Chronic Lymphocytic Leukemia Phenotype Research Articles

B-Cell Receptor Function and Gene Expression Profiling at Single-Cell Level across the Spectrum from Normal to Neoplastic CD5-Positive B-Cell Compartments in Humans

Antigen-independent, autonomous signalling of the B-cell receptor (BCR) is an oncogenic driver common to all cases and subtypes of chronic lymphocytic leukemia (CLL). We have recently shown that the dominant clonal population of low-count (<500 cells/µL) monoclonal B lymphocytosis (MBL) with CD5 +CD20 lowCD79B low CLL phenotype identified prospectively in healthy siblings of CLL patients also exhibit autonomous BCR signalling, albeit with lower signalling strength than CLL BCR (Quinten et al., Haematologica 2023). To gain further insight into the mechanisms of clonal expansion within the CD5 + B-cell compartment as the possible origin of CLL, we performed single-cell gene expression profiling and exemplary analyses of BCR function in normal CD5 + B cells (CD20 hiCD79B hi) and in CLL-phenotype B cells (CD5 +CD20 lowCD79B low) of healthy siblings of CLL patients in comparison to CLL and high-count MBL cells. Screening of blood samples of 16 sibling stem cell donors of CLL patients by flow cytometry revealed the presence of CD5 +CD20 lowCD79B low B cells at low counts in 5 donors. These cells and normal CD5 +CD20 hiCD79B hi B cells were sorted in parallel, pooled at equal ratios, and analyzed together with CLL B cells from their sibling stem cell recipients and 2 unrelated high-count MBL B cells on two 10X Genomics chips for single-cell 5‘ gene expression and BCR sequencing. Data deconvolution per individual was performed based on expressed SNPs and on the individuals’ HLA types. In a combined 2-dimensional t-SNE plot based on the 2000 most variably expressed genes, the normal CD5 +CD20 hiCD79B hi B cells from all 5 donors clustered distinctly from CLL-phenotype cells from all sources (dotted lines, Figure) and indeed showed significantly higher expression of CD20 and CD79B. Gene set enrichment analysis revealed significant upregulation of the hallmark oxidative phosphorylation gene set in all CLL samples compared to non-CLL B-cell populations. BCR analysis demonstrated clonal dominance of >99% for both Ig heavy and light chains in the 5 CLL samples. In accordance with the indication for allogeneic stem cell transplantation, these CLL BCR were unmutated. In marked contrast, normal CD5 +CD20 hiCD79B hi B cells from the healthy SC donors were also unmutated but completely polyclonal, i.e. no identical BCR was detected in any two cells of this compartment. In 4/5 donors, the CLL-phenotype CD5 +CD20 lowCD79B low compartment contained multiple clonal expansions of various degrees. In one high-count MBL, 17 other B-cell clones with various degrees of low-level expansion coexisted with the >90% dominant MBL clone. BCR from the most dominant and one or more of the less expanded CD5 +CD20 lowCD79B low clones of the healthy donors were tested for autonomous signaling as quantified by BCR-induced calcium flux without experimental cross-linking in murine TKO pre-B cells and compared to BCR from nonexpanded CD5 +CD20 hiCD79B hi cells and from their CLL siblings. Autonomous BCR signaling strength was calculated from the fraction of cells with spontaneous calcium flux above test background and calibrated against calcium flux after BCR crosslinking. CLL and high-count MBL BCR had robust median autonomous BCR signaling of 0.326. Expanded CD5 +CD20 lowCD79B low clones of the healthy donors showed significantly weaker BCR signalling strength of 0.163 (p<0.01; Scatter Plot). BCR signalling in nonexpanded CD5 + B cells appeared weaker than in expanded clones but was clearly above the background of BCR from mature B cells isolated from lymph node biopsies expressing a mutated IgG. In conclusion, the low-count CD5 +CD20 lowCD79B low CLL-phenotype B-cell compartment in healthy adults is composed of multiple, predominantly mutated B-cell clones. Therefore, the term “monoclonal B lymphocytosis” is highly misleading for these B-cell populations and should be avoided. Normal CD5 +CD20 hiCD79B hi B cells are exclusively unexpanded and express unmutated BCR, indicating a naïve status. Unexpectedly, these nonexpanded CD5 + B cells express BCR with relatively weak but quantifiable autonomous signalling. Globally, autonomous BCR signalling strength across the CD5 + B-cell compartment appears to be correlated to the degree of clonal expansion. Fully malignant CLL cells overexpress genes of the oxidative phosphorylation pathway, presumably to meet increased energy demand from alternative sources.

Abstract 3814: In CLL the U1 snRNA driver mutation alters splicing in multiple genes and pathways

Abstract 5-10% of patients with the IGHV wild type form of chronic lymphocytic leukemia (CLL) carry a g.3A>C driver mutation in the U1 small nuclear RNA (snRNA). We investigatedthe patterns of mis-splicing and their pathway consequences in three U1-mutant CLL cellline models using long read sequencing in order to better understand the mechanisms ofoncogenicity in tumors carrying this mutation. CLL cell lines (HG3, JVM3 and MEC1) expressing the U1 mutation and their wild-type counterparts were submitted to Oxford Nanopore sequencing to generate full-length RNA transcripts. This transcriptomic datawas evaluated, together with previously obtained short-read data. The g.3A>C mutation occurs at a specific location of the U1 snRNA, a core component of the eukaryotic spliceosome, and acts by altering the 5' splice site (5'SS) recognition sequence to cause consistent patterns of mis-splicing. Long read analysis identified multiple instances of intron retention and suppression of exon skipping, and in silico translation of these mis-splicing events predicted stop-gain and other loss of function mutations in several expressed genes, as well as widespread changes in gene expression levels. We performed a pathway overrepresentation analysis of the altered gene expression patterns in the mutant cells using the Reactome knowledgebase and identified an enrichment in the processes of translation, non-sense mediated decay (NMD) and immune signaling, including interferon signaling. In particular, there was a marked down-regulation of genes related to ribosomal assembly and the translational machinery. A more comprehensiveanalysis of the U1 mutation phenotype in CLL may accelerate the development ofbetter therapeutic and diagnostic approaches in patients. Citation Format: Andrea Senff-Ribeiro, Fatemeh Almodaresi, Quang Trinh, Shimin Shuai, David Spaner, Xose S. Puente, Elias Campo, Lincoln D. Stein. In CLL the U1 snRNA driver mutation alters splicing in multiple genes and pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3814.

