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Antibody-directed targeting of chemotherapeutic nanoparticles to primary human cancers holds promise for improving efficacy and reducing off-target toxicity. However, clinical responses to targeted nanomedicines are highly variable. Herein, we prepared and examined a matrix of 9 particles (organic and inorganic particles of three surface chemistries with and without antibody functionalization) and developed an ex vivo model to study the person-specific targeting of nanoparticles in whole blood of 15 patients with chronic lymphocytic leukemia (CLL). Generally, anti-CD20-functionalized poly(ethylene glycol) (PEG) nanoparticles efficiently targeted CLL cells, leading to low off-target phagocytosis by granulocytes and monocytes in the blood. However, there was up to 164-fold patient-to-patient variability in the CLL targeting. This was further exemplified through using clinically relevant PEGylated doxorubicin-encapsulated liposomes, which showed high interpersonal differences in CLL targeting (up to 234-fold differences) and off-target phagocytosis (up to 65- and 112-fold differences in granulocytes and monocytes, respectively). Off-target phagocytosis led to almost all monocytes being killed within 24 h of treatment. Variance of the off-target association of PEGylated liposomes with granulocytes and monocytes significantly correlated to anti-PEG immunoglobulin G levels in the blood of CLL patients. A negative correlation between CLL targeting of PEG particles and anti-PEG immunoglobulin M levels was found in the blood. Taken together, our study identifies anti-PEG antibodies as key proteins in modulating patient-specific targeting of PEGylated nanoparticles in human leukemia blood. Other factors, such as the antigen expression of targeted cells and fouling properties of nanoparticles, also play an important role in patient-specific targeting. The human leukemia blood assay we developed provides an ex vivo model to evaluate interpersonal variances in response to targeted nanomedicines.

Background and Objective: The DNA load of EBV may play a part in CLL pathogenesis and prognosis. The objective of this cross-sectional study was to examine the prognostic value of EBV viral load in CLL patients in comparison with other common laboratory prognostic factors., Materials and Methods: Whole blood and sera from forty untreated CLL patients were collected. Next, DNA was extracted from total white blood cells (WBC), and TaqMan real-time PCR was performed to determine the EBV-DNA load by amplifying a specific fragment in the BNRF1 gene. In addition, parameters such as complete blood counts (CBC) and lactate dehydrogenase (LDH) were determined using an automated clinical laboratory analyzer., Results: Twenty-one patients (52.5%) were positive for EBV by real-time PCR analysis (ranged 20 to 30000 copies/µL). The difference in LDH mean levels between EBV positive and negative patients was marginally significant (P = 0.05). Furthermore, platelet (PLT) count (P = 0.03) and CD5 + /CD19 + count (P = 0.04), between EBV positive and negative subgroups, were substantially different. In addition, individuals with a severe form of illness, as defined by an increase in LDH, a decrease in PLT, and an 11q deletion, had considerably higher EBV-DNA copy numbers (the ranges of viral loads were 9966.66 ± 20033 in the severe form vs. 137.13 ± 245.41 in the mild form)., Conclusion: The EBV-DNA load could be used as a prognostic factor in the initial examination of CLL patients to better characterize the disease outcome and prognosis., (© 2024. The Author(s).)

Background: We employed Mendelian randomization (MR) to investigate the causal relationship between the gut microbiota and lymphoid leukemia, further exploring the causal relationships among immune cells, lymphoid leukemia, and potential metabolic mediators., Methods: We utilized data from the largest genome-wide association studies to date, encompassing 418 species of gut microbiota, 713 types of immune cells, and 1,400 serum metabolites as exposures. Summary statistics for lymphoid leukemia, acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL) were obtained from the FinnGen database. We performed bidirectional Mendelian analyses to explore the causal relationships among the gut microbiota, immune cells, serum metabolites, and lymphoid leukemia. Additionally, we conducted a two-step mediation analysis to identify potential intermediary metabolites between immune cells and lymphoid leukemia., Results: Several gut microbiota were found to have causal relationships with lymphoid leukemia, ALL, and CLL, particularly within the Firmicutes and Bacteroidetes phyla . In the two-step MR analysis, various steroid hormone metabolites (such as DHEAS, pregnenolone sulfateprogestogen derivatives, and androstenediol-related compounds) were identified as potential intermediary metabolites between lymphoid leukemia and immune cells. In ALL, the causal relationship between 1-palmitoyl-2-docosahexaenoyl-GPE (16:0/22:6) and ALL was mediated by CD62L-plasmacytoid DC%DC (mediated proportion=-2.84%, P =0.020). In CLL, the causal relationship between N6,n6,n6-trimethyllysine and CLL was mediated by HLA DR+ CD8br AC (mediated proportion=4.07%, P =0.021)., Conclusion: This MR study provides evidence supporting specific causal relationships between the gut microbiota and lymphoid leukemia, as well as between certain immune cells and lymphoid leukemia with potential intermediary metabolites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhuang, Yin, Yang, Man, Wang, Geng and Shi.)

Random lasing (RL) is an optical phenomenon that arises from the combination of light amplification with optical feedback through multiple scattering events. In this paper, we present our investigations of RL generation from human blood samples. We tested mixtures of rhodamine B dye solutions with different blood components, including platelets, lymphocytes, erythrocytes, and whole blood. Intense coherent RL was obtained in all cases at relatively low pump thresholds, except for erythrocytes. We also studied the potential of RL signal analysis for biosensing applications using blood samples from healthy individuals and patients suffering from Chronic Lymphocytic Leukemia (CLL). CLL is a blood disease characterized by a high count of lymphocytes with significant morphological changes. A statistical analysis of the RL spectra based on principal component and linear discriminant analyses was conducted for classification purposes. RL-based sample discrimination was conducted for whole blood, platelet, and lymphocyte samples, being especially successful (86.7%) for the latter. Our results highlight the potential of RL analysis as a sensing tool in blood.

The open reading frame 8 (ORF8) protein, encoded by the SARS-CoV-2 virus after infection, stimulates monocytes/macrophages to produce pro-inflammatory cytokines. We hypothesized that a positive ex vivo monocyte response to ORF8 protein pre-COVID-19 would be associated with subsequent severe Coronavirus disease 2019 (COVID-19). We tested ORF8 ex vivo on peripheral blood mononuclear cells from 26 anonymous healthy blood donors and measured intracellular cytokine/ chemokine levels in monocytes by flow cytometry. The percentage of positive monocyte staining in the sample and change in mean fluorescence intensity (ΔMFI) after ORF8 were used to calculate the adjusted MFI for each cytokine. We then tested pre-COVID-19 peripheral blood mononuclear cell samples from 60 chronic lymphocytic leukemia (CLL) patients who subsequently developed COVID-19 infection. Severe COVID-19 was defined as hospitalization due to COVID-19. In the 26 normal donor samples, the adjusted MFI for interleukin (IL)-1β, IL-6, IL-8, and CCL-2 were significantly different with ORF8 stimulation versus controls. We next analyzed monocytes from pre-COVID-19 PBMC samples from 60 CLL patients. The adjusted MFI to ORF8 stimulation of monocyte intracellular IL-1β was associated with severe COVID-19 and a reactive ORF8 monocyte response was defined as an IL-1β adjusted MFI ≥0.18 (sensitivity 67%, specificity 75%). The median time to hospitalization after infection in CLL patients with a reactive ORF8 response was 12 days versus not reached for patients with a non-reactive ORF8 response with a hazard ratio of 7.7 (95% confidence interval: 2.4-132; P=0.005). These results provide new insight on the monocyte inflammatory response to virus with implications in a broad range of disorders involving monocytes.

