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Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries and is an example of hematological disease where cooperation between genetic defects and tumor microenvironmental interaction is involved in pathogenesis. CLL is a disease that is considered as "addicted to the host"; indeed, the crosstalk between leukemic cells and the tumor microenvironment is essential for leukemic clone maintenance supporting CLL cells' survival, proliferation, and protection from drug-induced apoptosis. CLL cells are not innocent bystanders but actively model and manipulate the surrounding microenvironment to their own advantage. Besides the different players involved in this crosstalk, nurse-like cells (NLC) resemble features related to leukemia-associated macrophages with an important function in preserving CLL cell survival and supporting an immunosuppressive microenvironment. This review provides a comprehensive overview of the role played by NLC in creating a nurturing and permissive milieu for CLL cells, illustrating the therapeutic possibilities in order to specifically target and re-educate them.

The tissue microenvironment in chronic lymphocytic leukaemia (CLL) plays a key role in the pathogenesis of CLL, but the complex blood microenvironment in CLL has not yet been fully characterised. Therefore, immunophenotyping of circulating immune cells in 244 CLL patients and 52 healthy controls was performed using flow cytometry and analysed by multivariate Patient Similarity Networks (PSNs). Our study revealed high inter-individual heterogeneity in the distribution and activation of bystander immune cells in CLL, depending on the bulk of the CLL cells. High CLL counts were associated with low activation on circulating monocytes and T cells and vice versa. The highest activation of immune cells, particularly of intermediate and non-classical monocytes, was evident in patients treated with novel agents. PSNs revealed a low activation of immune cells in CLL progression, irrespective of IgHV status, Binet stage and TP53 disruption. Patients with high intermediate monocytes (> 5.4%) with low activation were 2.5 times more likely (95% confidence interval 1.421-4.403, P = 0.002) to had shorter time-to-treatment than those with low monocyte counts. Our study demonstrated the association between the activation of circulating immune cells and the bulk of CLL cells. The highest activation of bystander immune cells was detected in patients with slow disease course and in those treated with novel agents. The subset of intermediate monocytes showed predictive value for time-to-treatment in CLL.

Background: Recent studies proved that metabolic changes in malignant disorders have an impact on protein glycosylation, however, only a few attempts have been made so far to use O-GlcNAc analysis as a prognostic tool. Glucose metabolism is reported to be altered in hematological malignancies thus, we hypothesized that monitoring intracellular O-GlcNAc levels in Rai stage 0-I (Binet A) CLL patients could give deeper insights regarding subtle metabolic changes of progression which are not completely detected by the routine follow-up procedures., Objective: In this proof of concept study we established a flow cytometric detection method for the assessment of O-GlcNAcylation as a possible prognostic marker in CLL malignancy which was supported by fluorescence microscopy., Methods: Healthy volunteers and CLL patients were recruited for this study. Lymphocytes were isolated, fixed and permeabilised by various methods to find the optimal experimental condition for O-GlcNAc detection by flow cytometry. O-GlcNAc levels were measured and compared to lymphocyte count and various blood parameters including plasma glucose level., Results: The protocol we developed includes red blood cell lysis, formalin fixation, 0.1% Tween 20 permeabilisation and employs standardized cell number per sample and unstained controls. We have found significant correlation between O-GlcNAc levels and WBC (R2= 0.8535, p< 0.0029) and lymphocyte count (R2= 0.9225, p< 0.0006) in CLL patients. Interestingly, there was no such correlation in healthy individuals (R2= 0.05664 for O-GlcNAc vs WBC and R2= 0.04379 for O-GlcNAc vs lymphocytes)., Conclusion: Analyzing O-GlcNAc changes in malignant disorders, specifically in malignant hematologic diseases such as CLL, could be a useful tool to monitor the progression of the disease.

Background: Chronic lymphocytic leukemia (CLL) is one of the most common hematologic malignancy in adults worldwide. This cancer has a poor prognosis at different stages. So, the identification of new biomarkers is important for diagnosis of B-CLL. Considering the oncogenic role of APRIL molecule in this leukemia as well as the regulatory role of miRNAs in different signaling pathways, the present study evaluated the miRNAs targeting APRIL gene in B-CLL., Methods: The miRNAs were predicted and selected using bioinformatics algorithms. A total of 80 plasma samples were subjected to RNA extraction and synthesis of cDNA. The expressions levels of predicted miRNAs and APRIL gene in plasma of B-CLL patients and healthy individuals were assessed by Real time PCR analysis. ROC analysis was performed to investigate the role predicted miRNAs as novel biomarkers in diagnosis of B-CLL., Results: The results of the prediction showed that miR-145-5p and miR-185-5p target the APRIL gene. The expression level of APRIL gene was strikingly higher in plasma of B-CLL patients than in the healthy individuals (102, P= 0.001). On the other hand, expression levels of miR-145-5p and miR-185-5p were strikingly lower in B-CLL patients than in the healthy individuals (0.07, P= 0.001) (0.29, P= 0.001). Also, ROC curve analyses demonstrated that miR-145-5p and miR-185-5p are specific and sensitive and may serve as new biomarkers for the detection of B-CLL. (AUC; 0.95, sensitivity; %90) (AUC; 0.87, sensitivity; %63)., Conclusion: These data suggest that miR-145-5p and miR-185-5p target the APRIL gene and might have a role in diagnosis of B-CLL. Therefore, these two miRNAs can be served as a novel and potential biomarker for detection of B-CLL.

