Cell Lymphocytosis Research Articles

CD20+ T cells in monoclonal B cell lymphocytosis and chronic lymphocytic leukemia: frequency, phenotype and association with disease progression.

In monoclonal B cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL), the expansion of malignant B cells disrupts the normal homeostasis and interactions between B cells and T cells, leading to immune dysregulation. CD20+ T cells are a subpopulation of T cells that appear to be involved in autoimmune diseases and cancer. Here, we quantified and phenotypically characterized CD20+ T cells from MBL subjects and CLL patients using flow cytometry and correlated our findings with the B-cell receptor mutational status and other features of the disease. CD20+ T cells were more represented within the CD8+ T cell compartment and they showed a predominant memory Tc1 phenotype. CD20+ T cells were less represented in MBL and CLL patients vs healthy controls, particularly among those with unmutated IGVH gene. The expansion of malignant B cells was accompanied by phenotypic and functional changes in CD20+ T cells, including an increase in follicular helper CD4+ CD20+ T cells and CD20+ Tc1 cells, in addition to the expansion of the TCR Vβ 5.1 in CD4+ CD20+ T cells in CLL.

TOX as a new diagnostic marker for T cell large granular lymphocytic leukaemia.

T cell large granular lymphocytic leukaemia (T-LGLL) is a rare disorder that may underlie otherwise unexplained cytopenias. The identification of T-LGLL cells in bone marrow biopsies can be a challenge, because a robust immunohistochemistry marker is lacking. The markers currently in use (granzyme B, TIA-1 and CD8) are difficult to interpret or lack specificity. Therefore, we investigated whether immunohistochemistry for thymocyte selection-associated high-mobility group box (TOX), a transcription factor that associates with chronic T cell stimulation, could be a reliable tool for the identification of T-LGLL cells. In this retrospective study, expression of TOX in CD8+ cells in bone marrow biopsies of T-LGLL patients (n = 38) was investigated and compared to bone marrow of controls with reactive T cell lymphocytosis (n = 10). All biopsies were evaluated for TOX staining within the CD8-positive T cell population. The controls were essentially negative for TOX, whereas all T-LGLL cases were positive (median = 80%, range = 10-100%), even when bone marrow involvement was subtle. TOX is a highly sensitive marker for the neoplastic cells of T-LGLL and we recommend its use, especially in the diagnostic work-up of patients with unexplained cytopenias.

Necrotic Ulcers from Occlusive Vasculopathy Associated with Monoclonal B Cell Lymphocytosis.

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Zeldzame neutrofiele dermatose met belangrijke hematologische implicaties

Rare neutrophilic dermatosis with significant hematologic implications A patient was seen in the dermatology department because of a solitary lesion on the right forearm. This lesion had an annular aspect with a raised, bright erythematous, inflammatory border of confluent papules with an oedematous aspect. Previously, similar lesions had developed, regressing spontaneously after a few weeks. Histological examination showed a marked oedema and a dens dermal predominant neutrophilic infiltrate with leucocytoclasia without vasculitis. Based on the clinical picture, the chronic recurrent course and the histology, the diagnosis of chronic recurrent annular neutrophilic dermatosis (CRAND) was made. Since CRAND belongs to the neutrophilic skin disorders, a comprehensive blood test was performed, showing a monoclonal B cell population. In consultation with a fellow haematologist, additional CT scans of the thorax and abdomen were performed. These showed neither adenopathies nor hepatosplenomegaly, upon which the diagnosis of monoclonal B cell lymphocytosis was made. This condition increases the risk of developing chronic lymphatic leukaemia. This case report thus shows a rare neutrophilic dermatosis that differs from Sweet syndrome given the absence of fever, systemic symptoms or neutrophilia in the blood. This case is specifically relevant because of the additional finding of a monoclonal B cell lymphocytosis, increasing the risk of developing chronic lymphatic leukaemia. This demonstrates the importance of further additional investigation to exclude possible (pre)malignancies when the diagnosis of CRAND is made.

