OF SGR‐1505 AS MONOTHERAPY IN SUBJECTS WITH MATURE B‐CELL MALIGNANCIES

Background: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of the nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. In particular, constitutive activation of the NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity, and demonstrates anti-proliferative activity in ABC-DLBCL cell lines, both BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3). SGR-1505 administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, demonstrates tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived xenograft and patient-derived xenograft models. Aims: These data suggest that SGR-1505-mediated MALT1 inhibition has therapeutic potential and may expand therapeutic options for patients with selected B-cell lymphomas supporting further evaluation of SGR-1505 in clinical trials. Methods: SGR-1505-101 (NCT05544019) is a phase 1, multicenter trial of SGR-1505 as monotherapy in subjects with mature B-cell malignancies. At present, the study has been activated in 3 sites in the United States. The primary objective is to evaluate safety and tolerability of SGR-1505 as monotherapy and to identify the maximum tolerated dose (MTD) and/or recommended dose (RD). Secondary objectives are to evaluate the pharmacokinetic (PK) profile, food effect, drug-drug interaction, and preliminary anti-tumor activity of SGR-1505. Key inclusion criteria are: history of mature B-cell malignancy (including aggressive and indolent B-cell lymphomas, Waldenström macroglobulinemia, and CLL); measurable or detectable disease according to the applicable disease-specific classification system (Lugano, iwCLL, WWM6); Eastern Cooperative Oncology Group (ECOG) performance status of ≤2. Patients with indolent B-cell lymphomas or CLL must have an indication for treatment and not require immediate cytoreductive therapy. Subjects with symptomatic or active CNS involvement, and other conditions or laboratory findings placing them at increased risk to the use of an investigational drug are excluded. SGR-1505 is initially dose-escalated using an accelerated titration design in cohorts of 1–6 subjects, and at higher dose levels using a conventional 3+3 design. Encore Abstract—previously submitted to EHA 2023 The research was funded by: Schrodinger Keywords: aggressive B-cell non-Hodgkin lymphoma, molecular targeted therapies, ongoing trials Conflicts of interests pertinent to the abstract. B. Yoo Employment or leadership position: Schrodinger Stock ownership: Schrodinger Z. Nie Employment or leadership position: Schrodinger Stock ownership: Schrodinger G. Krilov Employment or leadership position: Schrodinger Stock ownership: Schrodinger J. Tan Employment or leadership position: Schrodinger Stock ownership: Schrodinger H. Wright Employment or leadership position: Schrodinger Stock ownership: Schrodinger D. Weiss Employment or leadership position: Schrodinger Stock ownership: Schrodinger W. Yin Employment or leadership position: Schrodinger Stock ownership: Schrodinger K. Akinsanya Employment or leadership position: Schrodinger Stock ownership: Schrodinger

Characterization of the genetic and epigenetic landscape of B‐cell neoplasms with IG::BCL3‐translocation