Long-term severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shedding was observed from the upper respiratory tract of a female immunocompromised individual with chronic lymphocytic leukemia and acquired hypogammaglobulinemia. Shedding of infectious SARS-CoV-2 was observed up to 70 days, and of genomic and subgenomic RNA up to 105 days, after initial diagnosis. The infection was not cleared after the first treatment with convalescent plasma, suggesting a limited effect on SARS-CoV-2 in the upper respiratory tract of this individual. Several weeks after a second convalescent plasma transfusion, SARS-CoV-2 RNA was no longer detected. We observed marked within-host genomic evolution of SARS-CoV-2 with continuous turnover of dominant viral variants. However, replication kinetics in Vero E6 cells and primary human alveolar epithelial tissues were not affected. Our data indicate that certain immunocompromised individuals may shed infectious virus longer than previously recognized. Detection of subgenomic RNA is recommended in persistently SARS-CoV-2-positive individuals as a proxy for shedding of infectious virus., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has profound activity in chronic lymphocytic leukemia (CLL) but limited curative potential by itself. Residual signaling pathways that maintain survival of CLL cells might be targeted to improve ibrutinib's therapeutic activity, but the nature of these pathways is unclear. Ongoing activation of IFN receptors in patients on ibrutinib was suggested by the presence of type I and II IFN in blood together with the cycling behavior of IFN-stimulated gene (ISG) products when IFN signaling was blocked intermittently with the JAK inhibitor ruxolitinib. IFN signaling in CLL cells from human patients was not prevented by ibrutinib in vitro or in vivo, but ISG expression was significantly attenuated in vitro. ISGs such as CXCL10 that require concomitant activation of NF-κB were decreased when this pathway was inhibited by ibrutinib. Other ISGs, exemplified by LAG3 , were decreased as a result of inhibited protein translation. Effects of IFN on survival remained intact as type I and II IFN-protected CLL cells from ibrutinib in vitro, which could be prevented by ruxolitinib and IFNR blocking Abs. These observations suggest that IFNs may help CLL cells persist and specific targeting of IFN signaling might deepen clinical responses of patients on ibrutinib., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Purpose: The development of highly effective targeted agents for chronic lymphocytic leukemia offers the potential for fixed-duration combinations that achieve deep remissions without cytotoxic chemotherapy., Patients and Methods: This phase II study tested a combination regimen of obinutuzumab, ibrutinib, and venetoclax for a total of 14 cycles in both patients with treatment-naïve (n = 25) and relapsed or refractory (n = 25) chronic lymphocytic leukemia to determine the response to therapy and safety., Results: The primary end point was the rate of complete remission with undetectable minimal residual disease by flow cytometry in both the blood and bone marrow 2 months after completion of treatment, which was 28% in both groups. The overall response rate at that time was 84% in treatment-naïve patients and 88% in relapsed or refractory patients. At that time, 67% of treatment-naïve patients and 50% of relapsed or refractory patients had undetectable minimal residual disease in both the blood and marrow. At a median follow-up of 24.2 months in treatment-naïve patients and 21.5 months in relapsed or refractory patients, the median progression-free and overall survival times were not yet reached, with only 1 patient experiencing progression and 1 death. Neutropenia and thrombocytopenia were the most frequent adverse events, followed by hypertension. Grade 3 or 4 neutropenia was experienced by 66% of patients, with more events in the relapsed or refractory cohort. There was only 1 episode of neutropenic fever. A favorable impact on both perceived and objective cognitive performance during treatment was observed., Conclusion: The combination regimen of obinutuzumab, ibrutinib, and venetoclax offers time-limited treatment that results in deep remissions and is now being studied in phase III cooperative group trials.

Background: There has been a concern that blood donations can increase the risk of hematological malignancies. We investigated if blood donations increase the risk of developing hematological malignancies, specifically acute lymphoblastic leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia, Hodgkin lymphoma, and myeloma, as well other non-Hodgkin lymphoma., Study Design and Methods: In total, the study included 1,021,433 Swedish blood donors, with 19.5 million person-years of follow-up. Two sets of analysis were performed. In the first cohort analysis, standardized incidence ratios (SIRs) were calculated, comparing the incidence of the different types of hematological cancers in blood donors to that of the general population. In the second analysis, a nested case-control study was conducted, investigating the association between number of donations and the risk of each type of malignancy., Results: Apart from a modestly elevated risk of CLL (SIR, 1.07; 95% confidence interval [CI], 1.01-1.15) and a modestly decreased risk of AML (SIR, 0.85; 95% CI, 0.77-0.83), the risk of hematological malignancies did not differ between blood donors and the general population. In the nested case-control study there were no convincing associations between number of prior whole blood donations and site-specific malignancy risk., Conclusions: There was no convincing evidence of an increased risk in any hematological malignancy when interpreting the results from both series of analyses., (© 2020 The Authors. Transfusion published by Wiley Periodicals LLC. on behalf of AABB.)

