Modeling the B-cell receptor signaling on single cell level reveals a stable network circuit topology between nonmalignant B cells and chronic lymphocytic leukemia cells and between untreated cells and cells treated with kinase inhibitors.

B-cell receptor (BCR) signaling is central for the pathomechanism of chronic lymphocytic leukemia (CLL), and inhibitors of BCR signaling have substantially improved treatment options. To model malignant and nonmalignant BCR signaling, we quantified five components of BCR signaling (ZAP70/SYK, BTK, PLCγ2, AKT, ERK1/2) in single cells from primary human leukemic cells and from nonmalignant tissue. We measured signaling activity in a time-resolved manner after stimulation with BCR crosslinking by anti-IgM and/or anti-CD19 and with or without inhibition of phosphatases with H2 O2 . The phosphorylation of BCR signaling components was increased in malignant cells compared to nonmalignant cells and in IGHV unmutated CLL cells compared to IGHV mutated CLL cells. Intriguingly, inhibition of phosphatases with H2 O2 led to higher phosphorylation levels of BCR components in CLL cells with mutated IGHV compared to unmutated IGHV. We modeled the connectivity of the cascade components by correlating signal intensities across single cells. The network topology remained stable between malignant and nonmalignant cells. To additionally test for the impact of therapeutic compounds on the network topology, we challenged the BCR signaling cascade with inhibitors for BTK (ibrutinib), PI3K (idelalisib), LYN (dasatinib) and SYK (entospletinib). Idelalisib treatment resulted in similar effects in malignant and nonmalignant cells, whereas ibrutinib was mostly active on CLL cells. Idelalisib and ibrutinib had complementary effects on the BCR signaling cascade whose activity was further reduced upon dasatinib and entospletinib treatment. The characterization of the molecular circuitry of leukemic BCR signaling will allow a more refined targeting of this Achilles heel.

MODIFICATION OF THE TUMOR/INDUCED BYSTANDER EFFECT BY IRRADIATION UNDER COCULTIVATION OF LYMPHOCYTES FROM PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA AND LYMPHOCYTES FROM HEALTHY DONORS. / MODYFIKATsIIa OPROMINENNIaM PUKhLYNNO/INDUKOVANOGO EFEKTU SVIDKA PRY SUMISNO/ROZDIL'NOMU KUL'TYVUVANNI LIMFOTsYTIV KhVORYKh NA KhRONIChNU LIMFOTsYTARNU LEIKEMIIu Z LIMFOTsYTAMY ZDOROVYKh DONORIV.

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.

THE EXPRESSION OF THE MAIN AND ALTERNATIVE TRANSCRIPT (SORL1 Delta2) OF THE SORL1 GENE IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS AFFECTED BY THE CHORNOBYL ACCIDENT. / DOSLIDZhENNIa OSNOVNOGO I ALTERNATYVNOGO TRANSKRYPTU (SORL1 Delta2) GENA SORL1 U KhVORYKh NA KhRONIChNU LIMFOTsYTARNU LEAKEMIIu, POSTRAZhDALYKh VNASLIDOK ChORNOBYLSKA KATASTROFY.

OBJECTIVE: to study clinical-hematological data and expression of the main and alternative transcripts of SORL1 genein chronic lymphocytic leukemia (CLL) patients affected by the Chornobyl catastrophe. METHODS: Analysis was performed in the main group of 34 CLL patients irradiated due to the Chornobyl NPP acci-dent (30 clean-up workers, and 4 evacuees) and in the control group of 27 non-irradiated CLL patients. Groups ofpatients were comparable by age, sex, stage of disease, mutational status of IGHV genes. Expression of the main andalternative transcripts of SORL1 gene was evaluated by Quantitative Real-time polymerase chain reaction (PCR). TheIGHV gene mutational status, TP53 and SF3B1 mutations were studied by PCR followed by direct sequencing. Data wereanalyzed with the SPSS software package, version 20.0. RESULTS: Relative expression level of the main transcript of SORL1 gene was low (mean 1.71 ± 0.55, median 0.57),did not correlate with the IGHV gene mutational status, TP53 and SF3B1 mutations, stage of disease. The expressionof B transcript was not detected, F transcript was expressed at a very low level in 9 patients. The average relativeexpression level of SORL1-Δ2 transcript was 14.1 ± 6.04 (median 3.48; range 0.01-90.51). The expression of SORL1-Δ2transcript above the median was more frequent among patients on C stage (p = 0.001), and in patients with unmu-tated IGHV genes was associated with an extremely negative course of CLL (median of overall survival 9 months vs61 months at low expression). Relative expression levels of the main and alternative transcripts of SORL1 gene inpatients of the main and the control groups did not differ. CONCLUSIONS: Our preliminary data suggest that increased expression of SORL1-Δ2 transcript in CLL patients withunmutated IGHV genes can be considered as a negative prognostic marker.

