Monoclonal B-cell Lymphocytosis Patients 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.

Immune cell kinetics and antibody response in COVID-19 patients with low-count monoclonal B-cell lymphocytosis.

Low-count monoclonal B-cell lymphocytosis (MBLlo ) has been associated with an underlying immunodeficiency and has recently emerged as a new risk factor for severe COVID-19. Here, we investigated the kinetics of immune cell and antibody responses in blood during COVID-19 of MBLlo versus non-MBL patients. For this study, we analyzed the kinetics of immune cells in blood of 336 COVID-19 patients (74 MBLlo and 262 non-MBL), who had not been vaccinated against SARS-CoV-2, over a period of 43 weeks since the onset of infection, using high-sensitivity flow cytometry. Plasma levels of anti-SARS-CoV-2 antibodies were measured in parallel by ELISA. Overall, early after the onset of symptoms, MBLlo COVID-19 patients showed increased neutrophil, monocyte, and particularly, plasma cell (PC) counts, whereas eosinophil, dendritic cell, basophil, and lymphocyte counts were markedly decreased in blood of a variable percentage of samples, and with a tendency toward normal levels from week +5 of infection onward. Compared with non-MBL patients, MBLlo COVID-19 patients presented higher neutrophil counts, together with decreased pre-GC B-cell, dendritic cell, and innate-like T-cell counts. Higher PC levels, together with a delayed PC peak and greater plasma levels of anti-SARS-CoV-2-specific antibodies (at week +2 to week +4) were also observed in MBLlo patients. In summary, MBLlo COVID-19 patients share immune profiles previously described for patients with severe SARS-CoV-2 infection, associated with a delayed but more pronounced PC and antibody humoral response once compared with non-MBL patients.

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.

Prevalence of Clonal Hematopoiesis of Indeterminate Potential in Patients with Monoclonal B-Cell Lymphocytosis Compared to Untreated Chronic Lymphocytic Leukemia

Background: Monoclonal B-cell lymphocytosis (MBL) is a benign precursor condition to chronic lymphocytic leukemia (CLL). Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition characterized by accumulation of somatic mutations in the blood. Both conditions involve low level clonal populations in the blood. Review of the literature identified one prior abstract evaluating CHIP in patients with MBL. This study found CHIP in 17% of 251 patients with CLL (median age 63), 12% of 74 patients with MBL (median age 61.5), and 8% of 246 unaffected relatives (median age 54). CHIP was not more common in patients with MBL compared to CLL (Boddicker. Blood. 2018;132(S1): 3116). In this study, we focused on myeloid CHIP (M-CHIP) specifically, to distinguish CHIP mutations from MBL/CLL related mutations. Our objective was to characterize the prevalence of M-CHIP in patients with MBL compared to patients with untreated CLL. Methods: We performed a retrospective study including 44 patients with MBL and 749 patients with untreated CLL enrolled in a prospective CLL biobank at Dana-Farber Cancer Institute who had a 95-gene targeted next-generation sequencing (NGS) panel performed (J Mol Diagn. 2016;18:507). We created a list of 56 M-CHIP candidate genes using published criteria on M-CHIP (Nat Med. 2021;27:1921) and excluded 8 overlap genes that were more common in CLL than M-CHIP (SF3B1, TP53, BRAF, KRAS, BCOR, NRAS, CREBBP, SETD2; Knisbacher. Nat Genetics. In press). We performed a sensitivity analysis in which we only excluded overlap genes among the top 5 driver genes in CLL (specifically, only SF3B1, TP53). We analyzed the association of M-CHIP in patients with MBL vs. untreated CLL patients using logistic regression controlling for IGHV mutational status. We also performed an exploratory landmark survival analysis from time of NGS panel to assess the relationship between CHIP and progression from MBL to CLL. Results: In our cohort of 793 patients, there were no significant differences in age at diagnosis or NGS panel, sex, and cytogenetics, for patients with MBL vs. untreated CLL (Table 1). Median age at time of NGS panel for patients with MBL vs. untreated CLL was 65 (interquartile range (IQR) 60-71) and 64 (IQR 57-72) respectively. 43% vs. 40% of patients were female, respectively. Patients with MBL and untreated CLL patients were equally likely to have pathogenic mutations (50% vs. 60%, p=0.2) or VUS (70% vs. 77%, p=0.3) on the NGS panel, and had a similar total number of mutations (Table 1). Among patients with MBL, the most common pathogenic mutations were in DNMT3A (27%) and NOTCH1 (9%) and the most common VUS were in ATM (18%) and BCOR (14%). Among patients with untreated CLL, the most common pathogenic mutations were in NOTCH1 (15%) and SF3B1 (13%) and the most common VUS were in ATM (17%), CREBBP (6%), and TET2 (6%). The prevalence of M-CHIP was significantly greater among patients with MBL compared to patients with untreated CLL (23% vs. 12%, OR 2.2, 95% CI 1.1-4.7, p=0.034). However, when we used the more sensitive definition of M-CHIP, it was no longer more common in patients with MBL than CLL (25% vs. 18%, OR 1.7, 95% CI 0.8-2.2, p=0.15). Notably, the median age of patients with M-CHIP with MBL vs. untreated CLL was similar (70 (IQR 65-74) vs. 67 (IQR 61-74) (p=0.53)). Among patients with M-CHIP, patients with MBL were more likely to have DNMT3A mutations compared to patients with CLL (100% vs. 46%, p=0.001). In an exploratory analysis with a median follow-up time of 152 days (IQR 28-639), CHIP as a binary predictor was not associated with progression from MBL to CLL (HR 0.9, 95% CI 0.2-4.4, p=0.9). This analysis was limited in power due to a low number of progression events (N=9) and limited follow-up duration. Conclusions: When using a strict definition for M-CHIP, we found that the prevalence of M-CHIP was significantly greater in patients with MBL (23%) than in patients with untreated CLL (12%). However, this association did not persist when using a more sensitive definition of M-CHIP allowing more overlap between CLL driver genes and M-CHIP. Patients with CLL are more likely to have CLL driver genes than patients with MBL, explaining this discrepancy. This illustrates the challenges in M-CHIP categorization for patients with MBL/CLL, and further research is ongoing in which blood from patients with MBL is sorted into myeloid and lymphoid compartments to allow more definite categorization. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