Blocking antibodies targeting immune checkpoint molecules achieved invaluable success in tumor therapy and amazing clinical responses in a variety of cancers. Although common treatment protocols have improved overall survival in patients with chronic lymphocytic leukemia (CLL), they continue to relapse and progress. In the present in vitro study, the application of anti-PD-1 and anti-TIM-3 blocking antibodies was studied to restore the function of exhausted CD8 + T cells in CLL. CD8 + T cells were isolated from peripheral blood of 20 patients with CLL, treated with blocking antibodies, and cocultured with mitomycin-frozen non-CD8 + T cell fraction as target cells. Cultures were stimulated with anti-CD3/CD28 antibodies to assess the proliferation of CD8 + T cells by MTT and stimulated with PMA/ionomycin to measure the levels of CD107a expression and cytokine production by flow cytometry and ELISA, respectively. Our results showed that the blockade of PD-1 and TIM-3 does not improve the proliferation of CD8 + T cells in CLL patients. No significant difference was found between control and blocked groups in terms of degranulation properties and production of IFN-γ, TNF-α, IL-2, and IL-10 by CD8 + T cells. We observed that pre-treatment of CD8 + T cells with blocking antibodies in CLL patients at early clinical stages had no effects on restoring their functional properties. Further in vitro and in vivo complementary studies are required to more explore the utility of checkpoint inhibitors for CLL patients.

Ibrutinib may inhibit intestinal CYP3A4 and induce CYP2B6 and/or CYP3A. Secondary to potential induction, ibrutinib may reduce the exposure and effectiveness of oral contraceptives (OCs). This phase I study evaluated the effect of ibrutinib on the pharmacokinetics of the CYP2B6 substrate bupropion, CYP3A substrate midazolam, and OCs ethinylestradiol (EE) and levonorgestrel (LN). Female patients (N = 22) with B-cell malignancies received single doses of EE/LN (30/150 μg) and bupropion/midazolam (75/2 mg) during a pretreatment phase on days 1 and 3, respectively (before starting ibrutinib on day 8), and again after ibrutinib 560 mg/day for ≥ 2 weeks. Intestinal CYP3A inhibition was assessed on day 8 (single-dose ibrutinib plus single-dose midazolam). Systemic induction was assessed at steady-state on days 22 (EE/LN plus ibrutinib) and 24 (bupropion/midazolam plus ibrutinib). The geometric mean ratios (GMRs; test/reference) for maximum plasma concentration (C max ) and area under the plasma concentration-time curve (AUC) were derived using linear mixed-effects models (90% confidence interval within 80%-125% indicated no interaction). On day 8, the GMR for midazolam exposure with ibrutinib coadministration was ≤ 20% lower than the reference, indicating lack of intestinal CYP3A4 inhibition. At ibrutinib steady-state, the C max and AUC of EE were 33% higher than the reference, which was not considered clinically relevant. No substantial changes were noted for LN, midazolam, or bupropion. No unexpected safety findings were observed. A single dose of ibrutinib did not inhibit intestinal CYP3A4, and repeated administration did not induce CYP3A4/2B6, as assessed using EE, LN, midazolam, and bupropion., (© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Richter syndrome (RS) is an uncommon evolution of chronic lymphocytic leukaemia (CLL) with a dismal prognosis. Clinical-biological features predicting outcome and best therapeutic approach for these patients remain to be established. In this study, 128 patients with RS, including 112 diffuse large B-cell lymphoma (DLBCL)-type RS, 15 Hodgkin lymphoma (HL)-type RS, and one plasmablastic lymphoma, were identified in 11 centres of the Spanish CLL Study Group (GELLC). The median overall survival (OS) was 5·9 months for DLBCL-type RS and 30·8 months for HL-type RS. Eastern Cooperative Oncology Group Performance Status, haemoglobin level, platelet count, serum lactate dehydrogenase and β2-microglobulin levels, tumour protein p53 (TP53) abnormalities in the CLL clone concomitant to RS, number of prior therapies, and clonal relationship between CLL and RS, were associated with OS in patients with DLBCL-type RS. A platelet count of <100 × 10 9 /l, prior CLL therapy (0 vs. ≥1), and presence of TP53 alterations maintained an independent prognostic impact in the multivariate analysis. Patients without any of these factors had a better clinical outcome, with a median OS of 75·3 months, while patients with one or two or more of these factors presented a median OS of 25·5 and 3 months, respectively. Although OS of patients with RS is generally poor, a proportion of patients achieved prolonged survival. Treatment of RS remains a medical need, and further therapeutic approaches with novel therapies are warranted., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

The relationship between chronic lymphocytic leukaemia (CLL) and qualitative/quantitative gammaglobulin abnormalities is well established. Nevertheless, in order to better understand this kind of connection, we examined 1505 patients with CLL and divided them into four subgroups on the basis of immunoglobulin (Ig) aberrations at diagnosis. A total of 73 (4·8%), 149 (10%), 200 (13·2%) and 1083 (72%) patients were identified with IgM monoclonal gammopathy (IgM/CLL), IgG monoclonal gammopathy (IgG/CLL), hypogammaglobulinaemia (hypo-γ) and normal Ig levels (γ-normal) respectively. IgM paraprotein was significantly associated with a more advanced Binet/Rai stage and del(17p)/TP53 mutation, while IgG abnormalities correlated with a higher occurrence of trisomy 12. Patients with any type of Ig abnormality had shorter treatment-free survival (TFS) but no significant impact affecting overall survival (OS) compared to those with normal Ig levels., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Chronic lymphocytic leukemia (CLL) is an incurable disease accounting for almost one-third of leukemias in the Western world. Aberrant expression of microRNAs (miRNAs) is a well-established characteristic of CLL, and the robust nature of miRNAs makes them eminently suitable liquid biopsy biomarkers. Using a nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC), the predictive values of five promising human miRNAs (hsa-miR-16-5p, hsa-miR-29a-3p, hsa-miR-150-5p, hsa-miR-155-5p and hsa-miR-223-3p), identified in a pilot study, were examined in serum of 224 CLL cases (diagnosed 3 months to 18 years after enrollment) and 224 matched controls using Taqman based assays. Conditional logistic regressions were applied to adjust for potential confounders. The median time from blood collection to CLL diagnosis was 10 years (p25-p75: 7-13 years). Overall, the upregulation of hsa-miR-150-5p, hsa-miR-155-5p and hsa-miR-29a-3p was associated with subsequent risk of CLL [OR 1∆Ct-unit increase (95%CI) = 1.42 (1.18-1.72), 1.64 (1.31-2.04) and 1.75 (1.31-2.34) for hsa-miR-150-5p, hsa-miR-155-5p and hsa-miR-29a-3p, respectively] and the strongest associations were observed within 10 years of diagnosis. However, the predictive performance of these miRNAs was modest (area under the curve <0.62). hsa-miR-16-5p and hsa-miR-223-3p levels were unrelated to CLL risk. The findings of this first prospective study suggest that hsa-miR-29a, hsa-miR-150-5p and hsa-miR-155-5p were upregulated in early stages of CLL but were modest predictive biomarkers of CLL risk., (© 2020 UICC.)