Subcutaneous panniculitis-like T-cell lymphoma in two unrelated individuals with BENTA disease

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare primary cutaneous non-Hodgkin lymphoma involving CD8+ T cells, the genetic underpinnings of which remain incompletely understood. Here we report two unrelated patients with B cell Expansion with NF-κB and T cell Anergy (BENTA) disease and a novel presentation of SPTCL. Patient 1 presented early in life with recurrent infections and B cell lymphocytosis, linked to a novel gain-of-function (GOF) CARD11 mutation (p.Lys238del). He developed SPTCL-like lesions and membranoproliferative glomerulonephritis by age 2, treated successfully with cyclosporine. Patient 2 presented at 13 months with splenomegaly, lymphadenopathy, and SPTCL with evidence of hemophagocytic lymphohistiocytosis. Genetic analysis revealed two in cis germline GOF CARD11 variants (p.Glu121Asp/p.Gly126Ser). Autologous bone marrow transplant resulted in SPTCL remission despite persistent B cell lymphocytosis. These cases illuminate an unusual pathological manifestation for BENTA disease, suggesting that CARD11 GOF mutations can manifest in cutaneous CD4+and CD8+ T cell malignancies.

B Cell Lymphocytosis in Juvenile Dermatomyositis.

In this study, we determined if B lymphocytosis may serve as a JDM biomarker for disease activity. Children with untreated JDM were divided into two groups based on age-adjusted B cell percentage (determined through flow cytometry): 90 JDM in the normal B cell group and 45 in the high B cell group. We compared through T-testing the age, sex, ethnicity, duration of untreated disease (DUD), disease activity scores for skin (sDAS), muscle (mDAS), total (tDAS), CMAS, and neopterin between these two groups. The patients in the high B cell group had a higher tDAS (p = 0.009), mDAS (p = 0.021), and neopterin (p = 0.0365). Secondary analyses included B cell values over time and BAFF levels in matched patients with JM (juvenile myositis) and concurrent interstitial lung disease (ILD); JM alone and healthy controls Patient B cell percentage and number was significantly higher after 3-6 months of therapy and then significantly lower on completion of therapy (p =< 0.0001). The JM groups had higher BAFF levels than controls 1304 vs. 692 ng/mL (p = 0.0124). This study supports B cell lymphocytosis as a JDM disease-activity biomarker and bolsters the basis for B cell-directed therapies in JDM.

Elevated expression of interleukin 16 in chronic lymphocytic leukemia is associated with disease burden and abnormal immune microenvironment

Interleukin-16 (IL-16) is a novel biomarker that has been implicated in many cancers as well as inflammatory diseases. In this study, we examined plasma levels of 30 cytokines and chemokines in chronic lymphocytic leukemia (CLL) and monoclonal B cell lymphocytosis (MBL) patients, and examined their association with disease stage, CLL biomarkers and T cell subsets. Interleukin 16 (IL-16) was identified as a relatively uncharacterized cytokine significantly elevated in CLL patients compared to healthy controls and MBL patients. Plasma levels of IL-16 were significantly elevated by Rai stage 0, increased by Rai stage 3–4, correlated strongly with lymphocyte count and were decreased after Ibrutinib treatment. CLL cells expressed IL-16 mRNA and spontaneously secreted IL-16 in vitro. CLL cells express IL-16 mRNA at significantly higher levels in lymphoid tissues than blood, and we observed that IL-16 release was increased in co-cultures of CLL and autologous CD4 + T cells. Elevated plasma IL-16 levels were associated with abnormalities in the immune microenvironment including multiple inflammatory cytokines and chemokines and expansion of type 1 follicular helper T cells. Taken together, our results identify IL-16 as a novel biomarker in CLL with potential functional roles in cellular interactions between CLL cells and T cells.

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

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

A heterozygous CARD11 variant in a patient with infantile atopic disease, humoral deficiency and lymphocytosis