High Frequency of Monoclonal B-cell Lymphocytosis with a High Prevalence of Biclonal Cases in the Colombian Population

The World Health Organization (WHO) defines monoclonal B-Cell Lymphocytosis (MBL) as the presence in Peripheral Blood (PB) of monoclonal populations of B-Lymphocytes (BL) of up to 5x109/L, with the phenotype of Chronic Lymphocytic Leukemia (CLL) typical, CLL atypical, or non-CLL, in the absence of B symptoms or clinical manifestations related to chronic B-Cell Lymphoproliferative Disorders (B-CLPD). It is classified as Low Count (LC) (<500 clonal BL/μL) and High Count (HC) (>500 clonal BL/μL) [1]. It has been described in up to 12% of the healthy adult population [1], a proportion that increases in relatives of patients with CLL/Small Lymphocytic Lymphoma (SLL) to around 15% [2]. It is usually a more frequent entity (28.5%) in people with Hepatitis C virus (HCV) [3] infection, and it tends to persist for a long time (90% of cases) with a progression rate of the MBL-HC to clinical manifest B-CLPD between 1-4% per year [1,2]. Currently, it continues to be an entity under study since its possible evolution to CLL or another B-CLPD is clearly unknown [4].

Antibodies Produced by CLL Phenotype B Cells in Patients With Myasthenia Gravis Are Not Directed Against Neuromuscular Endplates.

Myasthenia gravis (MG) can in rare cases be an autoimmune phenomenon associated with hematologic malignancies such as chronic lymphocytic leukemia (CLL). It is unclear whether in patients with MG and CLL, the leukemic B cells are the ones directly driving the autoimmune response against neuromuscular endplates. We identified patients with acetylcholine receptor antibody-positive (AChR+) MG and CLL or monoclonal B-cell lymphocytosis (MBL), a precursor to CLL, and described their clinical features, including treatment responses. We generated recombinant monoclonal antibodies (mAbs) corresponding to the B-cell receptors of the CLL phenotype B cells and screened them for autoantigen binding. A computational immune cell screen revealed a subgroup of 5/38 patients with MG and 0/21 healthy controls who displayed a CLL-like B-cell phenotype. In follow-up hematologic flow cytometry, 2 of these 5 patients were diagnosed with an MBL. An additional patient with AChR+ MG as a complication of manifest CLL presented at our neuromuscular clinic and was successfully treated with the anti-CD20 therapy obinutuzumab plus chlorambucil. We investigated the specificities of expanding CLL-like B-cell clones to assess a direct causal link between the 2 diseases. However, we observed no reactivity of the clones against the AChR, antigens at the neuromuscular junction, or other common autoantigens. Our study suggests that AChR autoantibodies are produced by nonmalignant, polyclonal B cells The new anti-CD20 treatment obinutuzumab might be considered in effectively treating AChR+ MG. This is a single case study and provides Class IV evidence that obinutuzumab is safe to use in patients with MG.

Chronic Lymphocytic Leukemia With Two B-Cell Populations of Discordant Light Chain Restrictions in Individual Patients: Parallel Development of Biclonal B-Cell Neoplasms or Clonal Evolution With Isotype Switch?

To evaluate clinicopathologic characteristics of biclonal chronic lymphocytic leukemia (CLL). Retrospectively analyze clinical data and pathologic features. Ten cases were identified in which flow cytometry demonstrated an abnormal B-cell population with a CLL-like immunophenotype but showed no definitive light chain restriction. All had cytogenetic abnormalities detected, including seven with two CLL-related abnormalities. Four of these showed features suggestive of clonal evolution, all having del(13q) as a "stem-line" abnormality and three showing del(11q) as a "side-line" abnormality. Five (50%) cases demonstrated deleterious NOTCH1 mutations, in contrast to 11.8% in a control group of monoclonal CLL (P < .05). Of the 10 patients, 5 received treatment, with good/partial response in three cases and therapeutic resistance in one case. The median treatment-free survival was estimated at 68 months. Despite a polytypic pattern of light chain expression, the neoplastic nature of biclonal CLL is suggested by a characteristic CLL phenotype and can be confirmed by cytogenetic and genomic analyses. The two clones with discordant light chain isotypes may share a "stem-line" cytogenetic abnormality, suggesting possible clonal evolution. Biclonal CLL is associated with NOTCH1 mutations, which may occur in a small subclone and gradually evolve in clonal size. Genomic analysis on light chain-sorted and/or chronologically collected samples may provide insight into clonal evolution in CLL.