Background: Within the hematopoietic compartment, fibromodulin (FMOD) is almost exclusively expressed in chronic lymphocytic leukemia (CLL) lymphocytes. We set out to determine whether FMOD could be of help in diagnosing borderline lymphoproliferative disorders (LPD)., Methods: We established 3 flow cytometry-defined groups (CLL [n = 65], borderline LPD [n = 28], broadly defined as those with CLLflow score between 35 and -20 or discordant CD43 and CLLflow, and non-CLL LPD [n = 40]). FMOD expression levels were determined by standard RT-PCR in whole-blood samples. Patients were included regardless of lymphocyte count but with tumor burden ≥40%., Results: FMOD expression levels distinguished between CLL (median 98.5, interquartile range [IQR] 37.8-195.1) and non-CLL LPD (median 0.012, IQR 0.003-0.033) with a sensitivity and specificity of 1. Most borderline LPDs were CD5/CD23/CD200-positive with no loss of B-cell antigens and negative or partial expression of CD43. 16/22 patients with available cytogenetic analysis showed trisomy 12. In 25/28 (89%) of these patients, FMOD expression levels fell between CLL and non-CLL (median 3.58, IQR 1.06-6.21)., Discussion: This study could suggest that borderline LPDs may constitute a distinct group laying in the biological spectrum of chronic leukemic LPDs. Future studies will have to confirm these results with other biological data. Quantification of FMOD can potentially be of help in the diagnosis of phenotypically complex LPDs., (© 2020 International Clinical Cytometry Society.)

Background: In chronic lymphocytic leukemia, a better understanding of leukemic cell characteristics after treatment would help to design specific therapeutic approaches aimed at preventing clinical relapse. Gene arrays have become a powerful approach to perform gene expression profiling; nevertheless, to work with residual cells entails an intensive labor. The aim of this study was to set forth an effective gene expression approach to analyze residual leukemic cells., Methods: Leukocytes from CLL patient's samples were sorted by flow cytometry using a 6-color panel. The quality and quantity of RNA isolated from different inputs of cells were compared by two silica column protocols: RNeasy Micro and RNeasy Mini. RNA amplifications were carried out according to two manufacturer's protocols: Ovation Pico SL and Ovation Pico WTA. A total of 3.5 μg of cDNA was labeled and hybridized to Human Gene 2.0 ST arrays., Results: RNA extracted from low number of input cells by RNeasy Micro showed similar RNA integrity number to that obtained from RNeasy Mini; however, the RNA quantity was higher using the RNeasy Micro Kit. In addition, those RNA samples obtained with RNeasy Micro and amplified with Ovation Pico WTA showed good quality to proceed for a gene array study, independently of the number of input cells (range: 1 × 10 4 -5 × 10 5 cells)., Conclusions: We observed that this workflow is a feasible approach to obtain genomic material extracted from leukemic cells as little as 1 × 10 4 cells and it can be useful to carry out gene expression profile experiments to characterize residual leukemic cells in chronic lymphocytic leukemia., (© 2020 John Wiley & Sons Ltd.)

Purpose: Survival of CLL cells due to the presence of Bcl-2 and Mcl-1 has been established. Direct inhibition of Bcl-2 by venetoclax and indirect targeting of Mcl-1 with transcription inhibitors have been successful approaches for CLL. AMG-176 is a selective and direct antagonist of Mcl-1, which has shown efficacy in several hematologic malignancies; however, its effect on CLL is elusive. We evaluated biological and molecular effects of AMG-176 in primary CLL cells., Experimental Design: Using samples from patients ( n = 74) with CLL, we tested effects of AMG-176 on CLL and normal hematopoietic cell death and compared importance of CLL prognostic factors on this biological activity. We evaluated CLL cell apoptosis in the presence of stromal cells and identified cell death pathway including stabilization of Mcl-1 protein. Finally, we tested a couplet of AMG-176 and venetoclax in CLL lymphocytes., Results: AMG-176 incubations resulted in time- and dose-dependent CLL cell death. At 100 and 300 nmol/L, there was 30% and 45% cell death at 24 hours. These concentrations did not result in significant cell death in normal hematopoietic cells. Presence of stroma did not affect AMG-176-induced CLL cell death. IGHV unmutated status, high β2M and Mcl-1 protein levels resulted in slightly lower cell death. Mcl-1, but not Bcl-2 protein levels, in CLL cells increased with AMG-176. Low concentrations of venetoclax (1-30 nmol/L) were additive or synergistic with AMG-176., Conclusions: AMG-176 is active in inducing CLL cell death while sparing normal blood cells. Combination with low-dose venetoclax was additive or synergistic., (©2020 American Association for Cancer Research.)