Chronic lymphocytic leukemia (CLL) is a B-cell-derived hematologic malignancy whose progression depends on active B-cell receptor (BCR) signaling. Despite the spectacular efficacy of Ibrutinib, an irreversible inhibitor of Bruton tyrosine kinase (BTK), resistance can develop in CLL patients, and alternative therapeutic strategies are therefore required. Cancer immunotherapy has revolutionized cancer care and may be an attractive approach in this context. We speculated that characterizing the immune responses of CLL patients may highlight putative immunotherapeutic targets. Here, we used high-dimensional spectral flow cytometry to compare the distribution and phenotype of non-B-cell immune populations in the circulating blood of CLL patients treated with Ibrutinib displaying a complete response or secondary progression. Although no drastic changes were observed in the composition of their immune subsets, the Ibrutinib-resistant group showed increased cycling of CD8+ T cells, leading to their overabundance at the expense of dendritic cells. In addition, the expression of 11 different surface checkpoints was similar regardless of response status. Together, this suggests that CLL relapse upon Ibrutinib treatment may not lead to major alterations in the peripheral immune response.

Impact: Immune dysregulation is thought to contribute to chronic lymphocytic leukemia (CLL) risk, but biological mechanisms are unclear. We discovered that increased serum levels of B-cell activating factor (BAFF), an important regulator of B-cell maturation, were associated with a decreased risk of CLL, even >10 years after blood draw. Our findings suggest that BAFF could be a useful biomarker to assess risk among individuals at high risk, such as those with monoclonal b-cell lymphocytosis., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Association between measurable residual disease (MRD) and survival outcomes in chronic lymphocytic leukemia (CLL) has often been reported. However, limited quantitative analyses over large datasets have been undertaken to establish the predictive power of MRD. Here, we provide a comprehensive assessment of published MRD data to explore the utility of MRD in the prediction of progression-free survival (PFS). We undertook two independent analyses, which leveraged available published data to address two complimentary questions. In the first, data from eight clinical trials was modeled via a meta-regression approach, showing that median PFS can be predicted from undetectable MRD rates at 3-6 months of post-treatment. The resulting model can be used to predict the probability of technical success of a planned clinical trial in chemotherapy. In the second, we investigated the evidence for predicting PFS from competing MRD metrics, for example baseline value and instantaneous MRD value, via a joint modeling approach. Using data from four small studies, we found strong evidence that including MRD metrics in joint models improves predictions of PFS compared with not including them. This analysis suggests that incorporating MRD is likely to better inform individual progression predictions. It is therefore proposed that systematic MRD collection should be accompanied by modeling to generate algorithms that inform patients' progression., (© 2024 ASTRAZENECA. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Introduction: The impact of chemoimmunotherapy (CIT) on immunoglobulin (Ig) quantities in patients with chronic lymphocytic leukemia (CLL) has not been extensively studied., Methods: We analyzed Ig levels in 45 stable patients with indolent CLL (without indication for treatment) and 87 patients with progressive disease before first-line treatment. Fifty-five patients were evaluated again after the treatment with CIT., Results: We observed significantly lower levels of all Ig classes and subclasses in patients with progressive disease compared to patients with indolent disease. After treatment, median IgA increased from 0.59 g/L to 0.74 g/L (p = 0.0031). In stable patients, lower IgA2 was associated with shorter time to first treatment, although it did not reach statistical significance (p = 0.056). Shorter overall survival was observed in patients with progressive disease and lower IgG2 (p = 0.043). Surprisingly, among the patients with progressive CLL, unmutated IGHV genes were associated with higher levels of IgG, IgG1 and IgM, while TP53 mutation and/or 17p deletion were associated with higher levels of IgA and IgA1., Conclusions: CIT may lead to increase in IgA levels. Hypogammaglobulinemia is more common in patients with progressive CLL and unmutated IGHV or TP53 dysfunction., (© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.)

Chronic lymphocytic leukemia (CLL) is a chronic lymphoproliferative disorder characterized by monoclonal B cell proliferation. Studies carried out in recent years suggest that extracellular vesicles (EVs) may be a potential biomarker in cancer. Tyro3-Axl-Mertk (TAM) Receptor Tyrosine Kinases (RTKs) and Phosphatidylserine (PS) have crucial roles in macrophage-mediated immune response under normal conditions. In the tumor microenvironment, these molecules contribute to immunosuppressive signals and prevent the formation of local and systemic antitumor immune responses. Based on this, we aimed to evaluate the amount of PS and TAM RTK in plasma and on the surface of EVs in CLL patients and healthy volunteers in this study. In this study, 25 CLL (11 F/14 M) patients in the Rai (O-I) stage, newly diagnosed or followed up without treatment, and 15 healthy volunteers (11 F/4 M) as a control group were included. For all samples, PS and TAM RTK levels were examined first in the plasma and then in the EVs obtained from the plasma. We detected a significant decrease in plasma PS, and TAM RTK levels in CLL patients compared to the control. Besides, we determined a significant increase in TAM RTK levels on the EV surface in CLL, except for PS. In conclusion, these receptor levels measured by ELISA in plasma may not be effective for the preliminary detection of CLL. However, especially TAM RTKs on the surface of EVs may be good biomarkers and potential targets for CLL therapies., (© 2024 John Wiley & Sons Ltd.)

Objective: To investigate the association between serum immunoglobulin G (IgG) concentrations and the incidence of infections in patients with chronic lymphocytic leukemia (CLL) and secondary immunodeficiency receiving treatment with Privigen., Materials and Methods: Data was analyzed from a non-interventional study conducted in 31 centers in Germany and 1 in Austria. Adult CLL patients with hypogammaglobulinemia and recurrent infections were allowed to enter the study upon signing informed consent, if a prior decision for treatment with Privigen had been made. All infections requiring an antimicrobial treatment were subject to analysis. Patients were stratified according to their mean post-baseline serum IgG trough levels in a group with lower IgG trough levels (≤ 5.0 g/L), and a group with higher IgG trough levels (> 5.0 g/L)., Results: Overall, 89 patients and 840 treatment cycles were analyzed. Up to 11 treatment cycles (average duration 29 days) were documented in each patient. In the group with higher IgG trough levels (> 5.0 g/L, N = 72), significantly fewer infections were observed than in the group with lower IgG trough levels (≤ 5.0 g/L, N = 17), including fewer severe and serious infections. The Privigen dosage was a major determinant of the post-baseline serum IgG levels. Overall tolerability of Privigen was assessed as very good or good in 91% of patients., Conclusion: This analysis confirms the association of serum IgG trough levels with the incidence of infections and highlights the importance of careful monitoring of IgG levels during treatment of secondary immunodeficiencies in CLL patients.