Introduction: The BCL3 gene (19q13) encodes a transcriptional coactivator of the nuclear factor-kappaB (NF-κB) family. It is recurrently deregulated in B-cell neoplasms by a juxtaposition to regulatory elements of one of the immunoglobulin loci, mostly the immunoglobulin heavy chain (IGH) locus (14q32). Two distinct subsets of IG::BCL3 translocation-positive B-cell neoplasms, differing in the number of chromosomal aberrations, IGHV mutation status and histopathology, have been described [Martín-Subero et al., 2007]. In B-cell chronic lymphocytic leukaemia (CLL), the IG::BCL3 translocation has been associated with younger age at diagnosis, a more aggressive clinical course and an atypical tumour cell phenotype [Michaux et al., 1997; Au et al., 2002]. Methods: A total of 89 B-cell neoplasms with IG::BCL3 translocation detected by fluorescence in situ hybridisation (FISH), including predominately cases diagnosed as CLL, were studied. Breakpoints at the BCL3 and IGH loci were sequenced in a subset of samples using targeted capture and breakpoint-spanning PCR approaches. Chromosomal aberrations detected by FISH as well as the IGHV mutation status were analysed. In addition, DNA methylation and copy number aberration (CNA) analyses were performed for 48 samples with CLL using the 850K EPIC BeadChip array. Results: Targeted capture-based sequencing data showed that in the majority of cases BCL3 translocations are associated with aberrant class-switch recombination at the IGH locus, often involving IGHA segments (n = 4/10). From the 69 IG::BCL3-translocated cases with available IGHV mutation status, a total of 51 (74%) were classified as unmutated and 18 (26%) as mutated. This indicates an overrepresentation of cases with unmutated IGHV status among the IG::BCL3-translocated cases compared to the general CLL population. Based on EPIC BeadChip data (total n = 48) 25 (52%) IG::BCL3-translocated cases carried a trisomy 12, 11 cases (23%) a deletion 13q, 7 cases (15%) a deletion 17p and 3 cases (6%) a deletion 11q. FISH analysis of a partially overlapping set of neoplasms (n = 54) was in line with the EPIC BeadChip data and revealed 17 IG::BCL3-translocated cases (32%) with trisomy 12, 7 cases (13%) with deletion 17p and deletion 13q each, and 4 cases (7%) with deletion 11q. Thus, IG::BCL3-translocated cases showed a striking skewing towards the presence of trisomy 12 compared to the general CLL population (p = 0.011). In the DNA methylation analyses, the IG::BCL3-translocated CLLs segregated separately from other CLLs with and without IGH translocations. Conclusions: Our large multi-OMICs study of IG::BCL3-translocated B-cell neoplasms corroborates previous reports on the pathogenetic heterogeneity of these lymphomas and moreover provides evidence that IG::BCL3-translocated CLL might form a genetically and epigenetically distinct subtype of B-cell neoplasms distinct from common CLL. Keywords: Chronic Lymphocytic Leukemia (CLL), Genomics, Epigenomics, and Other -Omics No conflicts of interests pertinent to the abstract.

IL16 PRODUCTION IS A MECHANISM OF RESISTANCE TO BTK INHIBITORS AND TO R‐CHOP

Introduction: Covalent and non-covalent BTK inhibitors (BTKi) are used or being explored for the treatment of patients with B-cell malignancies, such as marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) of the activated B-cell type (ABC). The identification of mechanisms of resistance can lead to improvements in the current therapeutic approaches. We had reported IL16 production in a MZL model of secondary resistance to ibrutinib (Arribas ENA2019). Here, we expand the initial observation to additional lymphoma types and treatments modalities, performing therapeutic interventions to block it and providing clinical validation data on its expression and role in patients. Methods: VL51 (marginal zone lymphoma, MZL) parental (PAR) and Ibrutinib-resistant (RES) cells underwent RNA (mRNA & miRNA by RNA-Seq), DNA (promoter methylation, WES) and secretome (Cytokine array) profiling. RES cells exhibited resistance to covalent and non-covalent BTK inhibitors. No DNA copy number variations or mutations were detected by WES in RES compared to PAR cells, including those affecting BTK or PLCG2 genes. Results: Multi-omics (DNA, mRNA, miRNA, secretome) characterization of RES and PAR cells identified hypomethylation, up-regulation and secretion of IL16 in RES but not in PAR cells. Elevated levels of p-AKT and p-ERK was observed in RES compared to PAR, suggesting a signaling activation in RES cells. Stimulation with recombinant IL16 reduced sensitivity to BTKi not only in VL51 PAR cells, but also in models derived from MCL (REC1) and CLL (MEC1). Conversely, the use of IL16 blocking antibody recovered sensitivity to BTKi in RES cells, along with other lines with high expression of IL16 and low sensitivity to ibrutinib derived from MCL (MAVER1) and MZL (SSK41). IL16 is part of the ABC-DLBCL signature. We observed that high IL16 expression was associated with shorter overall survival in ABC but not in germinal center like DLBCL patients (n = 233, log-rank test p = 0.013, multivariate Cox: HR (95% CI) 0.43 (0.22–0.85), p = 0.016). IL16 stimulation also decreased sensitivity to RCHOP in ABC DBCL cells (OCILy10, HBL1). Finally, secreted levels of IL16 in the serum of CLL patients (n = 17) with resistance to ibrutinib (in absence of BTK or PLCG2 mutations) were significantly higher compared to patients responding to the drug and paired for clinical features. Conclusions: Starting from a MZL model of secondary resistance to ibrutinib, we showed that IL16 can sustain resistance to BTK inhibitors also in CLL and MCL. Furthermore, increased expression of IL16 associated with shorter outcome in ABC DLBCL patients and the interleukin induced in vitro resistance to RCHOP. The demonstration that IL16 blocking therapies recovered sensitivity to therapies can lead to novel therapeutic approaches to overcome resistance in lymphoma patients. The research was funded by: Swiss National Science Foundation (SNSF 31003A_163232/1) Keyword: molecular targeted therapies Conflicts of interests pertinent to the abstract A. Arribas Educational grants: Astra Zeneca

