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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.)

Prognostication in patients with chronic lymphocytic leukemia (CLL) is challenging due to heterogeneity in clinical course. We hypothesize that constitutional genetic variation affects disease progression and could aid prognostication. Pooling data from seven studies incorporating 842 cases identifies two genomic locations associated with time from diagnosis to treatment, including 10q26.13 (rs736456, hazard ratio (HR) = 1.78, 95% confidence interval (CI) = 1.47-2.15; P = 2.71 × 10 -9 ) and 6p (rs3778076, HR = 1.99, 95% CI = 1.55-2.55; P = 5.08 × 10 -8 ), which are particularly powerful prognostic markers in patients with early stage CLL otherwise characterized by low-risk features. Expression quantitative trait loci analysis identifies putative functional genes implicated in modulating B-cell receptor or innate immune responses, key pathways in CLL pathogenesis. In this work we identify rs736456 and rs3778076 as prognostic in CLL, demonstrating that disease progression is determined by constitutional genetic variation as well as known somatic drivers.

We report a cell recognition chromatography approach for blood cancer cell separation from healthy peripheral blood mononuclear cells (PBMCs) based on size-matched functionalized particle imprints. Negative imprints were prepared from layers of 15 μm polymeric microbeads closely matching the size of cultured human leukemic cells (HL60). We replicated these imprints on a large scale with UV curable polyurethane resin using nanoimprinting lithography. The imprints were functionalized with branched polyethylene imine (bPEI) and passivated by Poloxamer 407 to promote a weak attraction toward cells. When a matching cell fits into an imprint cavity, its contact area with the imprint is maximized, which amplifies the attraction and the binding selectivity. We tested these imprints specificity for depleting myeloblasts from a mixture with healthy human PBMCs in a cell recognition chromatography setup hosting the imprint. The mixture of fixed HL60/PBMCs ratio was circulated over the imprint and at each step the selectivity toward HL60 was assessed by flow cytometry. The role of the imprint length, flow rate, channel depth, and the bPEI coating concentration were examined. The results show that HL60 cells, closely matching the imprint cavities, get trapped on the imprint, while the smaller PBMCs are carried away by the drag force of the flow. Lower flow rates, longer imprints, and interim channel depth favor HL60 specific retention. The bPEI concentration higher than 1 wt % on the imprint made it less selective toward the HL60 because of indiscriminate attraction with all cells. Particle imprint based cell recognition chromatography was able to achieve selective myeloblast depletion from initial 11.7% HL60 (88.3% PBMC) to less than 1.3% HL60 for 3 h of circulation. The cell recognition chromatography with size-matched microbead imprints can be employed as an efficient cell separation technique and potentially lead to alternative therapies for myeloblasts removal from peripheral blood of patients with acute myeloid leukemia.

The clustering of different types of B-cell malignancies in families raises the possibility of shared aetiology. To examine this, we performed cross-trait linkage disequilibrium (LD)-score regression of multiple myeloma (MM) and chronic lymphocytic leukaemia (CLL) genome-wide association study (GWAS) data sets, totalling 11,734 cases and 29,468 controls. A significant genetic correlation between these two B-cell malignancies was shown (R g  = 0.4, P = 0.0046). Furthermore, four of the 45 known CLL risk loci were shown to associate with MM risk and five of the 23 known MM risk loci associate with CLL risk. By integrating eQTL, Hi-C and ChIP-seq data, we show that these pleiotropic risk loci are enriched for B-cell regulatory elements and implicate B-cell developmental genes. These data identify shared biological pathways influencing the development of CLL and, MM and further our understanding of the aetiological basis of these B-cell malignancies.

ARCTIC was a multicenter, randomized-controlled, open, phase IIB non-inferiority trial in previously untreated chronic lymphocytic leukemia (CLL). Conventional frontline therapy in fit patients is fludarabine, cyclophosphamide and rituximab (FCR). The trial hypothesized that including mitoxantrone with low-dose rituximab (FCM-miniR) would be non-inferior to FCR. A total of 200 patients were recruited to assess the primary end point of complete remission (CR) rates according to IWCLL criteria. Secondary end points were progression-free survival (PFS), overall survival (OS), overall response rate, minimal residual disease (MRD) negativity, safety and cost-effectiveness. The trial closed following a pre-planned interim analysis. At final analysis, CR rates were 76 FCR vs 55% FCM-miniR (adjusted odds ratio: 0.37; 95% confidence interval: 0.19-0.73). MRD-negativity rates were 54 FCR vs 44% FCM-miniR. More participants experienced serious adverse reactions with FCM-miniR (49%) compared to FCR (41%). There are no significant differences between the treatment groups for PFS and OS. FCM-miniR is not expected to be cost-effective over a lifetime horizon. In summary, FCM-miniR is less well tolerated than FCR with an inferior response and MRD-negativity rate and increased toxicity, and will not be taken forward into a confirmatory trial. The trial demonstrated that oral FCR yields high response rates compared to historical series with intravenous chemotherapy.