Background: Measurable residual disease (MRD) is an important prognostic indicator of chronic lymphocytic leukemia (CLL). Different flow cytometric panels have been developed for the MRD assessment of CLL in Western countries; however, the application of these panels in China remains largely unexplored., Methods: Owing to the requirements for high accuracy, reproducibility, and comparability of MRD assessment in China, we investigated the performance of a flow cytometric approach (CD45-ROR1 panel) to assess MRD in patients with CLL. The European Research Initiative on CLL (ERIC) eight-color panel was used as the "gold standard.", Results: The sensitivity, specificity, and concordance rate of the CD45-ROR1 panel in the MRD assessment of CLL were 100% (87/87), 88.5% (23/26), and 97.3% (110/113), respectively. Two of the three inconsistent samples were further verified using next-generation sequencing. In addition, the MRD results obtained from the CD45-ROR1 panel were positively associated with the ERIC eight-color panel results for MRD assessment (R = 0.98, p < 0.0001). MRD detection at low levels (≤1.0%) demonstrated a smaller difference between the two methods (bias, -0.11; 95% CI, -0.90 to 0.68) than that at high levels (>1%). In the reproducibility assessment, the bias was smaller at three data points (within 24, 48, and 72 h) in the CD45-ROR1 panel than in the ERIC eight-color panel. Moreover, MRD levels detected using the CD45-ROR1 panel for the same samples from different laboratories showed a strong statistical correlation (R = 0.99, p < 0.0001) with trivial interlaboratory variation (bias, 0.135; 95% CI, -0.439 to 0.709). In addition, the positivity rate of MRD in the bone marrow samples was higher than that in the peripheral blood samples., Conclusions: Collectively, this study demonstrated that the CD45-ROR1 panel is a reliable method for MRD assessment of CLL with high sensitivity, reproducibility, and reliability., (© 2024 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

Objective: To assess the association of serum protein electrophoresis abnormalities with clinicopathological characteristics, and its impact on overall survival in chronic lymphocytic leukaemia patients., Methods: The prospective study was conducted at Haematology and Immunology departments of the University of Health Sciences, Lahore, Pakistan, from 2019 to 2022, and comprised newly diagnosed chronic lymphocytic leukaemia patients. Lactate dehydrogenase and beta-2 microglobulin levels were measured by spectrophotometric principle, whereas serum protein electrophoresis was determined through commercially available capillary electrophoresis systems. Patients were followed up for 2 years post-diagnosis. Data was analysed using SPSS 21., Results: Of the 50 patients, 40(80%) were males and 10(20%) were females. The overall mean age was 60±11 years. Serum protein electrophoresis was available for 40(80%) patients, and, among them, 12(30%) patients had abnormal levels, while 29(72.5%) required treatment. Overall response rate was 25(86.2%), and median two-year overall survival was 16.5 months (95% confidence interval: 10-20 months). Abnormal serum protein electrophoresis was significantly associated with Binet stage C, lower mean haemoglobin levels and higher median levels of lactate dehydrogenase and beta-2 microglobulin (p<0.05)). Regarding overall survival, the survival curves of chronic lymphocytic leukaemia patients with normal and abnormal serum protein electrophoresis status differed significantly (p=0.04)., Conclusion: Abnormal serum protein electrophoresis could be considered a surrogate marker for advanced chronic lymphocytic leukaemia disease.

Despite considerable treatment advances with targeted therapies for patients with chronic lymphocytic leukemia (CLL) deemed high-risk [del(17p) and/or TP53 mutation], the outcome is still inferior compared with other CLL patients. Combining multiple agents with distinct mechanisms of action may further improve outcomes. CLL2-GIVe is an open-label, multicenter trial which enrolled patients with previously untreated CLL with del(17p) and/or TP53 mutation. Patients received induction therapy with obinutuzumab (GA-101), ibrutinib, and venetoclax (GIVe) for cycles 1 through 6 and consolidation therapy with venetoclax and ibrutinib for cycles 7 through 12. Ibrutinib monotherapy was continued for cycles 13 through 36 in patients not reaching a complete response (CR) with serial undetectable minimal residual disease (uMRD) after consolidation. The primary endpoint was CR rate at cycle 15 (final restaging). Secondary endpoints included MRD, survival, and safety. All 41 patients enrolled between September 2016 and August 2018 received study treatment and were included in efficacy and safety populations. With a CR rate of 58.5% at cycle 15, the primary endpoint was met (95% CI: 42.1-73.7; P < .001). At final restaging, 78.0% of patients had uMRD in peripheral blood (PB); 65.9% of patients had uMRD in bone marrow (BM). Estimated progression-free survival (PFS) and overall survival (OS) rates at 24 months were both 95.1%. Adverse events were reported in all patients; most were low grade (grade ≥3: 23.9%). Two deaths were reported (cardiac failure and ovarian carcinoma), neither related to study treatment. The CLL2-GIVe treatment regimen has a manageable safety profile and is a first-line treatment of good efficacy for patients with high-risk CLL., (© 2022 by The American Society of Hematology.)

Patients with chronic lymphocytic leukemia (CLL) have an impaired antibody response to coronavirus disease 2019 (COVID-19) vaccination. Here, we evaluated the antibody response to a third BNT162b2 mRNA vaccine in patients with CLL/small lymphocytic lymphoma (SLL) who failed to achieve a humoral response after standard 2-dose vaccination regimen. Anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies were measured 3 weeks after administration of the third dose. In 172 patients with CLL, the antibody response rate was 23.8%. Response rate among actively treated patients (12.0%; n = 12/100) was lower compared with treatment-naïve patients (40.0%; n = 16/40; OR = 4.9, 95% CI 1.9-12.9; P < .001) and patients off-therapy (40.6%; n = 13/32; OR = 5.0, 95% CI 1.8-14.1; P < .001), (P < .001). In patients actively treated with Bruton's tyrosine kinase (BTK) inhibitors or venetoclax ± anti-CD20 antibody, response rates were extremely low (15.3%, n = 9/59, and 7.7%, n = 3/39, respectively). Only 1 of the 28 patients (3.6%) treated with anti-CD20 antibodies <12 months prior to vaccination responded. In a multivariate analysis, the independent variables that were associated with response included lack of active therapy (OR = 5.6, 95% CI 2.3-13.8; P < .001) and serum immunoglobulin A levels ≥80 mg/dL (OR = 5.8, 95% CI 2.1-15.9; P < .001). In patients with CLL/SLL who failed to achieve a humoral response after standard 2-dose BNT162b2 mRNA vaccination regimen, close to a quarter responded to the third dose of vaccine. The antibody response rates were lower during active treatment and in patients with a recent exposure (<12 months prior to vaccination) to anti-CD20 therapy. This trial was registered at www.clinicaltrials.gov as #NCT04862806., (© 2022 by The American Society of Hematology.)

AIOLOS/IKZF3 is a member of the IKAROS family of transcription factors. IKAROS/IKZF1 mutations have been previously associated with different forms of primary immunodeficiency. Here we describe a novel combined immunodeficiency due to an IKZF3 mutation in a family presenting with T and B cell involvement, Pneumocystis jirovecii pneumonia, and/or chronic lymphocytic leukemia. Patients carrying the AIOLOS p.N160S heterozygous variant displayed impaired humoral responses, abnormal B cell development (high percentage of CD21low B cells and negative CD23 expression), and abrogated CD40 responses. Naive T cells were increased, T cell differentiation was abnormal, and CD40L expression was dysregulated. In vitro studies demonstrated that the mutant protein failed DNA binding and pericentromeric targeting. The mutant was fully penetrant and had a dominant-negative effect over WT AIOLOS but not WT IKAROS. The human immunophenotype was recapitulated in a murine model carrying the corresponding human mutation. As demonstrated here, AIOLOS plays a key role in T and B cell development in humans, and the particular gene variant described is strongly associated with immunodeficiency and likely malignancy., Competing Interests: Disclosures:   C.M. Dutmer reported personal fees from Horizon Therapeutics and personal fees from Enzyvant Therapeutics outside the submitted work. No other disclosures were reported., (© 2021 Kuehn et al.)