Autonomous BCR Signaling and Genetic Aberrations in CLL-Phenotype Monoclonal B Lymphocytosis in Siblings of CLL Patients

Clonal expansion of CD5-expressing B cells, termed monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL) with an increased incidence in CLL siblings. In app. 1% of MBL carriers, MBLprogresses eventually to clinically overt CLL. The mechanisms driving benign MBL expansion and subsequent malignant progression are unknown. Autonomous signaling of the clonotypic BCR of CLL cells, i.e. signaling without stimulation by cognate antigen, is an indispensable oncogenic signal in CLL. In order to investigate the early stages in CLL ontogeny, we sought to clarify whether MBL cells express BCR with autonomous signaling capacity and to compare the prevalence of inherited risk loci and acquired genetic aberrations in MBL-CLL sibling pairs. Peripheral blood of 191 siblings of CLL patients was screened with informed consent by six-color flow cytometry. A discrete CD19+CD5+CD20lowCD79low CLL-phenotype population of 0.2 to 1863 cells / µL blood was detected in 34 siblings (17.8%) of 26 CLL patients; 32 siblings (94%) had <500 clonal CLL-phenotype cells / µL. Clonal BCR were identified from highly purified MBL cells of 17 CLL siblings by ARTISAN PCR. Five MBL clones could be assigned to CLL stereotyped subsets, including two MBL BCR belonging to CLL subset #2 with expression of the IGLV3-21 gene with a G110R mutation and one IgG-expressing MBL belonging to CLL subset #4. Eleven MBL BCR, including all 5 assigned to a CLL stereotyped subset, were transduced into murine TKO cells arrested at the pro-B-cell stage by genetic deficiency of rag2, lambda5, and slp65. Slp65 function is reconstituted by a slp65-ERT2 fusion gene that enables BCR signaling in the presence of 4-hydroxytamoxifen. Calcium mobilization was measured in Indo-1 AM-loaded, live-gated TKO cells by flow cytometry as the ratio of signal intensities (SIR) at 485 and 405 nm prior and after tamoxifen supplementation and after BCR crosslinking with isotype-specific antibody. Robust calcium mobilization was observed in all MBL BCR-transduced TKO cells in the absence of external antigen and without experimental cross-linking, regardless of assignment to a CLL subtype. Calibrated quantification of this autonomous BCR signal, however, revealed lesser intensity in MBL BCR compared to the BCR of their CLL sibling (p=0.015, Wilcoxon's matched-pair signed rank test). BCR signaling strength was correlated to the degree of MBL clonal expansion (p=0.034). According to genotyping by single nucleotide polymorphism array, whole exome sequencing (WES), and targeted panel sequencing, a polygenic CLL risk score (PRS) of 24 risk loci was higher in both CLL and MBL siblings than in reference population data, but we found no evidence for a higher PRS in CLL cases in a matched-pair comparison to their MBL siblings. With respect to acquired genetic changes, quantitative analysis of SNP array data revealed recurrent CLL-associated copy number variations (CNV) at clonal levels in all of 11 informative CLL cases. In contrast, individual CNV were present in only a minor fraction of the highly purified MBL cells in six of ten samples (p=0.003; Fisher's exact test). Likewise, variant allele frequencies (VAF) of non-shared variants as detected by WES was significantly lower in MBL samples than in CLL cases (p<0.0001). In particular, subclonal variants defined by a VAF of 0.1-0.33 were more prevalent in MBL compared to CLL siblings (p=0.014), whereas clonal variants (VAF 0.43-0.55) were equally abundant (p=0.65) in both conditions. In this comprehensive comparison of MBL and CLL in siblings, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, suggesting that CLL risk loci predispose to clonal expansion of CLL phenotype cells in both low-count MBL and CLL. Vice versa, we did not find evidence for a particular association of CLL risk alleles with malignant progression from MBL to clinical CLL. Autonomous BCR signaling operates in both MBL and CLL as predicted by BCR stereotype, albeit at lesser strength in MBL. Acquired CLL-genetic changes, both copy number and sequence variants, are frequently subclonal in MBL but not in CLL. These findings support a stepwise CLL pathogenetic model wherein autonomous BCR signaling causes a non-malignant expansion of clonal CD5+ B cells in genetically predisposed individuals, followed by malignant progression to CLL after gradual acquisition of pathogenic genetic variants.

A clinical case of the effective combined use of BCL-2 and PI3K inhibitors in the treatment of a patient with an unfavorable chronic lymphocytic leukemia with transformation into diffuse large B-cell lymphoma (Richter’s syndrome)

Understanding the molecular biological basis of chronic lymphocytic leukemia (CLL) pathogenesis and stratification of patients into risk groups has now led to significant advances in treatment. New targeted drugs with different mechanisms of action (bruton’s tyrosine kinase inhibitors, bCL-2 inhibitors, pI3K inhibitors) have significantly improved the prognosis of high-risk CLL patients. In some CLL cases the nodular tumor component can change to a more aggressive subtype of lymphoma (often diffuse large b-cell) with preservation of the small-cell leukemic component with the CLL phenotype (Richter’s syndrome), usually characterized by rapid progression and poor prognosis. The issue of treatment efficacy in patients with Richter’s syndrome still remains unresolved. The results of new drugs clinical trials are often contradictory and cannot yet be recommended for routine use in clinical practice. The low incidence of Richter’s syndrome, the lack of a unified view of the pathogenesis and therapy approaches make the search for effective drugs an urgent task, so each clinical observation is of undoubted interest.A clinical case of CLL patient with unfavorable molecular cytogenetic risk and transformation into diffuse large b-cell lymphoma (Richter’s syndrome) is presented. The combined use of bCL-2 inhibitors (venetoclax) and pI3K (duvelisib) led to the achievement of partial remission followed by a gradual increase in the positive antitumor effect.

Rare Germline ATM Variants Influence the Development of Chronic Lymphocytic Leukemia.