Humoral and cellular immune responses after three or four doses of BNT162b2 in patients with hematological malignancies.

Initial responses to coronavirus disease 2019 vaccination are impaired in patients with hematological malignancies. We investigated immune responses after three or four doses of BNT162b2 in patients with myeloid and lymphoid malignancies compared to controls, and identified risk factors for humoral and cellular nonresponse 1 year after first vaccination. In 407 hematological patients (45 myeloid, 362 lymphoid) and 98 matched controls, we measured immunoglobulin G (IgG) and neutralizing antibodies specific for the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at baseline, 3 weeks, 2, 6, and 12 months, and interferon-γ release at 12 months. In patients with lymphoid malignancies, SARS-CoV-2 receptor-binding domain IgG concentration and mean neutralizing capacity was lower than in controls at all time points. A diagnosis of chronic lymphocytic B-cell leukemia (CLL) or lymphoma was associated with humoral nonresponse at 12 months compared to having multiple myeloma/amyloidosis (p < .001 and p = .013). Compared to controls, patients with lymphoid malignancies had increased risk of cellular nonresponse. A lymphoma diagnosis was associated with lower risk of cellular nonresponse compared to patients with multiple myeloma/amyloidosis, while patients with CLL had comparable response rates to patients with multiple myeloma/amyloidosis (p = .037 and p = .280). In conclusion, long-term humoral and cellular immune responses to BNT162b2 were impaired in patients with lymphoid malignancies.

Preliminary analysis of double-negative T, double-positive T, and natural killer T-like cells in B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the expansion of CD5+ malignant B lymphocytes. Recent discoveries have shown that double-negative T (DNT) cells, double-positive T (DPT) cells, and natural killer T (NKT)-cells may be involved in tumor surveillance. A detailed immunophenotypic analysis of the peripheral blood T-cell compartment of 50 patients with B-CLL (classified in three prognostic groups) and 38 healthy donors (as controls) matched for age was performed. The samples were analyzed by flow cytometry using a stain-lyse-no wash technique and a comprehensive six-color antibody panels. Our data confirmed a reduction in percentage values and an increase in absolute values of T lymphocytes in patients with B-CLL, as already reported. In particular, DNT, DPT, and NKT-like percentages were significantly lower than in the controls, except for NKT-like in the low-risk prognostic group. Moreover, a significant rise in the absolute counts of DNT cells in each prognostic group and in the low-risk prognostic group of NKT-like cells was found. A significant correlation of the absolute values of NKT-like cells in the intermediate-risk prognostic group versus B cells was observed. Furthermore, we analyzed whether the increase in T cells was related to the subpopulations of interest. Only DNT cells were positively correlated with the increase in CD3+ T lymphocytes, regardless of the stage of the disease, supporting the hypothesis that this T-cell subset plays a key role in the immune T response in B-CLL. These early results supported that DNT, DPT, and NKT-like subsets may be related to disease progression and should encourage further studies aimed at identifying the potential immune surveillance role of these minority T subpopulations.

B Cell Chronic Lymphocytic Leukemia Development in Mice with Chronic Lung Exposure to Coccidioides Fungal Arthroconidia.