The in vivo rituximab effects in B cell malignancies are only partially understood. Here we analyzed in a large chronic lymphocytic leukemia (CLL) cohort (n = 80) the inter-patient variability in CLL cell count reduction within the first 24 h of rituximab administration in vivo, and a phenomenon of blood repopulation by malignant cells after anti-CD20 antibody therapy. Larger CLL cell elimination after rituximab infusion was associated with lower pre-therapy CLL cell counts, higher CD20 levels, and the non-exhausted capacity of complement-dependent cytotoxicity (CDC). The absolute amount of cell-surface CD20 molecules (CD20 density x CLL lymphocytosis) was a predictor for complement exhaustion during therapy. We also describe that a highly variable decrease in CLL cell counts at 5 h (88 %-2%) following rituximab infusion is accompanied in most patients by peripheral blood repopulation with CLL cells at 24 h, and in ∼20 % of patients, this resulted in CLL counts higher than before therapy. We provide evidence that CLL cells recrudescence is linked with i) CDC exhaustion, which leads to the formation of an insufficient amount of membrane attack complexes, likely resulting in temporary retention of surviving rituximab-opsonized cells by the mononuclear-phagocyte system (followed by their release back to blood), and ii) CLL cells regression from immune niches (CXCR4 dim CD5 bright intraclonal subpopulation). Patients with major peripheral blood CLL cell repopulation exhibited a longer time-to-progression after chemoimmunotherapy compared to patients with lower or no repopulation, suggesting chemotherapy vulnerability of CLL cells that repopulate the blood., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Introduction: Leukemia is a malignant and progressive disease of hematopoiesis. The disease arises due to abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow. Chronic lymphoblastic leukemia (CLL) is a subtype of blood cancers, with the origin of B lymphocytes and the involvement of bone marrow, blood and lymph nodes. MicroRNAs (miRNAs) are small non-coding RNAs with pivotal roles in cellular and molecular processes related to different malignancies, including CLL. In this way, we aimed to evaluate the expression of miR-32-5p, miR-98-5p, and miR-374b-5p in CLL patients. We also investigated the signaling pathways regulated by the studied miRs and also frequently disturbed miRs in CLL., Methods: Blood samples were collected from 32 CLL patients from Kermanshah province, Iran and 34 age and sex-matched healthy individuals. RNA was extracted from PBMCs and then was subjected to cDNA synthesis. Using specifically designed primers and Real-Time PCR method the expression of miRNAs was detected and was statistically analyzed. Using mirPath v.3, systematic pathway enrichment analysis was performed for the three studied miRNAs here along with the frequently disturbed miRNAs in CLL., Results: The experiments indicated a significant reduction in the expression of all three miRs (p-value<0.0001) in CLL patients compared with healthy individuals. ROC analysis suggested that the three studied miRs can serve as potential biomarkers for early diagnosis of CLL. The in silico analysis suggested proteoglycans in cancer as a pathway regulated by the studied miRs and frequently dysregulated miRs in CLL., Conclusion: The observed reduction in expression of miR-32-5p, miR-98-5p, and miR-374b-5p in treatment naïve CLL patients here might be suggestive of their modulatory protective role in CLL progression. Moreover, the candidate peripheral miRNAs could potentially serve as diagnostic biomarkers which warrant further investigation in a larger sample size., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: Study the tumor-induced bystander effect of blood cells from chronic lymphocytic leukemia (CLL)patients on non-transformed bystander cells (peripheral blood lymphocytes (PBL) of conditionally healthy individ-uals) and the possibility of its modification after the impact of ionizing radiation., Materials and Methods: We carried out cocultivation and separate cultivation of blood samples from conditionallyhealthy volunteers and patients with CLL according to our technique. Using the Comet assay, the relative level ofDNA damage was evaluated., Results: A statistically significant increase (р < 0.001) in the level of DNA damage in PBL culture of conditionallyhealthy individuals after co-cultivation with malignant cells of CLL patients was observed. After irradiation, a drop in the level of cells with a high degree of DNA damage was noted, which was connected with an increase in the frequency of cells that were delayed in division at the S stage of the cell cycle. An increase in apoptotic activity in cultures of bystander cells was observed in all variants of the experiment (р < 0.001)., Conclusion: The influence of irradiated blood cells of patients with CLL results in an enhancement of the tumor-induced bystander effect manifestation in the PBL of conditionally healthy individuals., (D. Кurinnyi, S. Rushkovsky, O. Demchenko, M. Romanenko, T. Liashchenko, M. Pilinska.)

Bacterial infections are a major cause of morbidity and mortality in chronic lymphocytic leukemia (CLL), and infection risk increases in patients treated with the Bruton's tyrosine kinase (Btk) inhibitor, ibrutinib. Btk and related kinases (like Tec) are expressed in non-leukemic hematopoietic cells and can be targeted by ibrutinib. In platelets, ibrutinib therapy is associated with bleeding complications mostly due to off-target effects. But the ability of platelets to respond to bacteria in CLL, and the potential impact of ibrutinib on platelet innate immune functions remain unknown. FcγRIIA is a tyrosine kinase-dependent receptor critical for platelet activation in response to IgG-coated pathogens. Crosslinking of this receptor with monoclonal antibodies causes downstream activation of Btk and Tec in platelets, however, this has not been investigated in response to bacteria. We asked whether ibrutinib impacts on FcγRIIA-mediated activation of platelets derived from CLL patients and healthy donors after exposure to Staphylococcus aureus Newman and Escherichia coli RS218. Platelet aggregation, α-granule secretion and integrin αIIbβ3-dependent scavenging of bacteria were detected in CLL platelets but impaired in platelets from ibrutinib-treated patients and in healthy donor-derived platelets exposed to ibrutinib in vitro . While levels of surface FcγRIIA remained unaffected, CLL platelets had reduced expression of integrin αIIbβ3 and GPVI compared to controls regardless of therapy. In respect of intracellular signaling, bacteria induced Btk and Tec phosphorylation in both CLL and control platelets that was inhibited by ibrutinib. To address if Btk is essential for platelet activation in response to bacteria, platelets derived from X-linked agammaglobulinemia patients (lacking functional Btk) were exposed to S. aureus Newman and E. coli RS218, and FcγRIIA-dependent aggregation was observed. Our data suggest that ibrutinib impairment of FcγRIIA-mediated platelet activation by bacteria results from a combination of Btk and Tec inhibition, although off-target effects on additional kinases cannot be discarded. This is potentially relevant to control infection-risk in CLL patients and, thus, future studies should carefully evaluate the effects of CLL therapies, including Btk inhibitors with higher specificity for Btk, on platelet-mediated immune functions., Competing Interests: DA has received honoraria for speaking from Gilead Pharmaceuticals, funding for conference attendance from Bayer and CSL Behring and research funding from Roche. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Naylor-Adamson, Chacko, Booth, Caserta, Jarvis, Khan, Hart, Rivero, Allsup and Arman.)