The etiology of a vast majority of allergic diseases is presumed to be multifactorial, with environmental and nutritional exposures playing a causal role in polygenically susceptible individuals. The expanding availability of sequencing has allowed the identification of monogenic causes in patients presenting with severe atopic disease with or without other co-morbidities. Here, we describe a case of severe atopic dermatitis and hypogammaglobinemia associated with a CARD11 loss-of-function. Patient is an otherwise healthy 7-month male who presented with severe atopic dermatitis and seborrheic dermatitis. Eczematous lesions developed at 3 months of age and progressed despite standard treatment with topical steroids. There was no history of immunodeficiency or atopy in the family. Physical exam was notable for extensive scaly and xerotic plaques with lichenification. Workup revealed hypereosinophilia, high IgE (179 kIU/L), normal IgA (10 mg/dL), low IgM (6 mg/dL) and IgG (41 mg/dL). Lymphocyte analysis was significant for mild B-cell lymphocytosis (1,063 cells/cm2) with appropriate subsets. Patient was initiated on subcutaneous replacement immunoglobulin. Whole exome trio sequencing identified a de novo CARD11 variant (c.221G>A, p.G74D). Confirmatory testing in a CARD11 deficient Jurkat T-cell line demonstrated loss-of-function and dominant interference of wild-type CARD11 signaling.CARD11 encodes a membrane-associated protein that partners with BCL10 and MALT1 to form the CBM complex responsible for integrating antigenreceptor engagement with downstream activation of NF-κB. Recently, heterozygous hypomorphic mutations in CARD11 have been discovered in patients with severe atopic manifestations, otherwise known as CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS). Our case highlights the need for genetic testing in the evaluation of patients with severe and/or unusual atopy. We discovered a novel CADINS variant (p.G74D) in an infant presenting with extensive atopic dermatitis, seborrheic dermatitis, and hypogammaglobulinemia. Curiously, our patient also developed B cell lymphocytosis in the first year of life which is typically associated with BENTA disease. However, no gain-of-function activity was detected on in vitro testing. Further studies are needed to elucidate if modulation of CARD11 signaling by this variant can explain both gain-of-function and loss-of-function phenotypic features, as noted in prior cases.

Novel Presentation of a Mosaic STAT5B Gain-of-Function Variant

Somatic gain-of-function (GOF) variants in STAT5B have been described in leukemias and lymphomas, and more recently in three patients presenting with nonclonal eosinophilia, atopic dermatitis, diarrhea, and urticaria. Here, we describe a 7-year-old middle-Eastern female presenting with autoimmune adrenal insufficiency and lower extremity weakness. Diagnostic studies revealed CNS demyelination and demyelinating polyneuropathy of the motor nerves. CBC was notable for leukocytosis with eosinophilia, with peak AEC at 3100 cells/cumm. Immunophenotyping demonstrated normal IgG and subclasses and IgA, elevated IgM (233.1 mg/dL), and mildly elevated IgE (135 IU/ml). T cell lymphocytosis was noted on flow cytometry. Extensive infectious and autoantibody testing was unremarkable, and flow cytometry and cytology from CSF, blood, and bone marrow were not concerning for malignancy. She was treated with IV methylprednisolone and steroid taper, followed by rituximab and IVIG. Additional imaging demonstrated an enlarged thymus thought to represent rebound thymic hyperplasia, and splenomegaly. Trio whole exome sequencing performed on a buccal sample was notable for a de novo STAT5B c.1883C>G (p.Thr628Ser) variant present in 13/111 (–12%) of sequencing reads. This STAT5B variant has been reported in T cell prolymphocytic leukemia and studies have shown this variant confers GOF. Clinical evaluation for the STAT5B variant detected mosaicism in buccal swab DNA and near 50% variant allele frequency (VAF) in blood and bone marrow. We then assessed the VAF in various cells on a research basis. The VAF in buccal DNA was 7–9%, 46–48% in whole blood, and 48% in the bone marrow. Cell sorting for CD4+ and CD8+ T cells revealed a VAF of 46–48%. A similar VAF was observed in CD56+ cells (48%) and CD14+ cells (–50%). A fibroblast line was generated from a skin punch biopsy, and the variant allele was absent, suggesting somatic origin. Preliminary data from peripheral blood flow cytometric analysis demonstrated a slight increase in Treg cells, and Th2 skewing of Tregs as implied by cell surface markers. Assessment of pSTAT5 at baseline and after cytokine stimulation suggest increased responsiveness. The patient was started on tofacitinib, a JAK inhibitor primarily targeting JAK1/JAK3. She improved clinically, weaned on steroids, and is being evaluated for HSCT.