Evidence for Non-Cancer-Specific T Cell Exhaustion in the Tcl1 Mouse Model for Chronic Lymphocytic Leukemia.

The reinvigoration of anti-cancer immunity by immune checkpoint therapies has greatly improved cancer treatment. In chronic lymphocytic leukemia (CLL), patients as well as in the Tcl1 mouse model for CLL, PD1-expressing, exhausted T cells significantly expand alongside CLL development; nevertheless, PD1 inhibition has no clinical benefit. Hence, exhausted T cells are either not activatable by simple PD1 blocking in CLL and/or only an insufficient number of exhausted T cells are CLL-specific. In this study, we examined the latter hypothesis by exploiting the Tcl1 transgenic CLL mouse model in combination with TCR transgene expression specific for a non-cancer antigen. Following CLL tumor development, increased PD1 levels were detected on non-CLL specific T cells that seem dependent on the presence of (tumor-) antigen-specific T cells. Transcriptome analysis confirmed a similar exhaustion phenotype of non-CLL specific and endogenous PD1pos T cells. Our results indicate that in the CLL mouse model, a substantial fraction of non-CLL specific T cells becomes exhausted during disease progression in a bystander effect. These findings have important implications for the general efficacy assessment of immune checkpoint therapies in CLL.

Physiological Fitness and the Pathophysiology of Chronic Lymphocytic Leukemia (CLL).

Chronic lymphocytic leukemia (CLL) is associated with physical dysfunction and low overall fitness that predicts poor survival following the commencement of treatment. However, it remains unknown whether higher fitness provides antioncogenic effects. We identified ten fit (CLL-FIT) and ten less fit (CLL-UNFIT) treatment-naïve CLL patients from 144 patients who completed a set of physical fitness and performance tests. Patient plasma was used to determine its effects on an in vitro 5-day growth/viability of three B-cell cell lines (OSU-CLL, Daudi, and Farage). Plasma exosomal miRNA profiles, circulating lipids, lipoproteins, inflammation levels, and immune cell phenotypes were also assessed. CLL-FIT was associated with fewer viable OSU-CLL cells at Day 1 (p = 0.003), Day 4 (p = 0.001), and Day 5 (p = 0.009). No differences between the groups were observed for Daudi and Farage cells. Of 455 distinct exosomal miRNAs identified, 32 miRNAs were significantly different between the groups. Of these, 14 miRNAs had ≤-1 or ≥1 log2 fold differences. CLL-FIT patients had five exosomal miRNAs with lower expression and nine miRNAs with higher expression. CLL-FIT patients had higher HDL cholesterol, lower inflammation, and lower levels of triglyceride components (all p < 0.05). CLL-FIT patients had lower frequencies of low-differentiated NKG2+/CD158a/bneg (p = 0.015 and p = 0.014) and higher frequencies of NKG2Aneg/CD158b+ mature NK cells (p = 0.047). The absolute number of lymphocytes, including CD19+/CD5+ CLL-cells, was similar between the groups (p = 0.359). Higher physical fitness in CLL patients is associated with altered CLL-like cell line growth in vitro and with altered circulating and cellular factors indicative of better immune functions and tumor control.

3D Bioprinting Allows the Establishment of Long-Term 3D Culture Model for Chronic Lymphocytic Leukemia Cells.