Background: High UGT2B17 is associated with poor prognosis in untreated chronic lymphocytic leukaemia (CLL) patients and its expression is induced in non-responders to fludarabine-containing regimens. We examined whether UGT2B17, the predominant lymphoid glucuronosyltransferase, affects leukaemic drug response and is involved in the metabolic inactivation of anti-leukaemic agents., Methods: Functional enzymatic assays and patients' plasma samples were analysed by mass-spectrometry to evaluate drug inactivation by UGT2B17. Cytotoxicity assays and RNA sequencing were used to assess drug response and transcriptome changes associated with high UGT2B17 levels., Results: High UGT2B17 in B-cell models led to reduced sensitivity to fludarabine, ibrutinib and idelalisib. UGT2B17 expression in leukaemic cells involved a non-canonical promoter and was induced by short-term treatment with these anti-leukaemics. Glucuronides of both fludarabine and ibrutinib were detected in CLL patients on respective treatment, however UGT2B17 conjugated fludarabine but not ibrutinib. AMP-activated protein kinase emerges as a pathway associated with high UGT2B17 in fludarabine-treated patients and drug-treated cell models. The expression changes linked to UGT2B17 exposed nuclear factor kappa B as a key regulatory hub., Conclusions: Data imply that UGT2B17 represents a mechanism altering drug response in CLL through direct inactivation but would also involve additional mechanisms for drugs not inactivated by UGT2B17.

Background: The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines., Methods: Sensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels., Results: LoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low., Conclusions: These studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.

B-cell Chronic Lymphocytic Leukemia (B-CLL) is the most common hematological disorder among middle-aged/elderly people in the Western countries. We have shown earlier that B-CLL cells exhibit elevated total amount and available activity of µ-calpain, belonging to a family of ubiquitous, strongly Ca-dependent proteases, involved in the control of proliferation and apoptosis. In this study we attempted to estimate a potential clinical value of μ-calpain in relation to B-CLL clinical staging in patients with extremely high lymphocytosis and studied the molecular mechanisms associating calpain activity with clinical progress of the disease. We observed significant correlations between the amounts of intracellular μ-calpain and clinical staging of the disease, with RAI stage 1 corresponding to the highest calpain amounts in the leukemic cells. There was also a positive, statistically significant correlation between the amount of μ-calpain and phosphorylated (p)ZAP-70 in B-CLL lymphocytes. Calpain activity in the B-CLL cells is associated with decreased activities of pro-apoptotic caspases -3 and -9, and reciprocally with an increased amount of anti-apoptotic Bcl-2. Together, all of these findings make calpain activity in B-CLL cells a promising target modifying the properties of these cells and facilitating therapy. Finally, the proportion of CD19+ B cells with elevated μ-calpain and pZap-70 was markedly reduced in patients after successful therapy.

Background: Lipid traits have been inconsistently linked to risk of non-Hodgkin lymphoma (NHL). We examined the association of genetically predicted lipid traits with risk of diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and marginal zone lymphoma (MZL) using Mendelian randomization (MR) analysis., Methods: Genome-wide association study data from the InterLymph Consortium were available for 2,661 DLBCLs, 2,179 CLLs, 2,142 FLs, 824 MZLs, and 6,221 controls. SNPs associated ( P < 5 × 10 -8 ) with high-density lipoprotein (HDL, n = 164), low-density lipoprotein (LDL, n = 137), total cholesterol (TC, n = 161), and triglycerides (TG, n = 123) were used as instrumental variables (IV), explaining 14.6%, 27.7%, 16.8%, and 12.8% of phenotypic variation, respectively. Associations between each lipid trait and NHL subtype were calculated using the MR inverse variance-weighted method, estimating odds ratios (OR) per standard deviation and 95% confidence intervals (CI)., Results: HDL was positively associated with DLBCL (OR = 1.14; 95% CI, 1.00-1.30) and MZL (OR = 1.09; 95% CI, 1.01-1.18), while TG was inversely associated with MZL risk (OR = 0.90; 95% CI, 0.83-0.99), all at nominal significance ( P < 0.05). A positive trend was observed for HDL with FL risk (OR = 1.08; 95% CI, 0.99-1.19; P = 0.087). No associations were noteworthy after adjusting for multiple testing., Conclusions: We did not find evidence of a clear or strong association of these lipid traits with the most common NHL subtypes. While these IVs have been previously linked to other cancers, our findings do not support any causal associations with these NHL subtypes., Impact: Our results suggest that prior reported inverse associations of lipid traits are not likely to be causal and could represent reverse causality or confounding., (©2020 American Association for Cancer Research.)

Despite significant therapeutic improvements chronic lymphocytic leukemia (CLL) remains an incurable disease and there is a persistent pursuit of new treatment alternatives. Lurbinectedin, a selective inhibitor of active transcription of protein-coding genes, is currently in phase II/III clinical trials for solid tumors such as small-cell lung cancer (SCLC). In this study, we aimed to evaluate the activity of Lurbinectedin on circulating mononuclear cells from CLL patients and to determine whether Lurbinectedin could affect the cross-talk between B-CLL cells and the tumor microenvironment. We found that Lurbinectedin induced a dose- and time-dependent death in all cell types evaluated, with B cells, monocytes and monocytic myeloid derived suppressor cells (Mo-MDSC) being the most susceptible populations. At sub-apoptotic doses, Lurbinectedin decreased the expression of CCR7 in B-CLL cells and impaired their migration towards CCL19 and CCL21. Furthermore, low concentrations of Lurbinectedin stimulated the synthesis of pro-IL1β in monocytes and nurse-like cells, without inducing the inflammasome activation. Altogether, these results indicate that Lurbinectedin might have antitumor activity in CLL due to its direct action on leukemic cells in combination with its effects on the tumor microenvironment. Our findings encourage further investigation of Lurbinectedin as a potential therapy for CLL.