Bi-allelic mutation of CARD11 resulting in fatal immune dysregulation with dramatic B cell expansion

Abstract Caspase recruitment domain family member 11 (CARD11) functions as a scaffold to bridge antigen receptors with downstream immune signaling pathways that enable antigen-dependent lymphocyte activation. Given its pleiotropic role in regulating adaptive immunity, different genetic variants in CARD11 can result in diverse phenotypic consequences in humans. Here we describe a patient harboring a novel bi-allelic CARD11 mutation that results in potentially fatal immune dysregulation with massive B cell expansion. He presented with splenomegaly, amenia, leukocytosis and thrombocytopenia within the first year of life. A sibling showed similar symptoms before passing away in infancy. Due to parental consanguinity, an autosomal recessive inborn error of immunity was suspected; whole exome sequencing revealed a homozygous missense mutation in the coiled-coil (CC) domain of CARD11 (p.Arg331Pro, R331P). Functional testing revealed the R331P mutation caused a mild gain-of-function effect relative to wild-type CARD11 in driving constitutive NF-kB activation, suggesting that R331P may disrupt interactions needed to maintain CARD11 autoinhibition in resting lymphocytes. Dramatic B cell lymphocytosis noted in the patient was not present in his heterozygous parents, indicating that 2 mutant alleles are required for this phenotype. In contrast, the patient’s T cells responded poorly to TCR stimulation with severely reduced proliferation. Collectively, these results indicate that the novel R331P CARD11 mutation manifests as the first known recessive form of B cell Expansion with NF-kB and T cell Anergy (BENTA disease), and illustrate how rare monogenic inborn errors of immunity provide a unique discovery platform for basic immunology.

Bronchoalveolar Lavage Cell Count and Lymphocytosis Are the Important Discriminators between Fibrotic Hypersensitivity Pneumonitis and Idiopathic Pulmonary Fibrosis

Fibrotic hypersensitivity pneumonitis (fHP) shares many features with other fibrotic interstitial lung diseases (ILD), and as a result it can be misdiagnosed as idiopathic pulmonary fibrosis (IPF). We aimed to determine the value of bronchoalveolar lavage (BAL) total cell count (TCC) and lymphocytosis in distinguishing fHP and IPF and to evaluate the best cut-off points discriminating these two fibrotic ILD. A retrospective cohort study of fHP and IPF patients diagnosed between 2005 and 2018 was conducted. Logistic regression was used to evaluate the diagnostic utility of clinical parameters in differentiating between fHP and IPF. Based on the ROC analysis, BAL parameters were evaluated for their diagnostic performance, and optimal diagnostic cut-offs were established. A total of 136 patients (65 fHP and 71 IPF) were included (mean age 54.97 ± 10.87 vs. 64.00 ± 7.18 years, respectively). BAL TCC and the percentage of lymphocytes were significantly higher in fHP compared to IPF (p < 0.001). BAL lymphocytosis >30% was found in 60% of fHP patients and none of the patients with IPF. The logistic regression revealed that younger age, never smoker status, identified exposure, lower FEV1, higher BAL TCC and higher BAL lymphocytosis increased the probability of fibrotic HP diagnosis. The lymphocytosis >20% increased by 25 times the odds of fibrotic HP diagnosis. The optimal cut-off values to differentiate fibrotic HP from IPF were 15 × 106 for TCC and 21% for BAL lymphocytosis with AUC 0.69 and 0.84, respectively. Increased cellularity and lymphocytosis in BAL persist despite lung fibrosis in HP patients and may be used as important discriminators between IPF and fHP.

Unexpected CD5+ B Cell Lymphocytosis during SARS-CoV-2 Infection: Relevance for the Pathophysiology of Chronic Lymphocytic Leukemia.

Recently, cases of fortuitous discovery of Chronic Lymphocytic Leukemia (CLL) during hospitalization for Coronavirus disease (COVID-19) have been reported. These patients did not show a monoclonal B cell expansion before COVID-19 but were diagnosed with CLL upon a sudden lymphocytosis that occurred during hospitalization. The (hyper)lymphocytosis during COVID-19 was also described in patients with overt CLL disease. Contextually, lymphocytosis is an unexpected phenomenon since it is an uncommon feature in the COVID-19 patient population, who rather tend to experience lymphopenia. Thus, lymphocytosis that arises during COVID-19 infection is a thought-provoking behavior, strikingly in contrast with that observed in non-CLL individuals. Herein, we speculate about the possible mechanisms involved with the observed phenomenon. Many of the plausible explanations might have an adverse impact on these CLL patients and further clinical and laboratory investigations might be desirable.