COVID-19 vaccine failure in chronic lymphocytic leukaemia and monoclonal B-lymphocytosis; humoural and cellular immunity.

Chronic lymphocytic leukaemia (CLL) is associated with immunocompromise and high risk of severe COVID-19 disease and mortality. Monoclonal B-cell lymphocytosis (MBL) patients also have immune impairment. We evaluated humoural and cellular immune responses in 181 patients with CLL (160) and MBL (21) to correlate failed seroconversion [<50AU/ml SARS-CoV-2 II IgG assay, antibody to spike protein; Abbott Diagnostics)] following each of two vaccine doses with clinical and laboratory parameters. Following first and second doses, 79.2% then 45% of CLL, and 50% then 9.5% of MBL patients respectively remained seronegative. There was significant association between post dose two antibody level with pre-vaccination reduced IgM (p<0.0001), IgG2 (p<0.035), and IgG3 (p<0.046), and CLL therapy within 12months (p<0.001) in univariate analysis. By multivariate analysis, reduced IgM (p<0.0002) and active therapy (p<0.0002) retained significance. Anti-spike protein levels varied widely and were lower in CLL than MBL patients, and both lower than in normal donors. Neutralisation activity showed anti-spike levels <1000AU/ml were usually negative for both an early viral clade and the contemporary Delta variant and 72.9% of CLL and 53.3% of MBL failed to reach levels ≥1000AU/ml. In a representative sample, ~80% had normal T-cell responses. Failed seroconversion occurred in 36.6% of treatment-naïve patients, in 78.1% on therapy, and in 85.7% on ibrutinib.