Links between repeated microbial infections and B cell chronic lymphocytic leukemia (B-CLL) have been proposed but not tested directly. This study examines how prolonged exposure to a human fungal pathogen impacts B-CLL development in Eµ-hTCL1-transgenic mice. Monthly lung exposure to inactivated Coccidioides arthroconidia, agents of Valley fever, altered leukemia development in a species-specific manner, with Coccidioides posadasii hastening B-CLL diagnosis/progression in a fraction of mice and Coccidioides immitis delaying aggressive B-CLL development, despite fostering more rapid monoclonal B cell lymphocytosis. Overall survival did not differ significantly between control and C. posadasii-treated cohorts but was significantly extended in C. immitis-exposed mice. In vivo doubling time analyses of pooled B-CLL showed no difference in growth rates of early and late leukemias. However, within C. immitis-treated mice, B-CLL manifests longer doubling times, as compared with B-CLL in control or C. posadasii-treated mice, and/or evidence of clonal contraction over time. Through linear regression, positive relationships were noted between circulating levels of CD5+/B220low B cells and hematopoietic cells previously linked to B-CLL growth, albeit in a cohort-specific manner. Neutrophils were positively linked to accelerated growth in mice exposed to either Coccidioides species, but not in control mice. Conversely, only C. posadasii-exposed and control cohorts displayed positive links between CD5+/B220low B cell frequency and abundance of M2 anti-inflammatory monocytes and T cells. The current study provides evidence that chronic lung exposure to fungal arthroconidia affects B-CLL development in a manner dependent on fungal genotype. Correlative studies suggest that fungal species differences in the modulation of nonleukemic hematopoietic cells are involved.

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.

How We Manage Patients with Indolent B-Cell Malignancies on Bruton’s Tyrosine Kinase Inhibitors: Practical Considerations for Nurses and Pharmacists

The most common forms of B-cell malignancy, non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), have seen a drastic shift in the treatment landscape over the last two decades with the introduction of targeted agents. Among them are Bruton's tyrosine kinase (BTK) inhibitors, which have demonstrated excellent efficacy in indolent B-cell NHLs and CLL. Although BTK inhibitors are generally thought to be more tolerable than chemoimmunotherapy, they are associated with a unique safety profile including varying rates of rash, diarrhea, musculoskeletal events, cardiovascular events, and bleeding. Ibrutinib was the first BTK inhibitor to gain a Health Canada indication, followed by second-generation BTK inhibitors acalabrutinib and zanubrutinib, which have better safety profiles compared to ibrutinib, likely due to their improved selectivity for BTK. As BTK inhibitors are oral agents given continuously until disease progression, long-term adverse event (AE) monitoring and management as well as polypharmacy considerations are important for maintaining patient quality of life. This paper intends to serve as a reference for Canadian nurses and pharmacists on dosing, co-administration, and AE management strategies when caring for patients with indolent B-cell NHL or CLL being treated with BTK inhibitors.

In-vitro Modulation of mTOR-HIF-1α Axis by TLR7/8 Agonist (Resiquimod) in B-Chronic Lymphocytic Leukemia

Targeting toll-like receptors (TLRs), via TLR agonists, has been implicated in the regulation of immunometabolism. B-chronic lymphocytic leukemia (B-CLL) represents a suitable model for B-cell derived malignancies with shifted metabolic adaptations. Several signaling pathways have been found to be critical in metabolic reprogramming of CLL, including mechanistic target of rapamycin- hypoxia inducible factor-1α (mTOR- HIF-1α) pathway, the main metabolic regulator of glycolysis. Here, we investigated the effect of TLR7/8 agonist (Resiquimod) on the expression of mTOR and HIF-1α in patients with CLL. B cells were purified using Rosettesep Human B cell Enrichment Cocktail (Stem cell Technologies, Vancouver, BC, Canada#15,024) from peripheral venous blood of CLL patients (n = 20) and healthy individuals (n = 15). Isolated B cells were then cultured in both presence and absence of Resiquimod. Gene expression of mTOR and HIF-1α were assessed using qRT-PCR. Resiquimod significantly decreased mTOR and HIF-1α gene expression in both CLL (p < 0.001and p < 0.001, respectively) and Normal B cells (p = 0.004 and p = 0.001, respectively). Resiquimod may reprogram immunometabolism of malignant B-CLL cells via down-regulation of key glycolytic metabolic actors, mTOR and HIF-1α genes. Accordingly, Resiquimod may be an adjuvant as a therapeutic tool for CLL, which needs to be studied further.