Germline missense variants of unknown significance in cancer-related genes are increasingly being identified with the expanding use of next-generation sequencing. The ataxia telangiectasia-mutated (ATM) gene on chromosome 11 has more than 1,000 germline missense variants of unknown significance and is a tumor suppressor. We aimed to determine if rare germline ATM variants are more frequent in chronic lymphocytic leukemia (CLL) compared with other hematologic malignancies and if they influence the clinical characteristics of CLL. We identified 3,128 patients (including 825 patients with CLL) in our hematologic malignancy clinic who had received clinical-grade sequencing of the entire coding region of ATM. We ascertained the comparative frequencies of germline ATM variants in categories of hematologic neoplasms, and, in patients with CLL, we determined whether these variants affected CLL-associated characteristics such as somatic 11q deletion. Rare germline ATM variants are present in 24% of patients with CLL, significantly greater than that in patients with other lymphoid malignancies (16% prevalence), myeloid disease (15%), or no hematologic neoplasm (14%). Patients with CLL with germline ATM variants are younger at diagnosis and twice as likely to have 11q deletion. The ATM variant p.L2307F is present in 3% of patients with CLL, is associated with a three-fold increase in rates of somatic 11q deletion, and is a hypomorph in cell-based assays. Germline ATM variants cluster within CLL and affect the phenotype of CLL that develops, implying that some of these variants (such as ATM p.L2307F) have functional significance and should not be ignored. Further studies are needed to determine whether these variants affect the response to therapy or account for some of the inherited risk of CLL.

Protein Phosphorylation and Redox Status: An as Yet Elusive Dyad in Chronic Lymphocytic Leukemia.

Simple SummaryPhosphorylation is one of the most crucial modifications of lipids and proteins, as it regulates virtually all cellular functions. Like other human diseases, chronic lymphocytic leukemia (CLL), the most common leukemia in Western developed countries, exhibits deranged phosphorylation, which is induced by stimuli within specific tissues (e.g., lymph nodes), promoting enhanced proliferation and survival. Importantly, a growing body of evidence shows that reactive oxygen species (ROS), altered forms of oxygens generated from metabolism and peculiar enzyme complexes, and generally considered highly harmful due to their reactivity toward critical biomolecules (proteins, lipids, and DNA), act in concert with phosphorylation in supporting the malignant phenotype in CLL. This complex interplay is now providing insights into potential novel Achilles heels of intracellular signals for the development of innovative treatments which might synergize with the drugs currently in use that target the principal players in phosphorylation, namely kinases.Malignant cells in chronic lymphocytic leukemia (CLL) are characterized by oxidative stress that is related to abundant generation of reactive oxygen species (ROS) by increased mitochondrial oxidative phosphorylation (OXPHOS). Lymphoid tissues have been shown to provide a protective microenvironment that antagonizes the effects of ROS, contributing to establishing redox homeostasis that supports the vitality of CLL cells. In the last few decades, a complex antioxidant machinery has been demonstrated to be activated in CLL cells, including the different superoxide dismutase (SOD) isoforms, the thioredoxin (Trx) system, and the enzyme cascade inducing glutathione (GSH) biosynthesis and recycling, to name a few. Their expression is known to be upregulated by the activation of specific transcription factors, which can be regulated by either oxidative stress or phosphorylation. These two latter aspects have mostly been explored separately, and only recently an increasing body of evidence has been providing reasonable inference that ROS and phosphorylation may cooperate in an interplay that contributes to the survival mechanisms of CLL cells. Here, we present an overview of how oxidative stress and phosphorylation-dependent signals are intertwined in CLL, focusing on transcription factors that regulate the balance between ROS production and scavenging.

CircZNF91 Promotes the Malignant Phenotype of Chronic Lymphocytic Leukemia Cells by Targeting the miR-1283/WEE1 Axis.

Background Circular RNAs (circRNAs) are frequently dysregulated in cancers and are implicated in tumorigenesis and tumor progression. In this study, we investigated the role of circZNF91 in regulating the malignant phenotype of chronic lymphocytic leukemia (CLL) cells and the underlying molecular mechanism. Material/Methods The expression of circZNF91 was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The binding sequences between circZNF91/miR-1283 and miR-1283/WEE1 were predicted by the bioinformatic database. The functional interactions were confirmed by the dual-luciferase reporter, RT-qPCR, and Western blot assays. The functional roles of the circZNF91/miR-1283/WEE1 axis in CLL progression were examined by cell proliferation, apoptosis, and EdU incorporation assays. Results circZNF91 was upregulated in CLL samples. Silencing circZNF91 attenuated CLL cell proliferation and induced apoptosis and cell cycle arrest. circZNF91 could sponge miR-1283 to suppress its activity, which in turn upregulated WEE1 expression. Silencing circ-TTBK2 reduced WEE1 expression, while the inhibitor of miR-1283 enhanced WEE1 expression. The miR-1283/WEE1 axis mediated the effects of circZNF91 on cell proliferation and apoptosis, as well as induced cell cycle regulation. Conclusions The circZNF91/miR-1283/WEE1 axis is engaged in the pathological phenotypes of CLL cells, which could serve as potential targets for future therapy development.