Chronic Lymphocytic Leukemia (CLL) represents the most common leukemia in the western world and remains incurable. Leukemic cells organize and interact in the lymphoid tissues, however what actually occurs in these sites has not been fully elucidated yet. Studying primary CLL cells in vitro is very challenging due to their short survival in culture and also to the fact that traditional two-dimensional in vitro models lack cellular and spatial complexity present in vivo. Based on these considerations, we exploited for the first time three-dimensional (3D) bioprinting to advance in vitro models for CLL. This technology allowed us to print CLL cells (both primary cells and cell lines) mixed with the appropriate, deeply characterized, hydrogel to generate a scaffold containing the cells, thus avoiding the direct cell seeding onto a precast 3D scaffold and paving the way to more complex models. Using this system, we were able to efficiently 3D bioprint leukemic cells and improve their viability in vitro that could be maintained up to 28 days. We monitored over time CLL cells viability, phenotype and gene expression, thus establishing a reproducible long-term 3D culture model for leukemia. Through RNA sequencing (RNAseq) analysis, we observed a consistent difference in gene expression profile between 2D and 3D samples, indicating a different behavior of the cells in the two different culture settings. In particular, we identified pathways upregulated in 3D, at both day 7 and 14, associated with immunoglobulins production, pro-inflammatory molecules expression, activation of cytokines/chemokines and cell-cell adhesion pathways, paralleled by a decreased production of proteins involved in DNA replication and cell division, suggesting a strong adaptation of the cells in the 3D culture. Thanks to this innovative approach, we developed a new tool that may help to better mimic the physiological 3D in vivo settings of leukemic cells as well as of immune cells in broader terms. This will allow for a more reliable study of the molecular and cellular interactions occurring in normal and neoplastic conditions in vivo, and could also be exploited for clinical purposes to test individual responses to different drugs.

Active Akt signaling triggers CLL toward Richter transformation via overactivation of Notch1.

Richter's transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase-associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and diffuse large B-cell lymphoma. Multiomics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed an S100 protein-defined subcluster of highly aggressive lymphoma cells that developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation toward aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.

[TIM-3/galectin-9 is involved in negative regulation of T cells in patients with chronic lymphocytic leukemia].

Objective To investigate the regulation and clinical significance of T cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3)/galectin-9 signaling pathway in regulatory T cells (Tregs) and Th17 cells in patients with chronic lymphocytic leukemia (CLL). Methods The study enrolled 36 healthy individuals and 40 newly diagnosed CLL patients. The Tregs, Th17 cells, TIM-3+ Tregs and TIM-3+ Th17 cells were detected by the flow cytometry in their peripheral blood. Concentrations of IL-10, IL-17 and galectin-9 in serum were measured by the ELISA. Results Compared with the healthy controls, the CLL group had the higher levels of TIM-3+ Tregs, Tregs/Th17 cell ratio, TIM-3+ Tregs/TIM-3+ Th17 cell ratio, IL-10, galectin-9, and IL-10/IL-17 ratio. The level of TIM-3 expression on T cells, galectin-9 and the IL-10/IL-17 ratio in the CLL patients increased with the advance of Binet stage. Conclusion The TIM-3/galectin-9 signaling pathway is involved in the negative regulation of T cells in patients with CLL.

Berberine affects mitochondrial activity and cell growth of leukemic cells from chronic lymphocytic leukemia patients.

B-cell chronic lymphocytic leukemia (CLL) results from accumulation of leukemic cells that are subject to iterative re-activation cycles and clonal expansion in lymphoid tissues. The effects of the well-tolerated alkaloid Berberine (BRB), used for treating metabolic disorders, were studied on ex-vivo leukemic cells activated in vitro by microenvironment stimuli. BRB decreased expression of survival/proliferation-associated molecules (e.g. Mcl-1/Bcl-xL) and inhibited stimulation-induced cell cycle entry, irrespective of TP53 alterations or chromosomal abnormalities. CLL cells rely on oxidative phosphorylation for their bioenergetics, particularly during the activation process. In this context, BRB triggered mitochondrial dysfunction and aberrant cellular energetic metabolism. Decreased ATP production and NADH recycling, associated with mitochondrial uncoupling, were not compensated by increased lactic fermentation. Antioxidant defenses were affected and could not correct the altered intracellular redox homeostasis. The data thus indicated that the cytotoxic/cytostatic action of BRB at 10-30 µM might be mediated, at least in part, by BRB-induced impairment of oxidative phosphorylation and the associated increment of oxidative damage, with consequent inhibition of cell activation and eventual cell death. Bioenergetics and cell survival were instead unaffected in normal B lymphocytes at the same BRB concentrations. Interestingly, BRB lowered the apoptotic threshold of ABT-199/Venetoclax, a promising BH3-mimetic whose cytotoxic activity is counteracted by high Mcl-1/Bcl-xL expression and increased mitochondrial oxidative phosphorylation. Our results indicate that, while CLL cells are in the process of building their survival and cycling armamentarium, the presence of BRB affects this process.