A prospective pilot study was carried out on 34 CLL patients treated with ibrutinib, evaluating the effects on symptoms and physical function with changes in plasma exosomes (EXs), β2-microglobulin (β2M) and 26 plasma cytokines. The revised Edmonton Symptom Assessment Scale (ESAS-R) demonstrated moderate fatigue, shortness of breath and a sense of unwellness before treatment, which significantly improved within 2 weeks of starting ibrutinib. These changes were associated with a rapid improvement in sit-to-stand and 4 m walking speeds. The plasma levels of CCL11, IL-7, -8 and -10 dropped initially while the levels of TNF-α/-β, CCL3, CCL4, CCL17, and IL-16 continued to decline for 12 months. Despite the initial lymphocytosis, plasma β2M levels fell but no consistent change in plasma EXs occurred. Thus, ibrutinib can produce a rapid and sustained improvement in symptoms and physical function in CLL, associated with a decline in multiple plasma cytokines., (Copyright © 2021. Published by Elsevier Ltd.)

We aimed at molecularly dissecting the anatomical heterogeneity of small lymphocytic lymphoma (SLL), by analysing a cohort of 12 patients for whom paired DNA from a lymph node biopsy and circulating cells, as well as plasma-circulating tumour DNA (ctDNA) was available. Notably, the analyses of the lymph node biopsy and of circulating cells complement each other since a fraction of mutations (20·4% and 36·4%, respectively) are unique to each compartment. Plasma ctDNA identified two additional unique mutations. Consistently, the different synchronous sources of tumour DNA complement each other in informing on driver gene mutations in SLL harbouring potential prognostic and/or predictive value., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

B-cell chronic lymphocytic leukaemia (CLL) is associated with immunosuppression and patients are at increased clinical risk following SARS-CoV-2 infection. Covid-19 vaccines offer the potential for protection against severe infection but relatively little is known regarding the profile of the antibody response following first or second vaccination. We studied spike-specific antibody responses following first and/or second Covid-19 vaccination in 299 patients with CLL compared with healthy donors. 286 patients underwent extended interval (10-12 week) vaccination. 154 patients received the BNT162b2 mRNA vaccine and 145 patients received ChAdOx1. Blood samples were taken either by venepuncture or as dried blood spots on filter paper. Spike-specific antibody responses were detectable in 34% of patients with CLL after one vaccine (n = 267) compared to 94% in healthy donors with antibody titres 104-fold lower in the patient group. Antibody responses increased to 75% after second vaccine (n = 55), compared to 100% in healthy donors, although titres remained lower. Multivariate analysis showed that current treatment with BTK inhibitors or IgA deficiency were independently associated with failure to generate an antibody response after the second vaccine. This work supports the need for optimisation of vaccination strategy in patients with CLL including the potential utility of booster vaccines., (© 2021. The Author(s).)

Pseudohyperkalemia, a false elevation of potassium level in vitro, can be observed in chronic lymphocytic leukemia (CLL) patients due to fragility of leukocytes along with a high leukocyte count. This retrospective, observational study included all patients diagnosed with CLL at our hospital who had at least one leukocyte count ≥ 50.0 × 10 9 /L during the years 2008-2018. All hyperkalemic episodes (including when leukocyte count was below 50.0 × 10 9 /L) during this period were assessed. Pseudohyperkalemia was defined as when a normal potassium level was measured in a repeated blood test or when known risk factors and ECG changes typical of hyperkalemia were absent. Of the 119 episodes of hyperkalemia observed, 41.2% were considered as pseudohyperkalemia. Pseudohyperkalemia episodes were characterized by significantly higher leukocyte counts as well as higher potassium and LDH levels compared to true hyperkalemia. Pseudohyperkalemia was documented in medical charts only in a minority of cases (n = 4, 8.1%). Treatment was administered in 17 of 49 (34.7%) cases and caused significant hypokalemia in 6 of those cases. The incidence of pseudohyperkalemia in this study was rather high, suggesting that physicians should be more aware of this phenomenon in patients with CLL.

Anti-CD20 antibody treatments, such as obinutuzumab, have been associated with infusion-related reactions (IRRs). In the phase 3 iLLUMINATE study of ibrutinib-obinutuzumab versus chlorambucil-obinutuzumab in first-line chronic lymphocytic leukemia/small lymphocytic lymphoma, IRRs were substantially reduced with ibrutinib-obinutuzumab versus chlorambucil-obinutuzumab. We prospectively analyzed inflammatory cytokines to evaluate the impact of ibrutinib on circulating cytokine levels following obinutuzumab infusion. In iLLUMINATE, ibrutinib or chlorambucil was given approximately 30-120 min before the first obinutuzumab infusion. Cytokines evaluated were IFNγ, IL-6, IL-8, IL-10, IL-18, MCP-1, MIP-1α, MIP-1β, and TNFα. Changes in peak cytokine levels from baseline (immediately before obinutuzumab) to post-obinutuzumab infusion were compared between arms and between patients with versus without IRRs using Wilcoxon rank sum test. Of 228 treated patients, 95 on ibrutinib-obinutuzumab (15 with IRRs, 80 without) and 88 on chlorambucil-obinutuzumab (45 with IRRs, 43 without) with cytokine data were included. Irrespective of IRR occurrence, median increase in cytokines was lower with ibrutinib-obinutuzumab versus chlorambucil-obinutuzumab for all cytokines (P < 0.01) except MIP-1β. Across treatment arms, post-obinutuzumab median increase in all cytokines except MIP-1β was greater in patients with versus without IRRs (P < 0.001). IL-6 and IL-8 elevations were associated with IRRs in both treatment arms. Among patients with IRRs, those receiving ibrutinib-obinutuzumab had lower post-obinutuzumab increases in IL-6, IL-8, IL-10, and MCP-1 (P < 0.04) than patients receiving chlorambucil-obinutuzumab. For patients in the ibrutinib-treatment arm, we observed a reduction in both the rate of clinically apparent IRRs and the levels of IRR-related cytokines and chemokines. This observation supports an immunomodulatory mechanism of action for ibrutinib. Clinical Trial Registration: NCT02264574.

Introduction: Pre-analytical and analytical errors can threaten the reliability of flow cytometry (FC) results. A potential solution to some of these is the use of dry, pre-mixed antibodies, such as the ClearLLab 10C system. The purpose of the present study was to compare the diagnostic performance of the ClearLLab 10C B cell tube with that of our standard laboratory practice., Methods: We compared the diagnoses made with the ClearLLab 10C B cell tube (experimental strategy) with those made with standard laboratory practice (standard strategy). Samples were selected aiming for representation of the full spectrum of B cell disorders, with an emphasis on mature B cell malignancies, as well as healthy controls., Results: We included 116 samples (34 normal controls, 4 acute lymphoblastic leukemias, 54 mature lymphoproliferative disorders in peripheral blood and bone marrow, 3 myelomas, 6 bone marrow samples with involvement by lymphoma and 1 with elevated hematogone count, 14 lymph node samples, 1 cerebrospinal fluid, and 1 pleural effusion). There were two diagnostic errors (1.7%). The agreement between the two strategies in the percentage of CD19 cells and fluorescence intensity of CD5, CD19, CD20, CD200, and CD10 was very good., Conclusions: In this study, the ClearLLab 10C B cell tube performed similarly to our standard laboratory practice to diagnose and classify mature B cell malignancies., (© 2020 International Clinical Cytometry Society.)