Characterisation of a New Clinical Presentation of Chronic Lymphocytic Leukemia with Symptomatic Nasopharyngeal Mucosa Involvement

Patients with chronic lymphocytic leukemia (CLL) are prone to infectious complications, including Ears, Nose and Throat (ENT) infections due to the humoral immunodepression and/or to the immunosuppression related to the therapy. However, specific CLL infiltration in non-lymphoid regions of the head and neck causing ENT symptoms but unrelated to an infection is not well described. Extra-nodal localizations of CLL cells, including involvement of the mucosa of the rhinopharynx is uncommon and poorly reported. Here, we retrospectively analyzed the clinical, histopathologic and molecular features of 25 CLL patients with specific head and neck involvement. To date, this is the largest cohort reporting this new entity of CLL also named Nasal Associated Lymphoid Tissue (NALT) CLL.All patients had proven CLL prior to symptoms. Median time between ENT manifestation and CLL was 3 years [1-11 years]. Symptoms included chronic coughing (44%), antero-posterior nasal discharge (44%), nasal congestion (33%) and pharyngitidis (33%). ENT examination evidenced cervical lymphadenopathies in 68 % of cases, a granular aspect of the mucosa of the pharynx in 56%, enlarged tonsil (37%) or adenoids (37%). All patients underwent a biopsy of the mucosa of the nose or the throat. Histology and immunochemistry analysis demonstrated an infiltration of small lymphocytes CD20+, CD5+ and CD23+, consistent with a phenotype of CLL. The infiltration was diffuse in 50% of biopsies and perivascular in 38%.Patients with NALT-CLL had a poor prognosis: the majority was IGHV unmutated (n=18/25, 72%). Furthermore, they all required a treatment according to IWCLL criteria few time after the first ENT symptoms (median time 2 years), indicating that NALT-CLL is associated with a more progressive disease.To characterize the genetic background of NALT-CLL, we analyzed the cytogenetic data and NGS sequencing of 13 CLL-associated mutations from peripheral CLL cells. The karyotype was normal in only 2/24 cases (8%) and complex (>3 abnormalities) in 5/24 cases (20%). Half of the patients had a trisomy 12(12/24; 50%), while 13q, 17p and 11q deletions were found in 29%, 8% and 4% respectively. Eleven patients harbored one mutation (44%) while 9 patients had 2 to 5. Mutation of the NOTCH1 pathway was found in half of the cases (11/25 NOTCH1 and 2/25 FBXW7 mutated cases). TP53 and SF3B1 mutations occurred respectively in 20% (5/25 cases) and in 12% (3/25 cases).To gain insight into the molecular mechanism associated with involvement of the rhinopahrynx, we studied the expression of 70 genes related to cell migration and cell adhesion pathways using a qPCR array in the peripheral CLL cells of patients with NALT (n=4) compare to no NALT-CLL (n=4). Genes significantly up regulated with a fold change >2 included the chemokine receptors CCR7 (p=0.05) and CCR5 (p=0.04), the receptor involved in leukocyte trafficking CXCR3 (p=0.01) or the chemoattractant chemokine-like factor CKLF. By targeted qPCR, we confirmed the up regulation of CCR7 (p=0.002), CXCR3 (p=0.04) and CCR5 (p=0.03) in the cohort NALT-CLL patient (n=25) as compared to 20 age-matched CLL patients without head and neck symptoms (77% with unmutated IGHV, 30% with NOTCH1 mutation and 30% with trisomy 12). Up regulation of those targets was independent of the presence of NOTCH1/trisomy12 aberration or of the IGHV mutation status. These results suggest an increased migratory capacity of the leukemic CLL cells into the rhynopharynx mucosa related to a higher expression of these receptors involved in cell trafficking and migration. In line with these results, immunohistochemistry analysis of 5 patients with nasal involvement showed a strong staining of the CCR7 marker on the membrane of CLL cells infiltrating the mucosa. Interestingly, the staining of CCL21, the cognate ligand of CCR7, was positive in the vessels of the mucosa, suggesting that the recruitment and the transendothelial migration of CLL cells into the mucosa occur through a local secretion of CCL21 by the vessels.In summary, we report here a new presentation of CLL associated with symptomatic and specific ENT localization. CLL cells are predominantly IGHV unmutated, harbor NOTCH1 mutation and/or trisomy12 and show a higher expression of the chemokine receptors CCR7, CCR5 and CXCR3. We are currently studying the expression of those receptors by flow cytometry and the enhanced migratory capacity toward CCL21 through an in vitro chemotaxis assay. DisclosuresLetestu: AbbVie: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Janssen: Research Funding, Speakers Bureau. Cymbalista: Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; ASTRA ZENECA: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Lilly-LOXO: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees.

AML-448: Overlapping B-Cell Non-Hodgkin Lymphoma with Myeloid Malignancies: A Case Series

Context: This case series is reported from the cases we give care to in our facility. Objective: The main objective of this study is to highlight the possible co-incidence of myeloid malignancy with mature B cell neoplasm. Design: This is a case series. Patients or Other Participants: The case series included 4 patients with mature B-cell neoplasms and developed myeloid malignancy at the time of first presentation without giving any history of previous chemotherapy or radiotherapy exposure. Main Outcomes Measures: The main outcome is to report the possible occurrence of this confusing association and to study the biology behind it. Results: We have 4 cases included in this series. Case 1: A 58-year-old male who was diagnosed with chronic myeloid leukemia and showed accelerated profile. His CBC film showed peripheral leukocytosis, which was mistaken initially as a sign of acceleration; however the cell morphology was suspicious. Flow cytometric studies showed a mantle cells phenotype. His cytogenetics showed t(9,11) and extra Philadelphia chromosome. This patient received high-dose cytarabine with tyrosine kinase inhibitors and showed good response. Case 2: A 47-year-old female who was diagnosed with chronic lymphocytic leukemia (CLL) and put under watchful waiting. Then, she developed pancytopenia and appearance of aggressive B symptoms. Bone marrow examination and flow cytometry confirmed the presence of two clones of cells, one of CLL phenotype and the other of myelomonocytic line, and she had normal karyotyping. She received the FLAG protocol and went into remission. Case 3: A 32-year-old male presented with enlarged cervical LNs and peripheral leukocytosis. Excisional LN biopsy confirmed follicular NHL; however, promyelocytes were seen in his peripheral blood smear. Further PML-RARA mutation was detected with FISH, confirming the diagnosis of coinciding acute promyelocytic leukemia. Unfortunately, this patient died during chemotherapy induction from sepsis. Case 4: A 56-year-old male presented with bleeding and anemia. Bone marrow aspiration showed acute myeloid leukemia with dysplastic features. Further trephine biopsy showed a CLL clone. He received induction chemotherapy and died with sepsis. Conclusions: This overlap of hematologic malignancies may suggest that there is a common molecular aberration behind them. Testing for TP53 mutations should have been done together with germline mutations.