THE ROLE OF LABORATORY EVALUATION IN THE DIAGNOSIS AND MANAGEMENT OF NLRP12- ASSOCIATED AUTOINFLAMMATORY DISEASE

<h3>Introduction</h3> This case highlights the relevance of including autoinflammatory diseases in the differential diagnosis of adults with recurrent fever or episodes of shock. The role of genetic testing and confirmatory functional testing in the diagnosis and therapeutic management of autoinflammatory diseases is also discussed. <h3>Case Description</h3> A 67-year-old man presented with a 2-year history of episodes of shock of unknown etiology. Symptoms included hypotension, fever and abdominal pain requiring vasopressor support and antibiotics. Extensive infectious, autoimmune and rheumatological work-up was negative. Bone marrow biopsy showed chronic myelomonocytic leukemia (CMML) that Oncology determined was non-contributory and did not require treatment. Immunological investigations demonstrated elevated IgE, CD8+ T cell lymphocytosis during episodes of shock and increased sCD163. A targeted NGS panel revealed a heterozygous VUS in the <i>NLRP12</i> gene. Functional studies were performed offline to evaluate inflammasome activity. PBMCs stimulated with LPS for 24h revealed substantially elevated levels of IL-1β, IL-6 and TNF-α compared to healthy controls suggesting inflammasome activation and NFκB dysregulation. This prompted initiation of IL-1β blockade with canakinumab, with symptom resolution. <h3>Discussion</h3> In adults presenting with a hyperinflammatory state of unknown etiology, immunological investigations should be pursued for a possible autoinflammatory syndrome. In this case, genetic testing identified a VUS in <i>NLRP12</i>. NLRP12 is an inflammasome in which LOF and GOF variants have been reported with disease manifestations, including cold-induced autoinflammatory syndrome (FCAS). NLRP12 also regulates NFκB, which may explain some of the observed hyperinflammation. This patient had complete response to IL-1β blockade. Functional evaluation of VUS may provide an opportunity for therapeutic intervention.

Acute toxicity and effects of methanolic extract of Moringa oleifera flower on haematological parameters of Wistar rats

Methanolic extract of Moringa oleifera flowers was investigated for its acute toxicity and effects on haematological parameters of Wistar rats. The acute toxicity study to determine the LD50 was evaluated using Lorke’s method. It involves first and second phases with six rats each phase. The rats were divided into three groups of two rats each of the phase. In the first, they were administered 10, 100 and 1000 mg/kg of the extract intraperitonially while in the second phase, they were administered 1600, 2900 and 5000 mg/kg and then observed for 24 hours. The haematological study was carried out according to standard procedures. Twenty (20) rats were divided into 4 groups with five rats in each group. The rats in groups B, C and D were treated orally with the methanolic extract of Moringa oleifera flowers using 200 mg/kg, 400 mg/kg, and 800 mg/kg respectively for 21 days while group A that served as control were administered sterile water for injection at 0.5 ml/kg. Blood sample was taken from all the rats and used to establish the baseline values prior to treatments with the extract. Blood samples were collected at the end of every week and analyzed for haematological values. The administration of the extract at the dose of 5000 mg/kg intraperitoneally to the rats resulted in death but was safe at the dose of 2900 mg/kg. This indicated that the LD50 is below 5000 mg/kg. There were significant increases (p&lt;0.05) in red blood cells, haemoglobin concentration and the packed cell volume values in all treated groups when compared to the baseline values (control group). There were also significant (p&lt;0.05) decrease and variation in the neutrophilic values and an increase in the lymphocytic values of the differential leukocytes counts. This research has revealed that the methanolic extract of Moringa oleifera flowers increased significantly the red blood cells, packed cell volume, haemoglobin concentration and lymphocytosis in Wistar rats.

A novel CARD11 germline mutation in a Chinese patient of B cell expansion with NF-κB and T cell anergy (BENTA) and literature review

Germline gain-of-function (GOF) mutations in the CARD11 gene lead to a rare primary immunodeficiency disease known as B cell expansion with NF-κB and T cell anergy (BENTA). Affected patients present with a polyclonal expansion of B cells, lymphadenopathy, and splenomegaly. Herein, we report a novel germline in-frame three base-pair deletion (c.1030_1032del, p.K344del) in the CARD11 gene in a patient with atypical BENTA, presenting with a recurrent fever and B cell lymphocytosis. This mutation was inherited from his mother, who is clinically asymptomatic and had a recurrent respiratory tract infection in her childhood. In vitro functional analysis demonstrated that this variant decreased the expression level of the CARD11 protein and activated the NF-κB signal pathway, leading to a higher expression of several NF-κB target gene transcripts in HCT116 cells transfected with mutant CARD11 (K344del-CARD11) as revealed by RNA sequencing analysis. To our knowledge, only 23 BENTA patients have been identified and carried seven distinct GOF mutations in CARD11. The clinical manifestations of patients are highly heterogeneous and there was no significant correlation between genotype and phenotype. In summary, we identified a novel in-frame three base-pair deletion that may be responsible for the pathogenesis of atypical BENTA in a Chinese family. Our study expands the mutational spectrum of the CARD11 gene and may be helpful in the understanding of diseases caused by CARD11 mutations and the clinical management of BENTA.