Hypogammaglobulinemia is the most frequently observed immune defect in chronic lymphocytic leukemia (CLL). Although CLL patients usually have low serum levels of all isotypes (IgG, IgM and IgA), standard immunoglobulin (Ig) preparations for replacement therapy administrated to these patients contain more than 95% of IgG. Pentaglobin is an Ig preparation of intravenous application (IVIg) enriched with IgM and IgA (IVIgGMA), with the potential benefit to restore the Ig levels of all isotypes. Because IVIg preparations at high doses have well-documented anti-inflammatory and immunomodulatory effects, we aimed to evaluate the capacity of Pentaglobin and a standard IVIg preparation to affect leukemic and T cells from CLL patients. In contrast to standard IVIg, we found that IVIgGMA did not modify T cell activation and had a lower inhibitory effect on T cell proliferation. Regarding the activation of leukemic B cells through BCR, it was similarly reduced by both IVIgGMA and IVIgG. None of these IVIg preparations modified spontaneous apoptosis of T or leukemic B cells. However, the addition of IVIgGMA on in vitro cultures decreased the apoptosis of T cells induced by the BCL-2 inhibitor, venetoclax. Importantly, IVIgGMA did not impair venetoclax-induced apoptosis of leukemic B cells. Overall, our results add new data on the effects of different preparations of IVIg in CLL, and show that the IgM/IgA enriched preparation not only affects relevant mechanisms involved in CLL pathogenesis but also has a particular profile of immunomodulatory effects on T cells that deserves further investigation.

The chemoattractant CXCL13 organizes the cellular architecture of B-cell follicles and germinal centers. During adaptive immune responses, CXCL13 plasma concentrations transiently increase and function as a biomarker for normal germinal center activity. Chronic lymphocytic leukemia (CLL) cells express high levels of CXCR5, the receptor for CXCL13, and proliferate in pseudofollicles within secondary lymphoid organs (SLO). Given the morphologic and functional similarities between normal and CLL B-cell expansion in SLO, we hypothesized that CXCL13 plasma concentrations would correlate with CLL disease activity and progression. We analyzed CXCL13 plasma concentrations in 400 CLL patients and correlated the findings with other prognostic markers, time to treatment (TTT), CCL3 and CCL4 plasma concentrations, and in vivo CLL cell proliferation. We found that CXCL13 plasma concentrations were higher in CLL patients with active and advanced stage disease, resulting in a significantly shorter TTT. Accordingly, high CXCL13 levels correlated with other markers of disease activity and CCL3 levels. Higher CLL cell birth rates in vivo also associated with higher CXCL13 plasma concentrations. Interestingly, elevated CXCL13 plasma levels normalized during ibrutinib therapy, and increased in ibrutinib resistance patients. Collectively, these studies emphasize the importance of CXCL13 in crosstalk between CLL cells and the SLO microenvironment.

Objective: 3' tRNA-derived fragments (3' tRFs) are important epigenetic regulators in normal and pathological conditions. In this study, we aimed to explore the potential value of a 3' tRF as a prognostic and/or screening biomarker for B-cell chronic lymphocytic leukemia (B-CLL)., Methods: Publicly available next-generation sequencing data from 20 B-CLL cases were analyzed, followed by prediction of targets of the most abundantly and ubiquitously expressed 3' tRFs, leading to selection of tRF-Leu AAG/TAG . PBMCs were isolated from blood samples of 91 B-CLL patients and 43 non-leukemic donors, followed by total RNA extraction, in-vitro polyadenylation, and first-strand cDNA synthesis. Next, a real-time quantitative PCR (qPCR) assay was developed for the accurate quantification of tRF-Leu AAG/TAG and applied in all samples, prior to biostatistical analysis., Results: High tRF-Leu AAG/TAG levels are associated with inferior overall survival (OS) of B-CLL patients. The unfavorable significance of tRF-Leu AAG/TAG was independent of established prognostic factors in B-CLL. Stratified Kaplan-Meier OS analysis uncovered the unfavorable prognostic role of high tRF-Leu AAG/TAG levels for patients in Binet A or Rai I stage, negative CD38 expression, mutated, or unmutated IGHV genomic locus., Conclusion: Our approach revealed the independent prognostic value of a particular 3' tRF, derived from tRNA LeuAAG and tRNA LeuTAG (tRF-Leu AAG/TAG ) in B-CLL., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This multicenter, open-label, phase I study assessed the safety and antitumor activity of acalabrutinib in Japanese patients with relapsed/refractory (r/r) B-cell malignancies. Parts 1 (dose confirmation) and 2 (dose expansion) of this three-part study are reported. Treatment was a single dose of 100 mg acalabrutinib (day 1), followed by a washout period and then twice daily 100 mg acalabrutinib in part 1, or twice daily 100 mg acalabrutinib in part 2. Patients from parts 1 and 2 with r/r chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), and r/r mantle cell lymphoma (MCL) were assessed as r/r CLL/SLL and r/r MCL cohorts, respectively. Twenty-five patients received treatment (part 1, n = 6). Median age was 71.0 years. Nine (one patient from part 1) and 13 (two patients from part 1) patients were included in the r/r CLL/SLL and r/r MCL cohorts, respectively. Treatment-related adverse events (AEs) occurred in 88% of patients (grade ≥3, 36%); the most common were headache (28%) and purpura (24%), both grade 1/2. No AEs resulted in treatment discontinuation or death. Median duration of treatment was 31, 20, and 7 months for part 1, r/r CLL/SLL cohort, and r/r MCL cohort, respectively. Overall response rate (ORR) was 89% and 62% for the r/r CLL/SLL and r/r MCL cohorts, respectively. The median progression-free survival (PFS) was not reached for the r/r CLL/SLL cohort and was 7 months for the r/r MCL cohort. Acalabrutinib (100 mg twice daily) was generally safe and well-tolerated in adult Japanese patients with B-cell malignancies., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Chimeric antigen receptor (CAR) T cell therapy has yielded unprecedented outcomes in some patients with hematological malignancies; however, inhibition by the tumor microenvironment has prevented the broader success of CART cell therapy. We used chronic lymphocytic leukemia (CLL) as a model to investigate the interactions between the tumor microenvironment and CART cells. CLL is characterized by an immunosuppressive microenvironment, an abundance of systemic extracellular vesicles (EVs), and a relatively lower durable response rate to CART cell therapy. In this study, we characterized plasma EVs from untreated CLL patients and identified their leukemic cell origin. CLL-derived EVs were able to induce a state of CART cell dysfunction characterized by phenotypical, functional, and transcriptional changes of exhaustion. We demonstrate that, specifically, PD-L1 + CLL-derived EVs induce CART cell exhaustion. In conclusion, we identify an important mechanism of CART cell exhaustion induced by EVs from CLL patients., Competing Interests: Declaration of interests S.S.K. is an inventor on patents in the field of CAR immunotherapy that are licensed to Novartis (through an agreement between the Mayo Clinic, the University of Pennsylvania, and Novartis). M.J.C., R.S., and S.S.K. are inventors on patents in the field of CAR immunotherapy that are licensed to Humanigen (through the Mayo Clinic). S.S.K. is an inventor on patents in the field of CAR immunotherapy that are licensed to Mettaforge (through the Mayo Clinic). S.S.K. receives research funding from Kite, Gilead, Juno, Celgene, Novartis, Humanigen, MorphoSys, Tolero, Sunesis, Leahlabs, and Lentigen. M.J.C., F.L., N.E.K., and S.S.K. are inventors on patents related to this work. N.E.K. receives research funding from Acerta Pharma, BMS, Pharmacyclics, MEI Pharma, and Sunesis. N.E.K. has participated in Advisory Board meetings of Cytomx Therapy, Janssen, Juno Therapeutics, AstraZeneca, and Oncotracker, and on the DSMC for Agios and Cytomx Therapeutics. S.A.P. receives research funding from Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, AbbVie, and Ascentage Pharma. S.A.P. has participated in Advisory Board meetings of Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie (he was not personally compensated for his participation)., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Most patients with chronic lymphocytic leukaemia (CLL) are nowadays diagnosed without any symptoms and do not require therapy. A prognostic score identifying patients within this large group who are at high risk of disease progression would be highly beneficial. The recently published International Prognostic Score for Early asymptomatic patients (IPS-E) uses combination of absolute lymphocyte count (ALC) >15 × 10 9 /l, palpable lymphadenopathy, and unmutated immunoglobulin heavy-chain variable-region (IGHV) gene to predict the time to first-line therapy (TTFT). Patients at low, intermediate, and high risk had estimated 5-year TTFT of 8%, 28%, and 61%. We performed an external validation of the IPS-E score using an unselected, consecutive group of 130 Binet A patients. The 5-year TTFT was 11%, 36%, and 78% (C-statistic 0·74). Furthermore, we propose an alternative system (AIPS-E) using cytogenetic aberrations instead of palpable lymphadenopathy. This system yielded 5-year TTFT of 14%, 40%, and 72%. These results were externally validated in 388 Binet A patients from five Czech centres; the 5-year TTFT was 16%, 37%, and 80% (C-statistic 0·74). In conclusion, we have successfully validated the IPS-E score for patients with early stage CLL. In addition, we propose a modified scoring system, the AIPS-E, combining IGHV, fluorescence in situ hybridisation, and ALC., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