Ulcerated cutaneous Richter syndrome.

Richter syndrome or Richter transformation comprises the conversion of chronic lymphocytic leukemia into an aggressive type of large cell lymphoma. Classically, patients have diffuse and abrupt lymphadenopathy and organomegaly, in addition to fever, weight loss, and fatigue. Cutaneous involvement is rare and often nonspecific. We report a patient with chronic lymphocytic leukemia who presented with a large and rapidly evolving ulcer, revealed to be a high-grade cutaneous lymphoma.

BDNF belongs to the nurse-like cell secretome and supports survival of B chronic lymphocytic leukemia cells.

Evading apoptosis and sustained survival signaling pathways are two central hallmarks of B-cell chronic lymphocytic leukemia (B-CLL) cells. In this regard, nurse-like cells (NLC), the monocyte-derived type 2 macrophages, deliver stimulatory signals via B-cell activating factor (BAFF), a proliferation-inducing ligand (APRIL), and the C-X-C Motif Chemokine Ligand 12 (CXCL12). Previously, we demonstrated that brain-derived neurotrophic factor (BDNF) protects B-CLL cells from spontaneous apoptosis by activating the oncogenic complex NTSR2-TrkB (neurotensin receptor 2-tropomyosin-related kinase receptor B), only overexpressed in B-CLL cells, inducing anti-apoptotic protein Bcl-2 (B-cell lymphoma 2) expression and Src kinase survival signaling pathways. Herein, we demonstrate that BDNF belongs to the NLC secretome and promotes B-CLL survival. This was demonstrated in primary B-CLL co-cultured with their autologous NLC, compared to B-CLL cells cultured alone. Inhibition of BDNF in co-cultures, enhances B-CLL apoptosis, whereas its exogenous recombinant activates pro-survival pathways in B-CLL cultured alone (i.e. Src activation and Bcl-2 expression), at a higher level than those obtained by the exogenous recombinant cytokines BAFF, APRIL and CXCL12, the known pro-survival cytokines secreted by NLC. Together, these results showed that BDNF release from NLC trigger B-CLL survival. Blocking BDNF would support research strategies against pro-survival cytokines to limit sustained B-CLL cell survival.

Australian and New Zealand consensus statement on the management of lymphoma, chronic lymphocytic leukaemia and myeloma during the COVID-19 pandemic.

The COVID-19 pandemic poses a unique challenge to the care of patients with haematological malignancies. Viral pneumonia is known to cause disproportionately severe disease in patients with cancer, and patients with lymphoma, myeloma and chronic lymphocytic leukaemia are likely to be at particular risk of severe disease related to COVID-19. This statement has been developed by consensus among authors from Australia and New Zealand. We aim to provide supportive guidance to clinicians making individual patient decisions during the COVID-19 pandemic, in particular during periods that access to healthcare resources may be limited. General recommendations include those to minimise patient exposure to COVID-19, including the use of telehealth, avoidance of non-essential visits and minimisation of time spent by patients in infusion suites and other clinical areas. This statement also provides recommendations where appropriate in assessing indications for therapy, reducing therapy-associated immunosuppression and reducing healthcare utilisation in patients with specific haematological malignancies during the COVID-19 pandemic. Specific decisions regarding therapy of haematological malignancies will need to be individualised, based on disease risk, risks of immunosuppression, rates of community transmission of COVID-19 and available local healthcare resources.

Leptomeningeal leukaemia misdiagnosed as tubercular meningitis.

Chronic meningitis is a common syndrome with multiple aetiological causes. It can be associated with visionproblems as well as multifocal involvement of the central nervous system. Often it presents with constitutional symptoms as well. The intervention commonly practised in a tropical country like India is starting antitubercular therapy with corticosteroids. This practice though may be correct in a majority of situations, may lead to diagnostic delay and may be fatal.

[Chronic lymphocytic leukemia: update on pathophysiology and management].