TP53 Aberrations and Outcomes in MBL and Untreated CLL

TP53 Aberrations and Outcomes in MBL and Untreated CLL

Utilization of a Targeted Next Generation Sequencing Assay to Identify Copy Number Alterations in Chronic Lymphocytic Leukemia and Monoclonal B-Cell Lymphocytosis

Utilization of a Targeted Next Generation Sequencing Assay to Identify Copy Number Alterations in Chronic Lymphocytic Leukemia and Monoclonal B-Cell Lymphocytosis

Increase of DNA Damage and Alteration of the DNA Damage Response in Monoclonal B-Cell Lymphocytosis and Chronic Lymphocytic Leukemia

The increase of DNA damage and the alteration of the DNA damage response (DDR) are critical features of genetic instability presumably implicated in the pathogenesis of monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL). Immunofluorescence microscopy of γH2AX and 53BP1 was performed for the analysis of DNA double-strand breaks (DSB) in CD19+ lymphocytes of 6 healthy individuals, 6 MBL patients, 21 CLL patients and in cells of the CLL cell line HG-3. In addition, immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 was used for the analysis of the DNA damage response (DDR). As compared to the γH2AX foci levels in healthy individuals (0.1 focus per CD19+ lymphocyte ± 0.0; mean ± standard error of mean) increased γH2AX foci levels were detected in MBL patients (0.3 focus per CD19+ lymphocyte ± 0.1), CLL patients (1.3 foci per CD19+ lymphocyte ± 0.3) and in cells of the cell line HG-3 (1.0 focus per cell). Particularly high γH2AX foci levels were observed in immunoglobulin heavy-chain variable-region (IGHV)-unmutated, 11q-deficient, 17p-deficient and TP53 -mutated CLL. Co-localization of γH2AX and 53BP1 indicated promotion of 53BP1-mediated nonhomologous end-joining (NHEJ) repair mechanisms at sites of DSB. Absent expression of p-CHK1 and p-TP53 suggested impaired G2/M checkpoint control and impaired apoptosis signaling in MBL and CLL. In summary, our results provide evidence for a continuous increase of DNA damage across the spectrum from MBL to CLL in conjunction with an impaired DDR. Particularly high γH2AX foci levels were detected in IGHV -unmutated, 11q-deficient, 17p-deficient and TP53 -mutated CLL. Moreover, abundant γH2AX/53BP1 foci suggested a critical role for (in)effective NHEJ repair mechanisms at sites of DSB in MBL and CLL.Figure legendsFig. 1. Analysis of γH2AX foci in MBL and CLL patient samples. (A) Fluorescence microscopic image of γH2AX foci (green, Alexa 488) in nuclei (blue, DAPI) of the sample CLL8. (B) γH2AX foci levels in CD19+ lymphocytes of healthy individuals, MBL and CLL patients. (C) γH2AX foci levels in CLL samples according to CD38 and ZAP-70 expression. (D) γH2AX foci levels in CLL samples according to the immunoglobulin heavy-chain variable-region mutational status. (E) γH2AX foci levels according to cytogenetic risk categories. The categories were defined by (1) harboring a normal karyotype, (2) a 13q deletion as sole aberration, (3) a 12q trisomy but not an 11q or 17p deletion, (4) an 11q deletion but not a 17p deletion and (5) a 17p deletion. (F) γH2AX foci levels according to gene mutation categories. The categories were defined (irrespective of the karyotype) by harboring (1) no mutations in SF3B1, NOTCH1 and TP53, (2) a SF3B1 mutation but not a TP53 mutation, (3) a NOTCH1 mutation but not a TP53 or SF3B1 mutation and (4) a TP53 mutation (irrespective of other mutations). Note: Each dot in the graphs B - F represents the mean γH2AX foci level of one sample. MBL, monoclonal B-cell lymphocytosis; CLL, chronic lymphocytic leukemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reduced expansion of CD94/NKG2C+ NK cells in chronic lymphocytic leukemia and CLL-like monoclonal B-cell lymphocytosis is not related to increased human cytomegalovirus seronegativity or NKG2C deletions.