Understanding Monoclonal B Cell Lymphocytosis: An Interplay of Genetic and Microenvironmental Factors.

The term monoclonal B-cell lymphocytosis (MBL) describes the presence of a clonal B cell population with a count of less than 5 × 109/L and no symptoms or signs of disease. Based on the B cell count, MBL is further classified into 2 distinct subtypes: ‘low-count’ and ‘high-count’ MBL. High-count MBL shares a series of biological and clinical features with chronic lymphocytic leukemia (CLL), at least of the indolent type, and evolves to CLL requiring treatment at a rate of 1-2% per year, whereas ‘low-count’ MBL seems to be distinct, likely representing an immunological rather than a pre-malignant condition. That notwithstanding, both subtypes of MBL can carry ‘CLL-specific’ genomic aberrations such as cytogenetic abnormalities and gene mutations, yet to a much lesser extent compared to CLL. These findings suggest that such aberrations are mostly relevant for disease progression rather than disease onset, indirectly pointing to microenvironmental drive as a key contributor to the emergence of MBL. Understanding microenvironmental interactions is therefore anticipated to elucidate MBL ontogeny and, most importantly, the relationship between MBL and CLL.

Humoral and cellular immune responses to recombinant herpes zoster vaccine in patients with chronic lymphocytic leukemia and monoclonal B cell lymphocytosis.

Monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL) are clonal B-cell disorders associated with an increased risk of infections and impaired vaccination responses. We investigated the immunogenicity of recombinant zoster vaccine (RZV) in these patients. Individuals with MBL/untreated CLL and Bruton tyrosine kinase inhibitor (BTKi)-treated CLL patients were given two doses of RZV separated by 2 months. Responses assessed at 3 and 12 months from the first dose of RZV by an anti-glycoprotein E ELISA antibody assay and by dual-color Interferon-γ and Interleukin-2FLUOROSPOT assays were compared to historic controls matched by age and sex. About 62 patients (37 MBL/untreated CLL and 25 BTKi-treated CLL) were enrolled with a median age of 68 years at vaccination. An antibody response at 3 months was seen in 45% of participants, which was significantly lower compared to historic controls (63%, p= .03). The antibody response did not significantly differ between MBL/untreated CLL and BTKi-treated CLL (51% vs. 36%, respectively, p= .23). The CD4+ T-cell response to vaccination was significantly lower in study participants compared to controls (54% vs. 96%, p< .001), mainly due to lower responses among BTKi-treated patients compared to untreated MBL/CLL (32% vs. 73%, p= .008). Overall, only 29% of participants achieved combined antibody and cellular responses to RZV. Among participants with response assessment at 12 months (n= 47), 24% had antibody titers below the response threshold. Hypogammaglobulinemia and BTKi therapy were associated with reduced T-cell responses in a univariate analysis. Strategies to improve vaccine response to RZV among MBL/CLL patients are needed.

High Prevalence of Multiple B Cell Clones in Patients with Chronic Lymphocytic Leukemia