Direct Cell Death Induced by CD20 Monoclonal Antibodies on B Cell Lymphoma Cells Revealed by New Protocols of Analysis

CD20 monoclonal antibodies (mAbs) eliminate B cells in several clinical contexts. At least two of these Abs, obinutuzumab (OBI) and rituximab (RTX), induce quick elimination of targets and put cancer patients at risk of tumor lysis syndrome (TLS) within 12-24 h of the first dose. The mechanisms of killing can require the recruiting of effector mechanisms from the patient's immune system, but they can induce direct killing as well. This can be more rapid than recruiting cellular effectors and/or complement. We showed here that OBI and RTX induce quick (<1 h) and high (up to 60% for OBI) killing of two different B cell lines. This was unveiled by using two different techniques that circumvent cell centrifugation steps: a Muse® Cell Analyzer-based approach and a direct examination of the cells' physical properties by using forward scatter (FS) area and side scatter (SS) area by flow cytometry. These results excluded the presence of aggregates and were also confirmed by developing a normalized survival ratio based on the co-incubation of RTX- and OBI-sensitive cells with MOLM-13, an insensitive cell line. Finally, this normalized survival ratio protocol confirmed the RTX- and OBI-direct killing on primary tumor B cells from B cell chronic lymphocytic leukemia (B-CLL) and Non-Hodgkin's lymphoma (NHL) patients. Moreover, we unveiled that direct killing is higher than previously expected and absent in patients' samples at relapse. We also observed that these mAbs, prior to increasing intracellular calcium levels, decrease calcium entry, although manipulating calcium levels did not affect their cytotoxicity. Altogether, our results show that direct killing is a major mechanism to induce cell death by RTX and OBI mAbs.

Systematic Evaluation of Antigenic Stimulation in Chronic Lymphocytic Leukemia: Humoral Immunity as Biomarkers for Disease Evolution.

In this study, we conducted a quantitative serum analysis of 7 immunoglobulins in CLL and monoclonal B-cell lymphocytosis (MBL) patients (bead-suspension protein arrays) and a serological profile (IgG and IgM) study of autoantibodies and antimicrobial antigens (protein microarrays). Significant differences in the IgA levels were observed according to disease progression and evolution as well as significant alterations in IgG1 according to IGHV mutational status. More representative IgG autoantibodies in the cohort were against nonmutagenic proteins and IgM autoantibodies were against vesicle proteins. Antimicrobial IgG and IgM were detected against microbes associated with respiratory tract infections. Quantitative differences in immunoglobulin serum levels could be potential biomarkers for disease progression. In the top 5 tumoral antigens, we detected autoantibodies (IgM and IgG) against proteins related to cell homeostasis and metabolism in the studied cohort. The top 5 microbial antigens were associated with respiratory and gastrointestinal infections; moreover, the subsets with better prognostics were characterized by a reactivation of Cytomegalovirus. The viral humoral response could be a potential prognosis biomarker for disease progression.

Paraparesis Caused by Chronic Lymphocytic Leukemia Mass Compressing the Spinal Cord

B-cell chronic lymphocytic leukemia (CLL-B) is the commonest adult-onset leukemia. Although extra-medullary disease has been reported, neurological consequences are seldom a concern. We aim to report an unusual case of a 66 years-old woman who developed subacute paraparesis due to dorsal (D2-D6) spinal cord compression by an extradural CLL mass, 2 years after CLL diagnosis.

THE COURSE OF CHRONIC LYMPHOCYTIC LEUKEMIA AFTER SARS-COV-2 VIRUS INFECTION.