Dysregulated NK cell-mediated immune responses contribute to tumor evasion in chronic lymphocytic leukemia (CLL), although the NK cell compartment in CLL-like monoclonal B-cell lymphocytosis (MBL) is poorly understood. In healthy individuals, human cytomegalovirus (HCMV) induces the expansion of NK cells expressing high levels of CD94/NKG2C NK cell receptor (NKR) specific for HLA-E. We analyzed the expression of NKG2A, NKG2C, ILT2, KIR, CD161, and CD57 in 24 MBL and 37 CLL. NKG2C was genotyped in these patients and in 81 additional MBL/CLL, while NKG2C gene expression was assessed in 26 cases. In 8 CLL patients with increased lymphocytosis (≥20×109 /L), tumor HLA-E and HLA-G expression was evaluated. NKR distribution did not significantly differ between MBL and CLL patients, although they exhibited reduced NKG2C+ NK cells compared with a non-CLL group (4.6% vs 12.2%, P=.012). HCMV+ patients showed increased percentages of NKG2C+ NK cells compared with HCMV- (7.3% vs 2.9%, P=.176). Frequencies of NKG2C deletions in MBL/CLL were similar to those of the general population. Low/undetectable NKG2C expression was found among NKG2C+/- (45%) and NKG2C+/+ (12%) patients. CLL cases with increased lymphocytosis displayed especially reduced NKG2C expression (1.8% vs 8.1%, P=.029) and tumor cells with high HLA-E (>98%) and variable HLA-G expression (12.4%, range: 0.5-56.4). CLL patients with low NKG2C expression (<7%) showed shorter time to first treatment (P=.037). Reduced percentages of CD94/NKG2C+ NK cells were observed in CLL and MBL patients independently of HCMV serostatus and NKG2C zygosity, particularly in CLL patients with increased lymphocytosis, which could potentially be related to the exposure to tumor cells.

Reduced Expression of the CD94/NKG2C NK Cell Receptor in Chronic Lymphocytic Leukemia (CLL) and CLL-like Monoclonal B-Cell Lymphocytosis (MBL)

Background. Dysregulated NK-cell responses have been reported in chronic lymphocytic leukemia (CLL) patients, but little is known about the NK cell compartment in CLL-like monoclonal B cell lymphocytosis (MBL). Human cytomegalovirus (HCMV) infection induces an adaptive reconfiguration of the NK cell compartment characterized by the differentiation and persistent expansion of a subset displaying the CD94/NKG2C NK receptor (NKR). Moreover, a deletion of the NKG2C (KLRC2) gene has been reported to modulate the magnitude of the NK cell repertoire redistribution. Little is known about the expression of NKG2C in CLL and MBL patients. Aims. To analyse the distribution of NKR, with special attention to NKG2C, in MBL and CLL patients, assessing the relation of the NK cell immunophenotype with clinical features. Methods. The study cohort included 61 patients, 24 were diagnosed with clinical MBL and 37 were treatment-naïve CLL (32/37 Binet A). The expression of NKG2C, NKG2A, ILT2 (LIR1, LILRB1), CD161, CD57 and KIRs (identified with a cocktail of monoclonal antibodies) was assessed by flow cytometry in peripheral blood NK cells. The NKG2C (KLRC2) genotype was analysed in a larger representative MBL/CLL cohort (n=135). Results. The proportions of NK cells were reduced in CLL patients compared to MBL (median 5.5% vs. 10%; P=0.003), whereas their absolute numbers were increased (median 0.85x109/L vs. 0.57x109/L; P=0.002). No significant differences between MBL and CLL were detected regarding the distribution of the different NKR: NKG2C (median: 2.7 vs. 5.9%, respectively), NKG2A (31.4 vs. 30.8%), ILT2 (18.0 vs.15.8%), KIRs (54.4 vs. 52.7%), CD161 (16.1 vs. 16.4%) and CD57 (40.4 vs. 38.9%). Though a reduced NKG2C expression was noticed in both entities, it was specially marked in patients with a greater (&gt;30x109 cells/L) lymphocytosis (1.4 vs. 7.7%, P=0.016). The proportions of NKG2C+ NK cells in HCMV+ patients (85%, 47/55) as compared to HCMV- individuals were not significantly different (6.3% vs. 2.9%, respectively). HCMV+ patients showed a significantly lower NKG2C expression when compared with two independent age-matched cohorts of HCMV+ non-CLL/-MBL individuals, including 43 non-metastatic breast cancer patients (4.2% vs. 15.3% , P&lt;0.001); and 30 renal transplantation donors (4.2% vs.12.4% in P=0.028). The frequencies of NKG2C+/+ (56%), NKG2C+/del (37%) and NKG2Cdel/del (7%) genotypes were comparable to those previously defined in healthy blood donors. Moreover, cases with very low (&lt;2%) or undetectable NKG2C expression were found in NKG2Cdel/del patients (100%, 6/6), but also among NKG2C+/- (45%, 10/22) and NKG2C+/+ (12%, 3/26) genotypes. Conclusions. 1. MBL and CLL exhibited low proportions of NKG2C+ NK cells. This immunophenotype was particularly evident in CLL patients with increased lymphocytosis and could not be explained by differences in HCMV seropositivity, NKG2C zygosity nor age. 2. Additional studies are required to define the mechanism(s) and putative implications of the reduced NKG2C expression in these lymphoproliferative disorders. Acknowledgements. PI11/1621; PI15/437; 2017/SGR437; Fundació La Caixa; Fundación Española de Hematología y Hemoterapia (FEHH). Disclosures Gimeno: JANSSEN: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau. Abrisqueta:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau. Bosch:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding.