Cancer incidence and mortality have both increased in the last decade and are predicted to continue to rise. Diagnosis and treatment of cancers are often hampered by the inability to specifically target neoplastic cells. Bioimprinting is a promising new approach to overcome shortfalls in cancer targeting. Highly specific recognition cavities can be made into polymer matrices to mimic lock-and-key actions seen in in vivo biological systems. Early studies concentrated on molecules and were inhibited by template size complexity. Surface imprinting allows the capture of increasingly complex motifs from polypeptides to single cell organisms and mammalian cells. Highly specific cell shape recognition can also be achieved by cell interaction with imprints that can be made into polymer matrices to mimic biological systems at a molecular level. Bioimprinting has also been used to achieve nanometre scale resolution imaging of cancer cells. Studies of bioimprint-based drug delivery on cancer cells have been recently trialled in vitro and show that this approach can potentially improve existing chemotherapeutic approaches. This review focuses on the possible applications of bioimprinting with particular regards to cancer understanding, diagnosis and therapy. Cell imprints, incorporated into biosensors can allow the limits of detection to be improved or negate the need for extensive patient sample processing. Similar cell imprinting platforms can be used for nanoscale imaging of cancer morphology, as well as to investigate topographical signalling of cancer cells in vitro. Lastly, bioimprints also have applications as selective drug delivery vehicles to tumours with the potential to decrease chemotherapy-related side effects.

Several chronic lymphocytic leukaemia (CLL) susceptibility loci have been reported; however, much of the heritable risk remains unidentified. Here we perform a meta-analysis of six genome-wide association studies, imputed using a merged reference panel of 1,000 Genomes and UK10K data, totalling 6,200 cases and 17,598 controls after replication. We identify nine risk loci at 1p36.11 (rs34676223, P=5.04 × 10 -13 ), 1q42.13 (rs41271473, P=1.06 × 10 -10 ), 4q24 (rs71597109, P=1.37 × 10 -10 ), 4q35.1 (rs57214277, P=3.69 × 10 -8 ), 6p21.31 (rs3800461, P=1.97 × 10 -8 ), 11q23.2 (rs61904987, P=2.64 × 10 -11 ), 18q21.1 (rs1036935, P=3.27 × 10 -8 ), 19p13.3 (rs7254272, P=4.67 × 10 -8 ) and 22q13.33 (rs140522, P=2.70 × 10 -9 ). These new and established risk loci map to areas of active chromatin and show an over-representation of transcription factor binding for the key determinants of B-cell development and immune response., Competing Interests: The authors declare no competing financial interests.

B-cell malignancies (BCM) originate from the same cell of origin, but at different maturation stages and have distinct clinical phenotypes. Although genetic risk variants for individual BCMs have been identified, an agnostic, genome-wide search for shared genetic susceptibility has not been performed. We explored genome-wide association studies of chronic lymphocytic leukaemia (CLL, N = 1,842), Hodgkin lymphoma (HL, N = 1,465) and multiple myeloma (MM, N = 3,790). We identified a novel pleiotropic risk locus at 3q22.2 (NCK1, rs11715604, P = 1.60 × 10 -9 ) with opposing effects between CLL (P = 1.97 × 10 -8 ) and HL (P = 3.31 × 10 -3 ). Eight established non-HLA risk loci showed pleiotropic associations. Within the HLA region, Ser37 + Phe37 in HLA-DRB1 (P = 1.84 × 10 -12 ) was associated with increased CLL and HL risk (P = 4.68 × 10 -12 ), and reduced MM risk (P = 1.12 × 10 -2 ), and Gly70 in HLA-DQB1 (P = 3.15 × 10 -10 ) showed opposing effects between CLL (P = 3.52 × 10 -3 ) and HL (P = 3.41 × 10 -9 ). By integrating eQTL, Hi-C and ChIP-seq data, we show that the pleiotropic risk loci are enriched for B-cell regulatory elements, as well as an over-representation of binding of key B-cell transcription factors. These data identify shared biological pathways influencing the development of CLL, HL and MM. The identification of these risk loci furthers our understanding of the aetiological basis of BCMs.