Objective - to investigate the course of B-cell chronic lymphocytic leukemia (CLL) in patients after SARS-CoV-2 virus infection taking into account anamnestic exposure to the ionizing radiation (IR).Methods. The study was performed in a group of 51 CLL patients who were admitted to the Department of Radiation hematology of the National Research Center for Radiation Medicine of NAMS of Ukraine, Kyiv, from January 2020 (the beginning of SARS-CoV-2 epidemic) to August 2023. The group included 19 (37.3 %) clean-up workers of the Chornobyl NPP accident, 15 (29.4 %) inhabitants of radionuclide contaminated areas and 17 (33.3 %) IR non-exposed patients. The diagnosis of CLL was based on clinical history, lymphocyte morphology, and immunophenotypic criteria. Statistical studies were performed using the SPSS software package, version 20.0.Results. The diagnosis of CLL was established for the first time in 14 patients, in seven of them, CLL was diagnosed after 2-17 months after SARS-CoV-2 infection. In contrast to patients who did not suffer from a coronavirus infection, they had pronounced lymphadenopathy, which in some cases was accompanied by hyperleukocytosis, and needed early treatment. Thirteen patients with a previously established CLL were diagnosed with COVID-19 by PCR test. In seven of them (53.8 %) starting treatment was needed, or CLL has progressed. Seven of 51 patients (13.5 %) were vaccinated against SARS-CoV-2. Then, four of them were diagnosed with SARS-CoV-2 infection, confirmed by a positive PCR test, and two patients had a relapse of CLL within 1-2 months after vaccination. Most of patients with signs of the influence of SARS-CoV-2 infection on CLL belonged to sufferers of the Chornobyl NPP accident Conclusions. The clinical features of CLL that developed after SARS-CoV-2 were characterized firstly. The negative impact of SARS-CoV-2 infection on previously established CLL was established. The question about vaccination of CLL patients remains debatable.

Application of modified Matutes score containing CD200 in diagnosis of chronic lymphocytic leukemia.

Immunophenotyping technique is a powerful tool for the diagnosis and differential diagnosis of chronic lymphocytic leukemia (CLL) and other B-cell chronic lymphoproliferative diseases (B-CLPD). CD200 is strongly expressed in CLL. This study aims to analyze the clinical value of modified Matutes score (MMS) containing CD200 in the diagnosis of CLL. We retrospectively analyzed 103 B-CLPD patients diagnosed from January 2020 to July 2021, including 64 CLL patients, 11 follicular lymphoma (FL) patients, 14 mantle cell lymphoma (MCL) patients, 6 marginal zone lymphoma (MZL) patients, 1 hairy cell leukemia (HCL) patient, and 7 lymphoplasmic lymphoma/Waldenstrom macroglobulinemia (LPL/WM) patients. The expression of CD markers between the CLL group and the non-CLL group was compared, and the sensitivity, specificity, and clinical consistency of MMS and Royal Marsden Hospital (RMH) immunophenotyping score system were analyzed. There were significant differences in the expressions of CD5, CD23, FMC7, CD22, CD79b, CD200, and sIg between the CLL group and the non-CLL group (χ2 values were 37.42, 54.98, 30.71, 11.67, 55.26, 68.48, and 17.88, respectively, all P<0.01). When the RMH immunophenotyping score≥4, the sensitivity was 79.7%, and the specificity was 100%. When the MMS≥3, the sensitivity was 95.3%, and the specificity was 100%. The Kappa coefficient of RMH immunophenotyping system was 0.677, and the Kappa coefficient of MMS system was 0.860. The MMS system containing CD200 has better sensitivity and same specificity compared with RMH immunophenotyping system, and MMS system may be more useful in the diagnosis of CLL.

Chimeric Antigen Receptor T Cells: Toxicity and Management Considerations

Chimeric Antigen Receptor T Cells: Toxicity and Management Considerations

Clinical Applications of Flow Cytometry in Cancer Immunotherapies: From Diagnosis to Treatments.