Developmental DNA Methylation Subtype Predicts Progression to Treatment and Survival in High-Count Monoclonal B Lymphocytosis

Developmental DNA Methylation Subtype Predicts Progression to Treatment and Survival in High-Count Monoclonal B Lymphocytosis

Tumor Mutational Load and Germline Polygenic Risk Score Predicts Time-to-First Treatment in Chronic Lymphocytic Leukemia (CLL) and High-Count Monoclonal B Cell Lymphocytosis (MBL)

Background The CLL international prognostic index (CLL-IPI) is a validated staging system for newly diagnosed CLL patients that combines age, clinical stage, serum Beta-2 microglobulin, TP53, and IGHV mutation status into a single score and stratifies CLL patients into four prognostic risk groups with a c-statistic=0.72. Next-generation sequencing identified ~60 putative driver genes recurrently mutated in CLL, and genome-wide association studies (GWAS) identified 41 single nucleotide polymorphisms (SNPs) associated with CLL risk. We previously showed that a polygenic risk score (PRS) of the weighted average of the number of risk alleles among these 41 SNPs is associated with risk of both CLL and MBL. We and others have also shown that the tumor mutational load (TML), i.e., the total number of somatically mutated driver genes, is associated with time to first treatment (TTT). Herein, we examine whether TML and PRS add prognostic value beyond CLL-IPI for disease progression in a cohort of CLL and high-count MBLs. Methods We used the Mayo Clinic CLL resource to identify newly diagnosed (&amp;lt;2 years) previously untreated CLL and high-count MBL patients based on the 2008 International Workshop CLL (IWCLL) criteria . We extracted DNA from CD5+/CD19+ clonal B-cells and sequenced the coding regions of 59 recurrently mutated CLL driver genes using a custom SureSelect panel and Illumina HiSeq 4000. The median coverage depth per sample was 2737X. Somatic mutations were called using MuTect2 in tumor-only mode. Germline variants were eliminated based on minor allele frequencies &amp;gt;0.01%, and by those identified in 1000 Genomes Project, ExAC and/or ESP6500 databases, unless present in known mutation hotspots or COSMIC. After filtering high/moderate impact mutations, we calculated the TML. Peripheral blood DNA was genotyped on Illumina Omni Express array; after extensive quality control metrics, we calculated the germline PRS as the weighted average of the number of risk alleles among the CLL susceptibility SNPs. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI) to test for associations with TTT. We analyzed the low/intermediate risk groups and the high/very high risk groups in stratified analyses and tested for interactions between CLL-IPI and TML or PRS. Survival curves are displayed using the Kaplan-Meier method using P-values from the log-rank test; the PRS is displayed by tertile categories. Results Of 166 patients (120 CLL, 46 MBL), 67% were male and the median age at diagnosis was 62 years (range: 28-85). The median follow-up was 5.3 years, and 73 were treated for progressive disease as defined by the 2018 IWCLL criteria. Low risk CLL-IPI comprised 51%, intermediate risk 27%, high risk 18%, and very-high risk 4%. The most commonly somatically mutated genes were TP53 (8%), CHD2 (8%), BIRC3 (8%), SF3B1 (7%), and NOTCH1 (7%). We found that 42%, 32%, and 26% of patients had 0, 1, or 2+ genes with somatic mutations, respectively. The median PRS was 8.3 (range: 6.1-11.3) which is consistent with our prior PRS study on CLL/MBL and higher than 7.53 found in 7983 controls from our prior work. When we modeled TML, PRS, sex, CLL/MBL status and CLL-IPI together with the joint effects of CLL-IPI with TML and PRS, both interactions were significant in the model (P=0.008 for TML*CLL-IPI and P=0.014 for PRS*CLL-IPI), with c-statistic=0.81 (CI:0.77-0.85). When we stratified by CLL-IPI, TML was associated with shorter TTT in the low/intermediate risk (N=129, continuous HR=2.36 per TML category, CI: 1.61-3.47, P=1.3x10-5, Figure 1.A), but there was no evidence of an association in the high/very-high risk (N=37, continuous HR=1.12 per TML category, CI: 0.70-1. 78, P=0.64, Figure 1.B), after adjusting for PRS, sex, and CLL/MBL status. However, the PRS was associated with a shorter TTT in the high/very-high risk (continuous HR=1.77 per SD, CI: 1.00-3.12, P=0.048, Figure 1.D), with no evidence of an association in the low/intermediate risk (continuous HR=0.95 per SD, CI: 0.95-1.29, P=0.74, Figure 1.C), after adjusting for TML, sex, and CLL/MBL status. Conclusions In our study, TML was prognostic among the low/intermediate CLL-IPI risk groups while the PRS was prognostic among the high/very-high CLL-IPI risk groups. Sequencing CLL driver genes and genotyping relevant germline SNPs at time of diagnosis may provide incremental value over the CLL-IPI in predicting TTT among patients with CLL/MBL. Disclosures Parikh: MorphoSys: Research Funding; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Janssen: Research Funding; Ascentage Pharma: Research Funding; Acerta Pharma: Research Funding. Ding:Merck: Research Funding; DTRM Biopharma: Research Funding. Cerhan:Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB.