DOCK10, a guanine-nucleotide exchange factor (GEF) for Rho GTPases, represents the example of a gene that gives rise to alternative first exon mRNA isoforms, named DOCK10.1 and DOCK10.2. Expression of human DOCK10.2 protein in cell lines, and its induction by interleukin-4 (IL-4) in normal B lymphocytes and chronic lymphocytic leukemia (CLL) cells, were previously demonstrated using an antiserum raised against a peptide encoded by sequences from exon 1.2. Here, expression of human DOCK10.1 protein was demonstrated using an antiserum raised against a peptide encoded by sequences from exon 1.1. Specificity of the DOCK10.1 and DOCK10.2 antisera for their respective isoforms was demonstrated using transfected human 293 T cells. Their specificity for endogenous DOCK10 was strongly suggested by the high significance of the correlations between the levels of their expected signals at the molecular size of 250 kDa and the levels of DOCK10.1 and DOCK10.2 mRNAs, respectively, in human hematopoietic cell lines. Specificity of the DOCK10.1 antiserum for DOCK10 was also demostrated in mouse using the DOCK10 knockout model. The DOCK10.1 protein was induced by IL-4 in CLL cells, which demonstrates that the mechanism by which IL-4 regulates DOCK10 is not isoform-specific. Last, to get insights into differential regulation of the DOCK10 isoforms, their protein levels in cell lines were compared with their gene expression profiles retrieved from the Cancer Cell Line Encyclopedia (CCLE), leading to the identification of BCL3 and KLF12 as potential transcriptional regulators of DOCK10.1 and DOCK10.2, respectively.

Diagnosis of hematological cancer requires complete white blood cell count, followed by flow cytometry with multiple markers, and cytology. It requires substantial time and specialized training. A dual-layer paper microfluidic chip was developed as a quicker, low-cost, and field-deployable alternative to detect ROR1+ (receptor tyrosine-like orphan receptor one) cancer cells from the undiluted and untreated buffy coat blood samples. The first capture layer consisted of a GF/D glass fiber substrate, preloaded with cancer specific anti-ROR1 conjugated fluorescent particles to its center for cancer cell capture and direct smartphone fluorescence imaging. The second flow layer was comprised of a grade 1 cellulose chromatography paper with wax-printed four channels for wicking and capillary flow-based detection. The flow velocity was used as measure of antigen concentration in the buffy coat sample. In this manner, intact cells and their antigens were separated and independently analyzed by both imaging and flow velocity analyses. A custom-made smartphone-based fluorescence microscope and automated image processing and particle counter software were developed to enumerate particles on paper, with the limit of detection of 1 cell/μL. Flow velocity analysis showed even greater sensitivity, with the limit of detection of 0.1 cells/μL in the first 6 s of assay. Comparison with capillary flow model revealed great alignment with experimental data and greater correlation to viscosity than interfacial tension. Our proposed device is able to capture and on-chip image ROR1+ cancer cells within a complex sample matrix (buffy coat) while simultaneously quantifying cell concentration in a point-of-care manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Therapy regimens for Chronic lymphocytic leukemia (CLL) commonly include chemotherapy and immunotherapy, which act through complement-mediated-cytotoxicity (CDC) and other mechanisms. CDC depends on several factors, including the availability and activity of the complement classical pathway (CP). Recently, a significant decrease in CP activity was shown to be associated with an immunoglobulin-C5a complex (Ig-C5a) and other markers of chronic CP activation in 40% of the patients. The study focused on the involvement of IgG-hexamers, an established CP activator, in the mechanism of chronic CP activation in CLL. Sera from 51 naïve CLL patients and 20 normal controls were collected. CP and alternative pathway (AP) activities were followed by the complement activity marker sC5b-9. Serum high molecular weight (HMW) proteins were collected by gel-filtration chromatography and their complement activation capacity was assessed. The levels of IgM, another established CP activator, were measured. Data were associated with the presence of Ig-C5a. Baseline levels of activation markers negatively correlated with CP and the AP activities, supporting chronic complement activation. In patients with Ig-C5a, HMW proteins that are not IgM, activated the complement. HMW proteins were identified as IgG-aggregates by affinity binding assays and Western blot analysis. The data indicate chronic CP activation, mediated by cell-free IgG-hexamers as a cause of decreased CP activity in part of the CLL population. This mechanism may affect immunotherapy outcomes due to compromised CP activity and CDC., Competing Interests: The authors have declared that no competing interests exist.

The literature is rich in proof of concept studies demonstrating the potential of Raman spectroscopy for disease diagnosis. However, few studies are conducted in a clinical context to demonstrate its applicability in current clinical practice and workflow. Indeed, this translational research remains far from the patient's bedside for several reasons. First, samples are often cultured cell lines. Second, they are prepared on non-standard substrates for clinical routine. Third, a unique supervised classification model is usually constructed using inadequate cross-validation strategy. Finally, the implemented models maximize classification accuracy without taking into account the clinician's needs. In this paper, we address these issues through a diagnosis problem in real clinical conditions, i.e., the diagnosis of chronic lymphocytic leukemia from fresh unstained blood smears spread on glass slides. From Raman data acquired in different experimental conditions, a repeated double cross-validation strategy was combined with different cross-validation approaches, a consensus label strategy and adaptive thresholds able to adapt to the clinician's needs. Combined with validation at the patient level, classification results were improved compared to traditional strategies.