The scope of flow cytometry is rapidly expanding in the diagnosis of various cancers, and it is being used routinely as an aid in classifying leukemias and lymphomas. There are several applications of flow cytometry to enumerate tumorigenic anomalies in patients. The unusual distribution of cells in various locations, their DNA content, cell proliferation rate, dysregulated expression of several surface receptors, and expression of tumor antigens are some examples that can be characterized by using different flow cytometry-based techniques. For instance, the differential diagnosis between chronic lymphocytic leukemia (CLL) and various other mature B-cell neoplasms can be made by immunophenotyping in combination with absolute counting of numerous cellular subsets or by enumerating their percent distributions. Flow cytometry has several advantages over conventional techniques which include the ability to acquire a multiparametric data in a relatively shorter time and facilitate the comparative analysis of specific cellular subsets in an efficient manner.In addition to diagnosis, there are several other applications of flow cytometry in the management of various cancers which include treatment monitoring or even selecting a personalized precision-based immunotherapy in synch with advanced genetic tests to increase the chances of favorable prognosis and complete remission. The detection of chimeric antigen receptors (CARs) on various engineered effector cells can also be determined along with their specificity in engaging the targets. Furthermore, the assessment of numerous immunological parameters, their effector functions and potencies including the proliferation dynamics, cytokine secretion profiles, and activation efficiencies can also be measured before starting immunotherapies in patients.This chapter is a brief overview of flow cytometry applications in the diagnosis and treatment strategies of various cancers.

Bcl-2 Family (Bcl-2, Mcl-1, Bim and Bax) and Btk Expression in B-Cell Chronic Lymphocytic Leukemia (B-CLL) Lymphocytes in Different Lymphoid Compartments and Changes during Btk Inhibitor Therapies (ibrutinib or acalabrutinib)

B-cell chronic lymphocytic leukemia (B-CLL) is characterized with variable infiltration by B -CLL lymphocytes of various lymphoid compartments, i.e peripheral blood (PB), bone marrow (BM) and lymph nodes (LN) and lymphoid organs. Interactions with various microenvironments may result in different disease activity and resistance to apoptosis. Novel agents targeting Btk and Bcl-2 show remarkable activity in B-CLL. However, they may have different activity in different lymphoid compartments while Btk inhibitors also may cause significant B-CLL lymphocyte redistribution between compartments. Aim of this study was to evaluate: 1) is there a different expression of Bcl-2 family of anti- and pro-apoptotic proteins (Bcl-2, Bax, Bim, mcl-1) and Btk in B-CLL lymphocytes from different lymphoid compartments; 2) relationship of observed expressions to disease parameters (TTM, TD, stage, B2MG, LDH); 3) changes during treatment with Btk inhibitors. We have included 28 B-CLL patients (18/10 M/F, median age 71 yr, range 48-85) treated with Btk inhibitors (ibrutinib 24, acalabrutinib 4). There were 15 previously treated patients and 13 treatment naïve patients. All evaluated patients had samples from PB and BM before treatment while 19 patients had also LN samples. At least one follow-up sample from PB had 20 patients. Median TTM score was 10.2 (range 3.4-23.9) and TD score was 0.68. Rai stage III/IV had 8 patients. Expression of Bcl-2, Mcl-1, Bim, Bax and Btk was determined by flow cytometry in CD19+CD5+ lymphocytes from fresh samples taken simultaneously by venipuncture (PB) or fine needle aspiration (BM and LN). There is higher expression of Bcl-2, Mcl-1 in LN compared to PB and BM (p<0.05), while there is no change in follow-up PB during ibrutinib/acalbrutinib treatment. There is no significant difference in Bim expression between compartments, while there is trend for higher expression in PB follow-up sample (p=0.054). There is no significant difference in Bax expression between PB, BM and LN compartments, while there is significant drop in Bax expression in PB follow-up sample during treatment (p<0.05). p-Btk has higher expression in LN compared to PB and BM (p<0.05). Follow-up PB p-Btk expression is comparable to pretreatment expression in PB (despite redistribution). We haven't found significant correlation of Bcl-2 family expression and p-Btk expression to the parameters of tumor mass (TTM) and tumor distribution (TD). In conclusion, there is a different pattern of expression of anti-apoptotic and pro-apoptotic Bcl-2 family members and Btk between lymphoid compartments. Our results may indicate higher BCR signaling and higher antiapoptotic activity in B-CLL lymphocytes in lymph node microenvironment. With redistribution during Btk inhibitor treatment B-CLL lymphocytes assume PB phenotype with downregulation of Btk and anti-apoptotic Bcl-2 and Mcl-1 proteins. Downregulation of pro-apoptotic Bax and possible upregulation of Bim protein indicate that there may be more complex interplay between various proteins resulting in different disease course. These results again emphasize the role of LN microenvironment in pathogenesis of B-CLL and may provide insights for treatment strategies combining targeted agents (Btk inhibitors and Bcl-2 antagonists).