Accumulation of DNA damage and alteration of the DNA damage response in monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia

Accumulation of DNA damage and alteration of the DNA damage response (DDR) are critical features of genetic instability that is presumed to be implicated in the pathogenesis of monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL). Here, we show increased numbers of γH2AX foci, a marker of DNA double-strand breaks (DSB), in CD19+ cells of CLL patients as compared to CD19+ cells of MBL patients and healthy individuals. Furthermore, numerous γH2AX/53BP1 foci in CLL cells suggest activation of error-prone non-homologous end-joining repair mechanisms. Signatures of DDR proteins further indicate alterations of the DDR in CLL in contrast to a largely regular activation in MBL and healthy controls. In summary, our results provide evidence for the stepwise accumulation of DNA damage in the progression of MBL towards CLL and suggest increased DNA damage, error-prone DNA repair and altered DDR signaling to be critical mechanisms of clonal evolution in MBL and CLL.

Richter transformation driven by Epstein-Barr virus reactivation during therapy-related immunosuppression in chronic lymphocytic leukaemia.

The increased risk of Richter transformation (RT) in patients with chronic lymphocytic leukaemia (CLL) due to Epstein-Barr virus (EBV) reactivation during immunosuppressive therapy with fludarabine other targeted agents remains controversial. Among 31 RT cases classified as diffuse large B-cell lymphoma (DLBCL), seven (23%) showed EBV expression. In contrast to EBV- tumours, EBV+ DLBCLs derived predominantly from IGVH-hypermutated CLL, and they also showed CLL-unrelated IGVH sequences more frequently. Intriguingly, despite having different cellular origins, clonally related and unrelated EBV+ DLBCLs shared a previous history of immunosuppressive chemo-immunotherapy, a non-germinal centre DLBCL phenotype, EBV latency programme type II or III, and very short survival. These data suggested that EBV reactivation during therapy-related immunosuppression can transform either CLL cells or non-tumoural B lymphocytes into EBV+ DLBCL. To investigate this hypothesis, xenogeneic transplantation of blood cells from 31 patients with CLL and monoclonal B-cell lymphocytosis (MBL) was performed in Rag2-/- IL2γc-/- mice. Remarkably, the recipients' impaired immunosurveillance favoured the spontaneous outgrowth of EBV+ B-cell clones from 95% of CLL and 64% of MBL patients samples, but not from healthy donors. Eventually, these cells generated monoclonal tumours (mostly CLL-unrelated but also CLL-related), recapitulating the principal features of EBV+ DLBCL in patients. Accordingly, clonally related and unrelated EBV+ DLBCL xenografts showed indistinguishable cellular, virological and molecular features, and synergistically responded to combined inhibition of EBV replication with ganciclovir and B-cell receptor signalling with ibrutinib in vivo. Our study underscores the risk of RT driven by EBV in CLL patients receiving immunosuppressive therapies, and provides the scientific rationale for testing ganciclovir and ibrutinib in EBV+ DLBCL. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

High LEF1 expression predicts adverse prognosis in chronic lymphocytic leukemia and may be targeted by ethacrynic acid.