MiR-155 is involved in various physiological processes in the cell, including hematopoiesis, immunity, inflammation and differentiation. Increased expression of miR-155 is observed in many malignant diseases, including lymphomas, acute myeloid leukemia and CLL. However, a comparative study of the miR-155 expression in the blood leukocytes in patients with chronic myeloid and lymphoproliferative diseases has not yet been carried out. To investigate the expression of miR-155 in the blood cells of patients with lympho- and ph-negative myeloproliferative neoplasms. MiR-155 expression were studied in the blood leukocytes of 28 patients with B-CLL, 52 patients with MPN and 51 donors by "real time" PCR method. The study revealed an increase in miR-155 in blood leukocytes in both patients with CLL and patients with MPN compared with the control group. In accordance with the results of the ROC analysis, the sensitivity and specificity of blood leukocytes testing on miR-155 expression level was 81.8% and 78.4%, respectively, for CLL and 55.1% and 82.4%, respectively, for MPN. At the same time, in patients with CLL who received therapy, the level of miR-155 was significantly lower compared with those who did not receive therapy. Thus, the involvement of miR-155 in the pathogenesis of chronic myeloid and lymphoproliferative diseases was demonstrated., Competing Interests: The authors declare no conflict of interest.

Monoclonal antibodies ofatumumab (anti-CD20) and alemtuzumab (anti-CD52) which are approved for usage in patients with chronic lymphocytic leukemia (CLL), efficiently activate the classical complement pathway. However complement is an exhaustible component and high doses of its activators may deplete complement-dependent cytotoxicity (CDC) potential, thus reducing the effect of repeated mAb dosing. Widely used method to measure CDC activity of patients' serum is hemolytic assay (CH50) on sheep erythrocytes. Despite its simplicity, such CH50 assay may not reflect pivotal interactions between patient serum and human complement inhibitors on the surface of target cells. We propose calcein release assay performed on tumor cells similar to those targeted by therapeutic antibodies as an alternative method. We analyzed serum samples collected from 12 patients participating in the clinical study, receiving s.c. 30 mg alemtuzumab three times per week combined with i.v. ofatumumab at an initial dose of 300 mg in week 3 further escalated to 2000 mg every other week. All serum samples were measured by hemolytic assay on sheep erythrocytes as well as using calcein release assay on CD20-positive Raji cells. Our data show that results obtained from both assays are related to each other at the level of the whole group (n = 96 samples, Spearman r = 0.504, p < .001) but may substantially differ when analyzing individual patients. Furthermore, by using CDC assay on Raji cells, we found that in the presented clinical study CDC serum potential was not significantly affected when measured before consecutive administrations in most of the patients., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The prevalence of thrombosis in lymphoma patients is reportedly high and ranges from 3-10%. Vascular malfunction and inflammatory processes further contribute to the thrombotic activation process in these patients. Andexanet alfa (AA) is an antidote for factor Xa inhibitors and its usage has been reported with thrombotic complications. This study was designed to compare the effect of AA on the thrombin generation (TG) potential. Blood samples from 78 patients with confirmed diagnosis of non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) and Chronic lymphocytic leukemia (CLL) were collected from the University of Belgrade Clinic, Serbia. Normal human plasma (NHP) was used for referencing purposes. Individual samples were supplemented with AA at 100 ug/ml. TG studies were carried out using a commercially available fluorogenic substrate method. TG parameters such as peak thrombin (PT), lag time (LT) and area under the curve (AUC) were compiled. Cumulatively, lymphoma patients showed an increase in LT compared to NHP which decreases with AA. The PT and AUC levels were decreased compared to NHP and increases with AA. Upon sub-grouping of lymphoma patients, PT levels for all sub-groups were increased with AA. The AUC values increased for HL and NHL and decreased for CLL with AA. Variations in lag time were noted in all 3 sub-groups. Lymphoma represents a heterogenous group of patients where both the hypercoagulable state and inflammatory responses simultaneously occur. Increased thrombin generation in post AA supplemented samples suggest that the use of this agent may potentially be associated with thrombotic complications.

Epidemiological studies have demonstrated an increased risk of non-Hodgkin lymphoma (NHL) in patients with chronic hepatitis C virus (HCV) infection. Therefore, we investigated the risk of extrahepatic malignancies associated with HCV infection. Inpatients diagnosed with lymphoma, breast, thyroid, kidney, or pancreatic cancer (research group, n = 17,925) as well as inpatients with no malignancies (control group, n = 16,580) matched by gender and age were enrolled from The First Affiliated Hospital of Nanjing Medical University between January 2008 and December 2016. A case-control study was conducted by retrospective analysis. The difference in HCV prevalence was analyzed between the research group and the control group. Also, the research group was compared to the 2006 National Hepatitis C sero-survey in China. A total of 86 cases were positive for anti-HCV in the research group. Compared with the control group (103 cases were anti-HCV positive), no significant associations between extrahepatic malignancies and HCV infection were observed. Meanwhile, compared to the 2006 National Hepatitis C sero-survey, we observed a significant association between the chronic lymphoma leukemia/small lymphocytic lymphoma (CLL/SLL) and HCV seropositivity in females in the research group aged 1-59 years old (OR = 14.69; 95% CI, 1.94-111.01). HCV infection had a potential association with CLL/SLL in females aged 1-59 years old. Our study did not confirm an association between HCV infection and the risk of extrahepatic malignancies. In regions with a low HCV prevalence, the association between HCV infection and extrahepatic malignancies needs further investigation.