NNV024, a Humanized Anti-CD37 Antibody with Enhanced ADCC and Extended Plasma Half-Life for the Treatment of B-Cell Malignancies

Background: There is a significant unmet clinical need for new therapeutic approaches for patients with B-cell non-Hodgkin lymphomas (B-NHL) who relapse or are refractory to anti-CD20 therapies. Moreover, certain B-cell malignancies do not express CD20. High expression of the CD37 antigen on malignant B-cells associated with limited or no expression on other hematopoietic cells makes CD37 a highly attractive alternative target for B-cell depleting therapies. We have therefore developed a humanized anti-CD37 monoclonal antibody (NNV024) engineered to exhibit a strong antibody-dependent cellular cytotoxicity (ADCC). Methods: Generation of NNV024 was performed by combining in silico CDR-grafting and structure-based back mutation. Candidates were then selected for their affinity to CD37 expressing cell line, Ramos. In silico immunogenicity prediction tools were integrated in the selection process to support the generation of a candidate with low immunogenicity potential (NNV023). Last, to increase ADCC, the selected candidate was afucosylated at the N-linked bi-antennary glycan at N297 of the Fc region (NNV024). The NNV024 was characterized in vitro and in vivo. Binding to human Fc gamma receptors (FcγRs), neonatal Fc receptor (FcRn), and complement component 1q (C1q) was confirmed by ELISA and SPR. ADCC and antibody-dependent cellular phagocytosis (ADCP) induction were measured using cell-based reporter assays with eleven cell lines derived from B-NHL subtypes such as Burkitt's lymphoma, diffuse large B-Cell lymphoma (DLBCL), and mantel cell lymphoma (MCL). All cell lines express both CD20 and CD37 on their cell surface. In addition, B cell chronic lymphocytic leukaemia (B-CLL) cells isolated from patient samples were used to test induction of ADCC, ADCP and complement-dependent cytotoxicity (CDC) in vitro. NNV024 plasma pharmacokinetics were assessed in Tg32 transgenic mice expressing human FcRn, which is considered a key regulator of IgG plasma half-life. A therapy study comparing NNV024 (n=10) and obinutuzumab (n=10) was performed using a disseminated tumor (Daudi) model in CB17-SCID mice (107 cells/mouse, i.v.). The anti-CD20 Abs rituximab and obinutuzumab as well as the anti-CD37 Ab, Duohexabody-CD37, were used for benchmarking. Results: As expected, the glyco-engineering increased affinity of NNV024 to FcγRIIIa (KD before afucosylation: 460.8 ng/mL; KD after: 104.1 ng/mL). Binding of Abs to FcγRIIIa activates immunological effector cells such as Natural Killer cells, macrophages, monocytes, and eosinophils. NNV024 demonstrated ADCC superior to that observed with obinutuzumab, rituximab, and Duohexabody-CD37. NNV024 exhibited a 2-fold prolonged plasma half-life in mice (8.9±0.8 days) compared to obinutuzumab (4.4±1.3 days) and Duohexabody-CD37 (4.1±1.2 days). Mice survival was enhanced up to ~ 40% when treated with NNV024 (10 µg/mouse - 70.5 days; 50 µg/mouse - 84 days) compared to obinutuzumab at a dose of 10 µg (70.5 days vs. 53 days) or 50 µg (84 days vs. 62 days). Conclusion: These encouraging preclinical results support further development of NNV024 as a potential therapeutic mAb candidate for the treatment of B-cell malignancies and B-cell driven autoimmune disorders.