INTRODUCTIONStudies suggest that the first clonal events in chronic lymphocytic leukemia (CLL) may be acquired at hematopoietic stem cell stage. Consistently, it has been reported that CD34+cells from CLL patients engrafted in immunodeficient mice give rise to clonal B cell lymphoproliferation while CD34+ cells from healthy donors did not (Kikushige, Cancer Cell 2011). Furthermore, in rare patients with early CLL or monoclonal B cell lymphocytosis two clonal populations can be demonstrated. We hypothesized that immunofixation electrophoresis could serve as a sensitive tool to assess the prevalence of a second, clonally distinct, lymphoproliferative event in CLL patients. METHODSWe studied 262 treatment-naïve patients with a diagnosis of chronic lymphocytic leukemia (CLL) enrolled in our natural history study (NCT00923507). All patients had diagnostic peripheral blood flow cytometry testing (Laboratory of Pathology, NCI) and serum immunofixation electrophoresis (Department of Laboratory Medicine, Clinical Center, NIH) done. RESULTSOf the 262 patients, 148 (56.49%) were male and 114 (43.51%) were female. The median age of the study population was 65 years. Amongst the 262 patients, 243 (92.74%) were Caucasians, 8 (3.05%) were African Americans, 3 (1.14%) were Asians and 3 (1.14%) were multiracial. The race was undisclosed for 5 patients. Two of the 262 CLL patients had another B-cell malignancy on diagnostic workup, Waldenstrom's macroglobulinemia (Lymphoplasmacytic Lymphoma) and Hairy Cell Leukemia. Nine (3.44%) patients had two distinct clones of CLL by flow cytometry. In 4 both kappa and lambda clones were present. In 5, the two clones expressed the same light chains but had a distinct immunophenotype resulting in bi-phenotypic clones. By serum IFE, 62 (23.66%) of 262 patients had monoclonal gammopathy; in 44 (16.8%) patients one monoclonal band was detected, while 18 (6.9%) patients had 2 or more bands. Among patients with one monoclonal band 25 were kappa and 19 lambda light chain. In 19 of 25 patients with kappa clonal CLL by flow cytometry, the monoclonal band on IFE was kappa (concordant flow and IFE): IgM Kappa (in 8), IgG Kappa (10), and IgA Kappa (1); and in 6 patients with kappa clonal CLL the monoclonal band was lambda (discordant flow and IFE): IgM Lambda (in 2), and IgG Lambda (4). In 15 of 19 patients with lambda clonal CLL, flow cytometry and IFE were concordant: IgM Lambda (in 6), IgG Lambda (6), IgA Lambda (1), free lambda light chain (2), and discordant in 4: IgM Kappa (in 2), IgG Kappa (2). Amongst the 18 patients with 2 or more bands on IFE, only 5 patients had concordant flow cytometry and IFE, while 13 had discordant findings. In summary, using IFE we detected a second clonal event in 23 (8.8%) of 262 CLL patients. Considering the 11 patients with two distinct clones by flow cytometry, 34 (13%) of 262 treatment-naive CLL patients had at least one additional clonal B cell process. In addition, 18 patients with concordant flow and IFE findings had IgG or IgA type gammopathy, consistent with a concurrent plasma cell dyscrasia of the same light chain restriction as the CLL clone. CONCLUSIONIn 13% of CLL patients we found evidence for a second B cell lymphoproliferative process. The prevalence of biclonal CLL by flow cytometry at 3.4% is comparable to findings by others, reporting between 0.7% and 3.4%. In contrast, the prevalence of monoclonal gammopathy (23.7%) in our patients (median age 65) was much higher than reported in the general population: 3.2 % at age 50 and above, and 5.3% at age 70 and above (Kyle, NEJM 2006). Importantly, in 8.8% of all patients the monoclonal gammopathy was discordant with the CLL clone. These data support the existence of a genetic event in an early progenitor cell that predisposes to the development of clonal B cell expansions.This research was supported by the Intramural Research Program of NHLBI and NCI. DisclosuresWiestner:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding.

Human pluripotent stem cells identify molecular targets of trisomy 12 in chronic lymphocytic leukemia patients

Identifying precise targets of individual cancers remains challenging. Chronic lymphocytic leukemia (CLL) represents the most common adult hematologic malignancy, and trisomy 12 (tri12) represents a quarter of CLL patients. We report that tri12 human pluripotent stem cells (hPSCs) allow for the identification of gene networks and targets specific to tri12, which are controlled by comparative normal PSCs. Identified targets are upregulated in tri12 leukemic cells from a cohort of 159 patients with monoclonal B cell lymphocytosis and CLL. tri12 signaling patterns significantly influence progression-free survival. Actionable targets are identified using high-content drug testing and functionally validated in an additional 44 CLL patient samples. Using xenograft models, interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitor is potent and selective against human tri12 CLL versus healthy patient-derived xenografts. Our study uses hPSCs to uncover targets from genetic aberrations and apply them to cancer. These findings provide immediate translational potential as biomarkers and targets for therapeutic intervention.