Continuous treatment vs fixed duration of monotherapies and combinations of targeted agents are treatment options in relapsed chronic lymphocytic leukemia. The optimal choice of relapse treatment is dependent on the prior frontline therapy, duration of remission after frontline, genetic markers, and patients' condition, including age and comorbidities. Combination therapies may result in deep responses with undetectable minimal residual disease (uMRD). Although uMRD is an excellent predictive marker for disease progression, it is rarely used in clinical practice and needs additional evaluation in clinical trials before discontinuation of therapy should be guided according to uMRD., Competing Interests: Conflict-of-interest disclosure: B.E. received honoraria, travel grants, and scientific grants from Roche, Abbvie, Janssen, Gilead, Novartis, AstraZeneca, Arqule, and Celgene. M.F. received travel grants from Roche and Janssen and honoraria, travel grants, and scientific grants from Roche, Abbvie, Janssen, and Gilead. M.H. declares no competing financial interests., (© 2019 by The American Society of Hematology. All rights reserved.)

Acid sphingomyelinase (aSMase) is involved in the generation of metabolites that function as part of the sphingolipid signaling pathway. It catalyzes the breakdown of sphingomyelin into ceramide, a bioactive lipid that, among other roles, is involved in regulation of apoptosis. Dry drop blood test (DBS) and colorimetric 2-step enzymatic assay were used to assess the activity of human blood aSMase, beta-galactosidase, and beta-glucosidase, these enzymes are lysosomal hydrolases that catalyze the degradation of related sphingolipids, of sphingolipid signaling molecules. Blood was collected from a group of healthy volunteers and patients that were diagnosed with multiple myeloma (MM) in various stages of the disease. Additionally, activity of those enzymes in patients diagnosed with other hematological cancers was also assessed. We found that aSMase activity in the blood of patients with MM (at the time of diagnosis) was 305.43 pmol/spot*20 h, and this value was significantly lower ( p < 0.030) compared to the healthy group 441.88 pmol/spot*20 h. Our collected data suggest a possible role of aSMase in pathogenesis of MM development.

Patients with chronic lymphocytic leukemia (CLL) who achieve blood or bone marrow (BM) undetectable minimal residual disease (U-MRD) status after first-line fludarabine, cyclophosphamide, and rituximab (FCR) have prolonged progression-free survival (PFS), when assessed by an assay with sensitivity 10-4 (MRD4). Despite reaching U-MRD4, many patients, especially those with unmutated IGHV, subsequently relapse, suggesting residual disease <10-4 threshold and the need for more sensitive MRD evaluation. MRD evaluation by next-generation sequencing (NGS) has a sensitivity of 10-6 (MRD6). To better assess the depth of remission following first-line FCR treatment, we used NGS (Adaptive Biotechnologies Corporation) to assess MRD in 62 patients, all of whom had BM U-MRD by multicolor flow cytometry (sensitivity 10-4) at end-of-FCR treatment. Samples from these patients included 57 BM samples, 29 peripheral blood mononuclear cell (PBMC) samples, and 32 plasma samples. Only 27.4% of the 62 patients had U-MRD by NGS. Rate of U-MRD by NGS was lowest in BM (25%), compared with PBMC (55%) or plasma (75%). No patient with U-MRD by NGS in BM or PBMC was MRD+ in plasma. Patients with mutated IGHV were more likely to have U-MRD by NGS at the end of treatment (EOT; 41% vs 13%, P = .02) than those with unmutated IGHV. Median follow-up was 81.6 months. Patients with U-MRD at EOT had superior PFS vs MRD+ patients, regardless of sample type assessed (BM, P = .02, median not reached [NR] vs 67 months; PBMC, P = .02, median NR vs 74 months). More sensitive MRD6 testing increases prognostic discrimination over MRD4 testing., (© 2019 by The American Society of Hematology.)

Background: The scoring system used for chronic lymphocytic leukemia (CLL) cannot make an accurate diagnosis in some cases. Novel markers are available for the differential diagnosis of CLL, especially from MCL. However, these markers are still not incorporated into diagnostic algorithms. We investigated the role of CD43, CD81, CD200, and ROR1 in the differential diagnosis of CLL and their expression in non-CLL cases., Methods: We investigated the role of CD43, CD81, CD20, and ROR1 in the differential diagnosis of CLL by incorporating them into the diagnostic panel after studying peripheral blood or bone marrow samples of 165 patients with 8-color flow cytometry., Results: CD43 positivity was a sensitive marker but had a lower specificity for CLL. CD43 had high diagnostic value for CLL (sensitivity 100%, specificity 88.5%, AUC 98.0%). CD200 was a specific marker for CLL (sensitivity 98%, specificity 90%, AUC: 96%). CD81 expression was highest in the MCL cases, with a median expression rate of 68.5% (range: 54 - 82.5%). It was negative in all the CLL cases. For CLL, CD81 negativity had a sensitivity of 95%, a specificity of 82% and an AUC of 92%. ROR1 was positive in all CLL and MCL cases. CD79b, on the other hand, was a fairly sensitive and specific marker for MCL., Conclusions: CD43, CD81, CD200, and ROR1 should be incorporated into diagnostic algorithms for the differential diagnosis of CLL, especially from MCL.