Chronic lymphocytic leukemia (CLL) is the most common hematological malignancy in the Western countries. Although a multi-step model has been proposed for the pathogenesis of CLL as well as other malignancies, the precise mechanism underlying the development of CLL remains complicated and unclear. In addition, several studies have investigated adverse prognostic factors, including gene abnormalities and the evolution of clones with the disease progression. FCR (fludarabine, cyclophosphamide, and rituximab) has been the standard first-line therapy for "fit" younger patients without TP53 abnormality/17p deletion. Patients with CLL with TP53 abnormality/17p deletion exhibit poor prognosis because of the resistance to chemoimmunotherapy such as FCR. However, several novel targeted therapies such as B-cell receptor inhibitors including Bruton's tyrosine kinase inhibitors, PI3 kinase inhibitors, and Bcl-2 antagonists have been developed and have proven effective for high-risk patients with CLL with TP53 dysfunction. Moreover, in patients with CLL with the IGHV somatic mutation, FCR therapy offers long-term disease-free survival. These promising findings suggest the possibility of a cure. Although the aim of CLL treatment has been disease control, it is gradually changing to the possibility of a cure. Furthermore, the evaluation of minimal residual disease is important for the cure of CLL.

Minimal residual disease in chronic lymphocytic leukemia: A consensus paper that presents the clinical impact of the presently available laboratory approaches.

Chronic lymphocytic leukemia (CLL) is a malignancy defined by the accumulation of mature lymphocytes in the lymphoid tissues, bone marrow, and blood. Therapy for CLL is guided according to the Rai and Binet staging systems. Nevertheless, state-of-the-art protocols in disease monitoring, diagnostics, and prognostics for CLL are based on the assessment of minimal residual disease (MRD). MRD is internationally considered to be the level of disease that can be detected by sensitive techniques and represents incomplete treatment and a probability of disease relapse. MRD detection has been continuously improved by the quick development of both flow cytometry and molecular biology technology, as well as by next-generation sequencing. Considering that MRD detection is moving more and more from research to clinical practice, where it can be an independent prognostic marker, in this paper, we present the methodologies by which MRD is evaluated, from translational research to clinical practice.

Critically Elevated Potassium in a 55-Year-Old Female With Chronic Lymphocytic Leukemia.

Hyperkalemia in specimens from patients with chronic lymphocytic leukemia (CLL) may be due to tumor lysis syndrome (TLS) or specimen processing. This report describes a 55-year-old Caucasian woman with CLL who presented to an outside hospital with hyperkalemia and was transferred to a second hospital. Initial evaluation on the core laboratory chemistry analyzer (the VITROS 5600) and the ABL90 FLEX blood gas analyzer showed markedly elevated levels of potassium (K+). TLS was subsequently diagnosed, and dialysis was initiated. However, follow-up K+ measurements in whole blood (WB) yielded low levels that were unexpected after a single dialysis treatment. We then discovered that the initially elevated K+ level was from centrifuged plasma specimens and concluded that it indicated pseudohyperkalemia, likely from centrifugation. This case demonstrates that medical teams need be alert to potentially false K+ results in patients with elevated white blood cell counts. WB specimens are preferable, and steps to minimize trauma to the specimen and immediate analysis using blood gas instruments are recommended.

Utility of CD200 expression and CD20 antibody binding capacity in differentiating chronic lymphocytic leukemia from other chronic lymphoproliferative disorders.

BACKGROUND: Chronic lymphoproliferative disorders (CLPDs) are heterogeneous group of disorders with variable clinical presentations and outcomes. Therefore, accurate classification is crucial for treatment planning. At present, flow cytometry immunophenotyping (FCM-IPT) is a useful tool for diagnosing these diseases. However, overlapping immunophenotypes do exist. Recently, differential expression of CD200 and variation in number of CD20 antibody bound per cell (ABC) in different CLPDs has been reported. MATERIALS AND METHODS: Seventy-seven CLPD cases were analyzed by FCM-IPT for CD200 expression, and Quantibrite bead was used to calculate CD20 ABC. RESULTS: Variability in CD200 expression can help in the differentiation of chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL) from other CLPDs. CD200 was brightly expressed in 100% CLL cases, having homogenous bright (2+) intensity. On the contrary, CD200 was uniformly negative in all Mantle cell lymphoma cases except 1, in which the intensity was dim, and the mean fluorescence intensity was significantly lower than CLL. Furthermore, all HCL cases showed bright expression of CD200, thereby making it useful in differentiation from other CLPD with villous lymphocytes. Evaluation of CD20 ABC showed that it differs among various CLPD and was significantly lowest in CLL and highest in HCL both on peripheral blood and bone marrow samples. CONCLUSION: Our results support the fact that CD200 can be added to routine CLPD panel as it is useful in subcategorizing them. However, inclusion of CD20 ABC to routine panel does not seem plausible but may be done for difficult diagnostic cases or where anti-CD20 therapy is planned.