The infiltration of chronic lymphocytic leukemia (CLL) cells into lymphoid organs correlates with disease severity. CXCL12 is a key chemotactic factor for the trafficking of CLL. Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor and plays a role in CXCL12-mediated hematopoietic stem cell homing. We aim to explore the role of TFPI in CXCL12-mediated migration of CLL cells. In this study, plasma TFPI concentrations were measured by ELISA. CLL cells were isolated from patients and used for trans-endothelial migration (TEM) assays. Quantitative RT-PCR and Western blotting were used to detect the expression of CXCR7, CXCR4 and β-catenin. Immunofluorescence and co-immunoprecipitation was used to detect the binding of TFPI and glypican-3 (GPC3). We found that plasma TFPI levels in CLL patients were higher than in healthy controls, particularly in the patients with advanced disease. TFPI enhanced CXCL12-mediated TEM of CLL cells by increasing the expression of the CXCL12 receptor CXCR7, but not of the CXCL12 receptor CXCR4. The effect of TFPI on TEM was abolished by the CXCR7 inhibitor, CCX771, while the CXCR4 inhibitor AMD3100 strongly increased TEM. TFPI co-localized with GPC3 on the cell surface. An antibody to GPC3, HS20, decreased CXCR7 expression and abolished the effect of TFPI on TEM. TFPI activated β-catenin and the Wnt/β-catenin inhibitor IWP4 repressed the effect of TFPI on CXCR7 expression and TEM. We conclude that TFPI may contribute to organ infiltration in CLL patients.

Objective: Fludarabine, cyclophosphamide and rituximab (FCR) is the standard regimen for fit patients with untreated CD20-positive chronic lymphocytic leukemia (CLL). However, this combination is unavailable in Japan because rituximab is not approved for CLL. We investigated the efficacy and safety of FCR in this single-arm, multicenter study designed as a bridging study to the CLL8 study by the German CLL Study Group., Methods: The study enrolled previously untreated patients with CLL of Binet stage B or C with active disease. Patients with a Cumulative Illness Rating Scale score of ≤6 and creatinine clearance of ≥70 ml/min were eligible. Patients received 6 cycles of FCR every 28 days and were followed for up to 1 year., Results: Seven patients were enrolled. The best overall response rate according to the 1996 NCI-WG Guidelines, the primary endpoint of the study, was 71.4% (95% confidence interval, 29.0-96.3%), with one patient achieving complete response. No deaths or progression occurred during follow-up. The main adverse event was hematotoxicity. CD4-positive T-cell count decreased in all patients; most patients showed no reduction in serum immunoglobulin G., Conclusion: Although the number of patients was limited, FCR appears to be effective with manageable toxicity for treatment-naïve fit Japanese patients with CD20-positive CLL., Clinical Trial Number: JapicCTI-132285., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

In a series of 349 patients with chronic lymphocytic leukaemia (CLL), we found lower levels of signalling lymphocytic activation molecule family member 1 (SLAMF1) expression in cases with highly complex karyotypes, as defined by the presence of five or more chromosomal abnormalities (CK5; P < 0·001) and with major chromosomal structural abnormalities (P < 0·001). SLAMF1 downregulation was significantly associated with advanced Binet Stage (P = 0·001), CD38 positivity (P < 0·001), high β 2 -microglobulin levels (P < 0·001), immunoglobulin heavy chain variable region gene (IGHV) unmutated status (P < 0·001), 11q deletion (P < 0·001), tumour protein p53 (TP53) disruption (P = 0·011) and higher risk CLL International Prognostic Index categories (P < 0·001). Multivariate analysis showed that downregulated SLAMF1 levels had independent negative prognostic impact on time-to-first treatment (P < 0·001) and overall survival (P < 0·001)., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Several studies in chronic lymphocytic leukemia (CLL) patients have reported impaired immune cell functions, which contribute to tumor evasion and disease progression. However, studies on CLL-like monoclonal B-cell lymphocytosis (MBL) are scarce. In the study described here, we characterized the immune environment in 62 individuals with clinical MBL, 56 patients with early-stage CLL, and 31 healthy controls. Gene expression arrays and quantitative reverse transcription polymerase chain reaction were performed on RNA from CD4 + peripheral blood cells; serum cytokines were measured with immunoassays; and HLA-DR expression on circulating monocytes, as well as the percentages of Th1, cytotoxic, exhausted, and effector CD4 + T cells, were evaluated by flow cytometry. In addition, cell cultures of clonal B cells and CD14-enriched or -depleted cell fractions were performed. Strikingly, MBL and early-stage CLL differed in pro-inflammatory signatures. An increased inflammatory drive orchestrated mainly by monocytes was identified in MBL, which exhibited enhanced phagocytosis, pattern recognition receptors, interleukin-8 (IL8), HMGB1, and acute response signaling pathways and increased pro-inflammatory cytokines (in particular IL8, interferon γ [IFNγ], and tumor necrosis factor α). This inflammatory signature was diminished in early-stage CLL (reduced IL8 and IFNγ levels, IL8 signaling pathway, and monocytic HLA-DR expression compared with MBL), especially in those patients with mutations in IGHV genes. Additionally, CD4 + T cells of MBL and early-stage CLL exhibited a similar upregulation of Th1 and cytotoxic genes and expanded CXCR3 + and perforin + CD4 + T cells, as well as PD1 + CD4 + T cells, compared with controls. Cell culture assays disclosed tumor-supporting effects of monocytes similarly observed in MBL and early-stage CLL. These novel findings reveal differences in the inflammatory environment between MBL and CLL, highlighting an active role for antigen stimulation in the very early stages of the disease, potentially related to malignant B-cell transformation., Competing Interests: Conflict of interest disclosure The authors have no financial conflicts of interest., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Background/aim: A proliferation-inducing ligand (APRIL) has been investigated as a prognostic marker in chronic lymphocytic leukemia (CLL) patients. However, there is no cut-off level for serum APRIL (sAPRIL) levels that predict time to treatment in CLL patients., Materials and Methods: Between May and December 2012, 94 consecutive CLL patients and 25 healthy controls were assessed. sAPRIL levels were measured by ELISA. Demographic data and prognostic markers were obtained from the patients’ files. Treatment-naïve patients were followed up for 6.5 years for any treatment need., Results: Patients were divided into 3 groups: Treatment-naïve (n = 47), chemotherapy receiving (n = 25), and those who had received chemotherapy previously (n = 22). There was no difference in median sAPRIL levels of patients who were receiving chemotherapy at the sampling time and the healthy controls, which indicates that sAPRIL levels might be influenced by treatment. For treatment-naïve patients, the best cut-off in predicting time to treatment was found at the sAPRIL level of 2.04 ng/mL, with 78% sensitivity and 63% specificity. Time to treatment was significantly earlier in the APRIL high group (n = 27) than in the APRIL low group (n = 20) (P = 0.010, log-rank test)., Conclusion: sAPRIL, a simple, promising blood test which can be measured by ELISA, will likely obtain a place in the wide range of prognostic markers in CLL. Prospective large-scale studies are required to validate and confirm the feasibility of the proposed cut-off level of 2.04 ng/mL as a predictor of time to treatment in treatment-naïve CLL patients., Competing Interests: The authors declare no competing interests., (This work is licensed under a Creative Commons Attribution 4.0 International License.)