Defining the prognosis of individual cancer sufferers remains a significant clinical challenge. Here we assessed the ability of high-resolution single telomere length analysis (STELA), combined with an experimentally derived definition of telomere dysfunction, to predict the clinical outcome of patients with chronic lymphocytic leukaemia (CLL). We defined the upper telomere length threshold at which telomere fusions occur and then used the mean of the telomere 'fusogenic' range as a prognostic tool. Patients with telomeres within the fusogenic range had a significantly shorter overall survival (P < 0·0001; Hazard ratio [HR] = 13·2, 95% confidence interval [CI] = 11·6-106·4) and this was preserved in early-stage disease patients (P < 0·0001, HR=19·3, 95% CI = 17·8-802·5). Indeed, our assay allowed the accurate stratification of Binet stage A patients into those with indolent disease (91% survival at 10 years) and those with poor prognosis (13% survival at 10 years). Furthermore, patients with telomeres above the fusogenic mean showed superior prognosis regardless of their IGHV mutation status or cytogenetic risk group. In keeping with this finding, telomere dysfunction was the dominant variable in multivariate analysis. Taken together, this study provides compelling evidence for the use of high-resolution telomere length analysis coupled with a definition of telomere dysfunction in the prognostic assessment of CLL., (© 2014 John Wiley & Sons Ltd.)

Genome-wide association studies (GWAS) of chronic lymphocytic leukemia (CLL) have shown that common genetic variation contributes to the heritable risk of CLL. To identify additional CLL susceptibility loci, we conducted a GWAS and performed a meta-analysis with a published GWAS totaling 1,739 individuals with CLL (cases) and 5,199 controls with validation in an additional 1,144 cases and 3,151 controls. A combined analysis identified new susceptibility loci mapping to 3q26.2 (rs10936599, P = 1.74 × 10(-9)), 4q26 (rs6858698, P = 3.07 × 10(-9)), 6q25.2 (IPCEF1, rs2236256, P = 1.50 × 10(-10)) and 7q31.33 (POT1, rs17246404, P = 3.40 × 10(-8)). Additionally, we identified a promising association at 5p15.33 (CLPTM1L, rs31490, P = 1.72 × 10(-7)) and validated recently reported putative associations at 5p15.33 (TERT, rs10069690, P = 1.12 × 10(-10)) and 8q22.3 (rs2511714, P = 2.90 × 10(-9)). These findings provide further insights into the genetic and biological basis of inherited genetic susceptibility to CLL.

Type 2N Von Willebrand's disease (2N VWD) is a rare, recessively inherited bleeding disorder, comprising 1-2% of all VWD patients, usually manifesting as a mild bleeding diathesis. Treatment includes desmopressin (DDAVP) or intermediate purity plasma-derived FVIII concentrates containing residual VWF. We present a case of a 75-year-old gentleman, incidentally diagnosed with type 2N VWD in 2002 on routine blood testing before surgery with an ISTH bleeding score of 1-2, who was treated with recombinant FVIII preoperatively.

Venous thromboembolism (VTE) is a major cause of morbidity and mortality in cancer patients. The literature is sparse on the incidence in the most common lymphoid malignancy, chronic lymphocytic leukaemia (CLL). We calculated the incidence rates for VTE in an unselected UK CLL clinic population at 1.45% per patient year. This represents a tenfold increase over previously published estimates of incidence in the general population and a twofold increase over that of the local hospital inpatient population. In our cohort, the risk of VTE was related to stage C disease. Clinicians should be aware that CLL patients are at risk of VTE., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

To identify new risk variants for chronic lymphocytic leukemia (CLL), we conducted a genome-wide association study of 299,983 tagging SNPs, with validation in four additional series totaling 2,503 cases and 5,789 controls. We identified four new risk loci for CLL at 2q37.3 (rs757978, FARP2; odds ratio (OR) = 1.39; P = 2.11 x 10(-9)), 8q24.21 (rs2456449; OR = 1.26; P = 7.84 x 10(-10)), 15q21.3 (rs7169431; OR = 1.36; P = 4.74 x 10(-7)) and 16q24.1 (rs305061; OR = 1.22; P = 3.60 x 10(-7)). We also found evidence for risk loci at 15q25.2 (rs783540, CPEB1; OR = 1.18; P = 3.67 x 10(-6)) and 18q21.1 (rs1036935; OR = 1.22; P = 2.28 x 10(-6)). These data provide further evidence for genetic susceptibility to this B-cell hematological malignancy.