SF3B1 Mutation Causes Aberrant Splicing and Downregulation of PPP2R5A in Chronic Lymphocytic Leukemia

The splicing factorSF3B1 is mutated in 10-20% of patients with chronic lymphocytic leukemia (CLL). SF3B1 mutation is associated with faster disease progression, shorter overall survival and fludarabine resistance. However, the molecular mechanism of how SF3B1 mutation contributes to leukemogenesis and the drug resistance phenotype in CLL remains elusive. Comparison of the transcriptome sequencing data of SF3B1 -mutated cancer patients with that of un-mutated samples identified a subset of genes that were aberrantly spliced, mainly at alternative 3‘ splicing sites, by the usage of cryptic 3‘ splice and branchpoint sites in CLL. In this study, we performed an integrated analysis on the transcriptome sequencing data of primary CLL patients with and without SF3B1 mutation available in our lab and those downloaded from Gene Ominibus (GEO) database, as well as those generated from a human B cell precursor leukemia cell line with a knock-in SF3B1K700E mutation. PPP2R5A ,encoding one of the regulatory subunits of tumor protein phosphatase-2A (PP2A B56α), stands out as one of the key genes that were most consistently affected by an aberrant spliced junction, and significantly down-regulated (>2 fold) in CLL patient samples with various SF3B1 hotspot mutations across several CLL transcriptome sequencing data sets. Splicing analyses by RT-PCR and Sanger sequencing confirmed that a 13-nucleotides sequence was added before the 5th exon of PPP2R5A via alternative splicing in CLL patients with SF3B1 mutation. Due to this 13-nucleotides addition, three consecutive premature stop codons were created by frame-shift at a position more than 55 bases upstream of an exon-exon junction, which is a canonical feature of the mRNAs degraded subsequently through non-sense mediated mRNA decay (NMD). The down-regulation of PPP2R5A was confirmed by quantitative PCR (p <0.05) and immunoblot at mRNA and protein levels in CLL patients with SF3B1 mutation as compared to CLL patients without SF3B1 mutation and normal CD19 B+ cell samples. Furthermore, overexpression of mutant SF3B1K700E in HEK 293T cells resulted in the same aberrant splicing pattern observed in CLL patients with SF3B1 mutation and downregulation of PPP2R5A (p <0.05) as compared to HEK 293T cells overexpressing wild type SF3B1. Treatment with cycloheximide, a translation inhibitor known to inhibit NMD, resulted in an increased abundance of alternatively spliced PPP2R5A transcript, suggesting that the aberrant PPP2R5A transcript was degraded by NMD. PP2A, when incorporated with PPP2R5A as the regulatory subunit, dephosphorylates BCL2 and MYC, and results in a decreased anti-apoptotic activity of BCL2 and the degradation of MYC and beta-catenin. Our results demonstrate that SF3B1 mutation causes aberrant splicing and downregulation of tumor suppressor protein PPP2R5A in vitro and in vivo, and reveal one of the consequential roles of SF3B1 mutation in leukemogenesis and the development of drug resistance phenotype. DisclosuresNo relevant conflicts of interest to declare.

NOTCH1 Aberrant Activation Is a Common Nonmutational Early Event in Chronic Lymphocytic Leukemia Hematopoietic Stem-Cells

Chronic lymphocytic leukemia (CLL) is a B cell disorder characterized by constitutive NOTCH1 activation. NOTCH1 mutations are recurrently associated with CLL providing a new clonal marker to track CLL origin. To investigate CLL-initiating cells, we assessed NOTCH1 mutational status and signaling in hematopoietic stem (HSCs) and progenitor cells from CLL bone marrow. We collected a total of 29 bone marrow (BM) samples including 22 CLL patients and 7 healthy donors (HDs). BM cells were sorted into CD34+CD38- HSCs (purity 94.23% ± 3.04% ) and CD34+CD38+ progenitor fraction (98.12% ± 1.34%). While Sanger sequencing analysis of the NOTCH1 failed to detect alterations in CD34+/CD38- HSCs, AS-PCR and Droplet digital PCR (ddPCR) indicated the presence of small HSCs mutated clones in 57% of cases. Altogether, these data confirm that NOTCH1 mutation is an early event in CLL hematopoiesis. The analysis of CD34+/CD38+ progenitors detected the NOTCH1 mutation in the majority of samples (66%). The NOTCH1 mutational burden progressively increased along specific stages of HSC differentiation (6.4%± 4.7 in CD34+CD38- to 14.9%±11.3 in CD34+CD38+CD10+CD19+ cells, 22.7%±6.5 in CD34-CD38+CD10+CD19+ cells and 40.5%±4.3 in neoplastic CD5+CD19+ cells). This suggests that the NOTCH1 lesion is selected and expands during HSC differentiation toward a B neoplastic cell, thus strengthening the hypothesis that the genetic alteration is an initial event associated with the stepwise malignant transformation of CLL. Conversely, CD34+ cells (1x106) from CLL patients carrying the mutation, when transplanted into NOD/SCID/IL2Rgnull (NSG) mice generated a mature CLL phenotype (the median percentage of CD5+CD19+ cells in hCD45+ cells was 38.5% and 43.8% in BM and spleen respectively) lacking NOTCH1 mutation (as showed by AS-PCR assay performed on enriched hCD45 cells). This data is more in line with the hypothesis that this genetic abnormality is acquired at the mature B cell stage as an additional leukemogenic event to transform into clinical CLL. Thus, we analyzed the NOTCH1 signaling status in HSCs and progenitor cells of NOTCH1-mutated and unmutated CLL samples. The flow-cytometric analysis of the active NOTCH1-ICN protein level showed unmutated and mutated CLL has a significantly higher NOTCH1-ICN level than HDs samples in both CD34+/CD38- HSCs and CD34+CD38+ (73.4%±22.9 and 83%±16.4 vs 33.3%±14.8; 94.4%±7.3 and 92.8%±4.3 vs 47.9%±13.8, p<0.01 and p<0.001, respectively). c-MYC expression was found significantly higher in HSCs cells from NOTCH1 mutated and unmutated CLL samples compared to HD (3.5±0.7 and 2.6±0.08 vs 1.3±0.1). Western blot analysis of the expression levels of the NOTCH1-TM subunit revealed that HSCs from mutated and unmutated CLL patients always expressed the NOTCH1-TM protein, compared to HDs where NOTCH1-TM was either absent or expressed at lower levels. The present study indicated that the pool of CD34+ cells, including HSC and progenitor compartments, have NOTCH1 aberrantly expressed and activated in CLL patients compared to HDs demonstrating a common nonmutational NOTCH1 activation occurring early in CLL hematopoiesis. This selective pressure might contribute to the onset of specific NOTCH1 mutations in a DNA context that is prone to spontaneous microdeletion. These data represent a rationale for the use of therapies targeting the NOTCH1 signaling in CLL aimed to inhibit the survival of CLL-initiating cells. DisclosuresNo relevant conflicts of interest to declare.