Flow cytometry immunophenotyping has an essential role in distinguishing chronic lymphocytic leukemia from other B-chronic lymphoproliferative disorders. Recently, CD200 is considered as a relatively consistent marker in chronic lymphocytic leukemia. We retrospectively assessed CD200 expression in 252 patients with B chronic lymphoproliferative disorders with four-color flow cytometry. CD200 expression estimation included the proportion of positive cells (≥30%) and the mean fluorescence intensity ratio. Additionally, we have incorporated CD200 into Matutes score, also replaced FMC7 and CD79b in an attempt to improve the score discriminative power. Of 252 patients enrolled, 199(79%) patients were classified as chronic lymphocytic leukemia and 53 (21%) as other B-chronic lymphoproliferative disorders. All chronic lymphocytic leukemia cases and 20 of 53 (37.7%) of other B-chronic lymphoproliferative disorders demonstrated high CD200 expression (≥30%). Further, CD200 (≥30%) revealed a higher accuracy in comparison to other markers in Matutes score (range: 51%-92.5%). Also, CD200 addition to the Matutes score has correctly recognized all 199 chronic lymphocytic leukemia cases including 10 atypical chronic lymphocytic leukemia cases. As for non-CLL cases, 20 of 53 attained a higher score, yet keeping the original diagnosis. Moreover, CD200 enhanced the diagnostic accuracy of Matutes score to 100%, and when included in a simplified 4-markers score, showed an accuracy of 99.8% compared to 99.4% of Matutes score. In conclusion, CD200 is an accurate diagnostic marker for chronic lymphocytic leukemia, and can refine the modified Matutes score accuracy when added with other markers., Competing Interests: The author has declared that no competing interests exist.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the western world. One of the treatments offered for CLL is immunotherapy. These treatments activate various cellular and biochemical mechanisms, using the complement system. Recently it was shown that the complement system in CLL patients is persistently activated at a low level through the classical pathway (CP). The mechanism of chronic CP activation involves the formation of IgG-hexamers (IgG-aggregates). According to recent studies, formation of ordered IgG-hexamers occurs on cell surfaces via specific interactions between Fc regions of the IgG monomers, which occur after antigen binding. The present study investigated the formation of IgG-hexamers in CLL patients and normal (non-malignant) controls (NC), their ability to activate complement, their incidence as cell-free and cell-bound forms and the identity of the antigen causing their formation. Sera from 30 patients and 12 NC were used for separation of IgG- aggregates. The obtained IgG- aggregates were measured and used for assessment of CP activation. For evaluation of the presence of IgG- aggregates on blood cells, whole blood samples were stained and assessed by flow cytometry. Serum levels of IgG- aggregates were higher in CLL and they activated the complement system to a higher extent than in NC. Alpha 2 macroglobulin (A2M) was identified as the antigen causing the hexamerization/aggregation of IgG, and was found to be part of the hexamer structure by mass spectrometry, Western blot and flow cytometry analysis. The presence of A2M-IgG-hexamers on B-cells suggests that it may be formed on B cells surface and then be detached to become cell-free. Alternatively, it may form in the plasma and then attach to the cell surface. The exact time course of A2M-IgG-hexamers formation in CLL should be further studied. The results in this study may be useful for improvement of current immunotherapy regimens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Naseraldeen, Michelis, Barhoum, Chezar, Tadmor, Aviv, Shvidel, Litmanovich, Shehadeh, Stemer, Shaoul and Braester.)

Purpose: Approximately 30% of patients with chronic lymphocytic leukemia (CLL) can be grouped into subsets with stereotyped B-cell receptor immunoglobulin (BcR IG) displaying remarkable similarity in the heavy complementarity-determining region 3 (VH CDR3). Here, we investigated whether the consensus VH CDR3 sequences from CLL stereotyped subsets can be exploited for immunotherapy approaches., Experimental Design: Immunogenic epitopes from the consensus VH CDR3 sequence of the clinically aggressive subsets #1 and #2 and from Eμ-TCL1 mice, which spontaneously develop CLL with BcR IG stereotypy, were identified and used to generate specific HLA class I- and II-restricted T cells in vitro . T-cell reactivity was assayed in vitro as IFNγ production. Bone marrow-derived dendritic cells loaded with the peptides were used as vaccination strategy to restrain leukemia development in the Eμ-TCL1 mouse model., Results: These stereotyped epitopes were naturally processed and presented by CLL cells to the VH CDR3-specific T cells. Furthermore, we validated the efficacy of VH CDR3 peptide-based immunotherapy in the Eμ-TCL1 transplantable mouse model. Immunization of mice against defined VH CDR3 peptide epitopes, prior to the challenge with the corresponding leukemia cells, resulted in the control of CLL development in a significant fraction of mice, and increased overall survival., Conclusions: Our data highlight the immunogenicity of stereotyped VH CDR3 sequences and support the feasibility and efficacy of their use for novel cancer vaccine in CLL. Such approach has the advantage to generate "off-the-shelf" therapeutic vaccines for relevant groups of patients belonging to stereotyped subsets. See related commentary by Seiffert, p. 659 ., (©2020 American Association for Cancer Research.)