We conducted a genome-wide association study of 299,983 tagging SNPs for chronic lymphocytic leukemia (CLL) and performed validation in two additional series totaling 1,529 cases and 3,115 controls. We identified six previously unreported CLL risk loci at 2q13 (rs17483466; P = 2.36 x 10(-10)), 2q37.1 (rs13397985, SP140; P = 5.40 x 10(-10)), 6p25.3 (rs872071, IRF4; P = 1.91 x 10(-20)), 11q24.1 (rs735665; P = 3.78 x 10(-12)), 15q23 (rs7176508; P = 4.54 x 10(-12)) and 19q13.32 (rs11083846, PRKD2; P = 3.96 x 10(-9)). These data provide the first evidence for the existence of common, low-penetrance susceptibility to a hematological malignancy and new insights into disease causation in CLL.

Bone marrow aspiration and biopsy is a procedure of central importance in the diagnosis of haematological disease and is one that can be safely performed in the outpatient clinic using only local anaesthesia. We evaluated the role of inhaled nitrous oxide (Entonox) as an adjuvant to local anaesthesia (LA) in patients requiring bone examination. We prospectively studied the role of Entonox in patients attending our outpatient department who required bone marrow examination. All patients received LA with injected 2% lignocaine but patients were then assigned to either LA alone or LA plus inhaled Entonox. Following the procedure patients were given a questionnaire and asked to score the discomfort associated with the procedure. Twenty-eight of 85 patients (33%) who received LA alone scored their pain as 3 (severe) compared with seven of 51 patients (14%) who received LA plus Entonox, P = 0.013. No adverse events were associated with Entonox use. Forty-three of the 51 patients who received Entonox stated that they would prefer to use this method of anaesthesia again. We conclude that inhaled Entonox is a useful adjuvant to local anaesthesia for patients undergoing bone marrow examination.

Hairy cells (HCs) are mature malignant B cells that contain a number of constitutively active signaling molecules including GTP-bound Rac1, protein kinase C, and Src family kinases. Because Rac1 is a component of the reactive oxidant species (ROS)-generating NADPH oxidase system, we investigated the role of this GTPase in ROS production in HCs. In this study, we show that ROS production in HCs involves a flavin-containing oxidase dependent on Ca2+, but not on GTP-Rac1 or protein kinase C. This suggests the involvement of the nonphagocytic NADPH oxidase NOX5, an enzyme found in lymphoid tissues, but not in circulating lymphocytes. By using RT-PCR and Southern and Western blotting and by measuring superoxide anion production in membrane fractions in the absence of cytosolic components, we demonstrate for the first time that HCs (but not circulating normal B cells or some other lymphoid cell types) express NOX5. We also demonstrate that inhibition of NADPH oxidase in HCs results in a selective increase in the activity of Src homology region 2 domain-containing phosphatase 1 (SHP-1). Furthermore, SHP-1 in HCs coimmunoprecipitates with tyrosine phosphorylated CD22 and localizes in the same cellular compartment as NOX5. This allows the inactivation of SHP-1 by NOX5-generated ROS and contributes to the maintenance of the constitutive activation of HCs.

Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which interaction of the malignant cells with antigen is thought to play a key role. Individual CLL-cell clones markedly differ in their ability to respond to B-cell receptor ligation, but the mechanism underlying the frequent hyporesponsiveness is incompletely understood. Our aim was to further clarify the extent and cause of the B-cell receptor signaling abnormality in CLL and to assign pathophysiologic relevance to the presence or absence of B-cell receptor responsiveness. We show that extracellular signal-regulated kinase-2 phosphorylation, intracellular Ca2+ increases, CD79a phosphorylation, and translocation of the B-cell receptor to lipid rafts in response to ligation with anti-immunoglobulin M (as a surrogate for antigen) are features of CLL cells with relatively unmutated VH genes (<5% deviation from germ line) and a poor prognosis. B-cell receptor stimulation in these cases also promoted cell survival. In clones with mutated VH genes (>5% deviation from germ line), surface immunoglobulin M ligation failed to induce receptor translocation to rafts or to prolong cell survival. This failure of receptor translocation observed in mutated CLL cells was associated with the constitutive exclusion of the B-cell receptor from rafts by a mechanism involving src-dependent interactions between the B-cell receptor and the actin cytoskeleton. We conclude that exposure to antigen promotes the survival of unmutated CLL clones, contributing to the poor prognosis of this group. In contrast, hyporesponsive mutated CLL clones may have developed into a stage where continuous exposure to antigen results in relative tolerance to antigenic stimulation mediated by the exclusion of the B-cell receptor from lipid rafts.

Hairy-cell leukaemia (HCL) is usually readily diagnosed by seeing typical hairy cells (HCs) in the blood film. The diagnosis is then confirmed by tartrate-resistant acid phosphatase staining, marker analysis, and bone marrow examination. HCs are clonal mature memory B cells with specific features of activation. This HC activation is responsible for many of the pathological features of the disease, including its distinctive bone marrow fibrosis, splenic red pulp invasion, and pseudo-sinus formation. Chlorodeoxyadenasine is the treatment of first choice. Deoxycoformycin and rituximab are useful for the treatment of relapsed/refractory disease. The nature of the primary oncogenic event(s) remains unknown and is the major unresolved issue in HCL.

The diagnosis of HCL is usually straightforward and is based on the identification of typical HCs in the blood and bone marrow. The suspected diagnosis is confirmed by a combination of TRAP cytochemistry, a distinctive immunophenotype and characteristic BM trephine appearances. Nucleoside treatment is highly effective in inducing prolonged remissions; relapsing patients can usually be successfully retreated with nucleoside. Monoclonal antibody therapy is a promising novel approach to the treatment of resistant disease.

The intermittent pneumatic compression device is a relatively new treatment for patients with venous ulcers. With the advent of this therapy being used by the patient in the home setting, the nurse is the primary point of contact for patient questions, concerns, and patient education. Nurses need to know the optimal compression pressure, inflation time, and sequencing time cycles to advise patients in the proper use of this therapy and how to screen patients for its safe use. The major contraindication for this therapy is the presence of deep venous thrombi. A review of the literature is presented, concluding with a recommended scientific basis for optimal compression pressure, inflation time, and sequencing time cycle pattern for the intermittent pneumatic sequential compression device in the venous ulcer patient population. Patient education strategies and topics are discussed.

Vascular endothelial cells from different parts of the circulation are known to show different functional responses, presumably corresponding to physiological roles. Previous studies have shown that ATP acts on P2 purinergic receptors of endothelial cells of major blood vessels, stimulating the formation of inositol phosphates. Here we have compared the action of ATP and congeners acting on endothelial cells of bovine thoracic aorta with cells derived from the microvasculature of bovine adrenal medulla. With measurement of total inositol phosphates, cells from the aorta showed a rank order of agonist potency of 2-methylthio-ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) greater than ADP greater than ATP greater than beta, gamma-imido-ATP greater than beta, gamma-methylene-ATP, consistent with action at receptors of the P2Y subtype. However, with adrenal cells the rank order of potency was ATP gamma S greater than ATP greater than beta, gamma-imido-ATP greater than ADP greater than beta, gamma-methylene-ATP = 2-methylthio-ATP. This profile is not consistent with either P2X or P2Y receptors. When the nature of this inositol phosphate response was analyzed with anion exchange chromatography, it was found that the aortic cells showed an inositol trisphosphate stimulation that peaked within a few seconds and rapidly declined, whereas the response of the adrenal medulla cells continued to rise through 5 min. Analysis of isomers of inositol phosphates revealed a different pattern of metabolism between the two cell types, which may account for the different time course of response. With adrenal cells, ATP at low micromolar concentrations caused a dose-dependent increase in levels of cyclic AMP and had a greater than additive effect on cyclic AMP levels when combined with submaximal stimulation by prostaglandin E2. These results suggest the presence of a P2Y receptor on aortic endothelial cells, with an 'atypical' purinocepter, i.e., neither P2X nor P2Y, on adrenal cells. Furthermore, they show that activation of P2 receptors on the two cell types has different functional consequences.