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.

Screening for monoclonal B-lymphocyte expansion in a hospital-based Chinese population with lymphocytosis: an observational cohort study.

ObjectivesScreening of monoclonal B-cell lymphocytosis (MBL) has improved the early detection of B-cell lymphoproliferative disorders (B-LPDs). This study was designed to find the most cost-effective way to screen for asymptomatic B-LPD.DesignObservational study.SettingA lymphocytosis screening project was conducted at a large-scale hospital among the Chinese population.ParticipantsFor 10 consecutive working days in 2018, 22 809 adult patients who received a complete blood count (CBC) were reviewed. These patients were selected from the outpatient, inpatient and health examination departments of a National Medical Centre in China.ResultsA total of 254 patients (1.1%, 254/22 809) were found to have lymphocytosis (absolute lymphocyte count (ALC) >3.5×109/L). Among them, a population of circulating monoclonal B-lymphocytes were detected in 14 patients, with 4 having chronic lymphocytic leukaemia (CLL) and 10 having MBL, indicating an overall prevalence of 5.5% for B-LPD (3.9% for MBL). The prevalence of CLL among the elderly patients with lymphocytosis (≥60 years) was determined to be 4.3% (4/92). In the patients over 60 years of age, the prevalence of MBL was found to be 8.7%. CD5 (−) non-CLL-like MBL was observed to be the most common subtype (8, 80%), followed by CLL-like phenotype (1, 10.0%) and atypical CLL phenotype (1, 10.0%). The receiver operating characteristic curve analysis for the CBC results revealed that the ALC of 4.7×109/L may serve as the optimal and cost-effective cut-off for screening for early-stage asymptomatic B-LPD.ConclusionIn Chinese patients with lymphocytosis, there was a relatively high proportion of patients with CLL among individuals over 60 years of age. MBL is an age-related disorder. Non-CLL-like MBL was the most common MBL subtype, almost all of whom displayed a pattern of ‘marginal zone lymphoma (MZL)-like’ MBL. Lymphocytosis screening among the elderly would be effective in the detection of B-LPD and MBL.

A 10-Color Flow Cytometry Panel for Both Diagnosis and Minimal Residual Disease Measurement in Chronic Lymphocytic Leukemia

A 10-Color Flow Cytometry Panel for Both Diagnosis and Minimal Residual Disease Measurement in Chronic Lymphocytic Leukemia

Evaluation of a 10color protocol as part of a 2tube screening panel for flow cytometric assessment of peripheral blood leukocytic subsets

Peripheral blood (PB) immunophenotyping is commonly required for initial evaluation of various suspected disease entities. Several approaches have been proposed. The objective of this work is to explore the value of a 10color protocol developed in our laboratory for flow cytometric assessment of PB leukocytic subsets, as part of a 2tube screening panel. A combination of CD16/CD56/CD34/CD33/CD19/CD4/CD8/CD3/CD20/CD45 antibodies in 1 tube was applied routinely during flow cytometric analysis of PB samples for diagnostic purposes. The protocol was systematically complemented by a 2nd tube with anti-kappa, anti-lambda, CD5, CD19, and CD45 antibodies for adults and selected pediatric patients, and specifically oriented panels when necessary. 25 samples with no detectable neoplastic PB involvement and 31 samples with a hematolymphoid disorder were investigated retrospectively. The contribution of CD33 in the separation of leukocytic populations, as well as the benefits from the simultaneous assessment of CD20/CD19/CD45, CD16/CD56 and the detection of CD34+ cells were examined. The gating strategy with the use of CD33 provided additional information in certain cases. The protocol enabled recognition of differential expression of CD20 and CD45 in CD19+ cells with chronic lymphocytic leukemia phenotype, overall evaluation of NK and NK like T cells, estimation of CD16− granulocytes and CD56/CD16 expression in monocytes, as well as identification of minor cell subsets, such as CD34+ cells. The proposed 10color combination of antibodies analyzed in a standardized manner can offer significant information in the initial evaluation of PB samples, thus, guiding subsequent investigation if needed.

Multiparametric analysis of CD8+ T cell compartment phenotype in chronic lymphocytic leukemia reveals a signature associated with progression toward therapy

ABSTRACTCD8+ T cells are frontline defenders against cancer and primary targets of current immunotherapies. In CLL, specific functional alterations have been described in circulating CD8+ T cells, yet a global view of the CD8+ T cell compartment phenotype and of its real impact on disease progression is presently elusive. We developed a multidimensional statistical analysis of CD8+ T cell phenotypic marker expression based on whole blood multi-color flow-cytometry. The analysis comprises both unsupervised statistics (hClust and PCA) and supervised classification methods (Random forest, Adaboost algorithm, Decision tree learning and logistic regression) and allows to cluster patients by comparing multiple phenotypic markers expressed by CD8+ T cells.Our results reveal a global CD8+ T cell phenotypic signature in CLL patients that is significantly modified when compared to healthy donors. We also uncover a CD8+ T cell signature characteristic of patients evolving toward therapy within 6 months after phenotyping. The unbiased, not predetermined and multimodal approach highlights a prominent role of the memory compartment in the prognostic signature. The analysis also reveals that imbalance of the central/effector memory compartment in CD8+ T cells can occur irrespectively of the elapsed time after diagnosis.Taken together our results indicate that, in CLL patients, CD8+ T cell phenotype is imprinted by disease clinical progression and reveal that CD8+ T cell memory compartment alteration is not only a hallmark of CLL disease but also a signature of disease evolution toward the need for therapy.

Integrated Genomic and Proteomic Analysis of Murine CLL-like Cells Reveals SF3B1 Mutation to Impact DNA Damage Response and BCR Signaling

Collective large-scale sequencing efforts have unexpectedly revealed the high frequency of mutations in the splicing factor genes (SF3B1, U2AF1, SRSF2, ZRSR2) in various solid and hematological cancers, suggesting the association of splicing dysregulation with tumorigenesis. Mutations in SF3B1 occur in 5-20% of patients with chronic lymphocytic leukemia (CLL) and are associated with poorer overall survival and chemotherapy resistance. These mutations are restricted to hotspots (>50% at K700E site) and strongly co-occur with ATM mutations (loss-of-function) and deletion of 11q (ATM minimal deleted region). Numerous studies including ours have demonstrated that somatic alterations in this gene cause RNA splicing dysregulation, however, how this splicing factor mutation alone and in combination with ATM deletion impacts cellular processes and contributes to CLL remains to be fully defined.To this end, we modeled the effects of these combined alterations by crossing mice with conditional knockout of Atm and mice with a conditional knock-in allele of SF3B1 mutation (Sf3b1-K700E). We achieved B cell-restricted expression of heterozygous Sf3b1 mutation and Atm deletion by breeding these mice with CD19-Cre homozygous transgenic mice. Conditional expression of heterozygous Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters B-cell development, and induces a state of cellular senescence. Combined with Atm deletion in B cells led to the overcoming of cellular senescence and the development of clonal CLL cells in elderly mice at low penetrance (6%). These malignant cells could be propagated by in vivo passaging, with detectable disease within 4 weeks following transfer, thus making this mouse line amenable to further drug discovery and biologic investigations.To fully understand the underlying mechanisms of how the combined alterations led to CLL, we performed integrated genome, transcriptome, and proteome analysis using mouse CLL (DM-CLL) cells and B cells with either Sf3b1 mutation or Atm deletion, or with double genetic lesions (DM). Whole-genome sequencing of paired DNA from B cells (or DM-CLL) and non-B cell tissue (kidney) revealed the somatic mutation rate in the CLL cells to be ~0.5 mutations/Mb. Few recurrent mutations were identified among the samples. However, copy number variation analysis of DM-CLL cells revealed recurrent amplifications of chromosomes 15 and 17. RNA-seq analysis revealed that these amplifications were associated with overexpression of 835 of 987 Chr15 and Chr17 genes detected in DM-CLL vs. DM cells. Of note, 146 genes were overexpressed in human CLLs with SF3B1 mutations (DFCI cohort), compared to normal B-cells (p<0.05).Integrated transcriptome and proteome analysis of the DM-CLL cells showed coordinated dysregulation of multiple CLL-associated cellular processes with B-cell receptor (BCR) signaling as the most dramatically downregulated compared to DM cells. Since BCR signaling is a therapeutic target in CLL and has critical roles in B cell biology, we asked how SF3B1 mutation contributes to gene expression of BCR signaling. Through RNA-seq data analysis derived from two independent patient cohorts (DFCI and ICGC), we identified downregulation of BCR gene expression in SF3B1 mutant CLL cells. In line with this, human CLLs harboring SF3B1 mutations exhibit greater sensitivity to in vitro treatment with ibrutinib, and altered response kinetics in vivo to ibrutinib, per analysis of patients with SF3B1 mutations treated with ibrutinib. These studies together highlight a role of SF3B1 mutation in BCR signaling.In summary, we have generated a genetically-engineered murine model that recapitulates human CLL genetics, and presents an informative model to functionally dissect the effects of mutant SF3B1 in a B cell context. Starting from computation-based identification of recurrent co-occurring events in CLL, our study employs murine lines that express genetic alterations in an lineage-specific fashion, utilizes integrated genomics and proteomics approaches to dissect pathways that are fundamental to CLL phenotype, and more importantly, links the dysregulated pathways back to human CLL gene expression data and clinical trials to reveal novel mechanisms underlying therapeutic response. DisclosuresWiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding. Wu:Neon Therapeutics: Equity Ownership.