Aberrant activation of lymphoid enhancer-binding factor-1 (LEF1) has been identified in several cancers, including chronic lymphocytic leukemia (CLL). As a key transcription factor of the Wnt/β-catenin pathway, LEF1 helps to regulate important genes involved in tumor cell death mechanisms. In this study, we determined LEF1 gene expression levels in CLL (n = 197) and monoclonal B-cell lymphocytosis (MBL) (n = 6) patients through real-time RT-PCR. LEF1 was significantly up-regulated in both MBL and CLL patients compared with normal B cells. Treatment-free survival (TFS) time and overall survival (OS) time were much longer in CLL patients with low LEF1 expression than in those with high LEF1 levels. Furthermore, Wnt inhibitor ethacrynic acid (EA) induced both apoptosis and necroptosis in primary CLL cells. EA also enhanced the cytotoxicity of both fludarabine and cyclophosphamide against CLL cells in vitro. Finally, we demonstrated that EA functions by inhibiting the recruitment of LEF1 to DNA promoters and restoring cylindromatosis (CYLD) expression in CLL cells. Our results showed, for the first time, that high LEF1 expression is associated with poor survival for CLL patients. Combined with other chemotherapeutic drugs, EA may be a promising therapeutic agent for CLL.

High‐throughput sequencing for the identification of NOTCH1 mutations in early stage chronic lymphocytic leukaemia: biological and clinical implications

NOTCH1 mutations have recently emerged as new genetic lesions significantly correlated with survival in chronic lymphocytic leukaemia (CLL). We performed deep next generation sequencing of the NOTCH1 mutation hotspot in 384 cases at diagnosis, including 100 monoclonal B cell lymphocytosis (MBL) and 284 Binet stage A CLL cases, enrolled in the Gruppo Italiano Studio Linfomi O-CLL1 multicentre trial. The NOTCH1 c.7541_7542delCT dinucleotide deletion was detected and confirmed by an extremely sensitive polymerase chain reaction-based approach in 11% of MBL and 13·4% of CLL patients. Remarkably, the NOTCH1 mutation was often observed at low clonal level, mainly in MBL patients. Sequential analyses in a fraction of cases showed that the NOTCH1 mutation generally does not occur during the disease course and that the mutational load in positive cases tends to be stable over time. NOTCH1-mutated cases, even at low clonal level, displayed a significant reduction in median progression-free survival, although NOTCH1 mutation lost its prognostic impact in a multivariate analysis including 11q and/or 17p deletion, IGHV mutational status, and MBL or CLL status. Our data highlight the importance of using highly sensitive methods to measure NOTCH1 mutations, in order to improve prognostic stratification and obtain useful information for potential therapeutic approaches.

Next-Generation Sequencing Reveals Oncogenic Driver Mutations In Both Progressive and Non-Progressive Monoclonal B Lymphocytosis

Essentially all cases of CLL are preceded by an asymptomatic expansion of a monoclonal B cell population, a condition that has been termed monoclonal B-cell lymphocytosis (MBL). MBL can be found in the peripheral blood of 3 to 5% of normal older adults. Although MBL is precursor state of CLL, the majority of MBL do not progress to CLL. Thus, MBL is being increasingly recognized and diagnosed in routine clinical practice, but the prognostic and treatment implications of MBL are unclear.The application of whole genome and exome sequencing has yielded important insights into somatic genetic mutations that contribute to the biology of CLL. Such work has identified a role for recurrent mutations in a number of novel genes including NOTCH1 and SF3B1 in CLL. However, the role of genetic mutations in the development of MBL and progression of MBL to CLL is completely unknown. Our central hypothesis is that acquired somatic mutations are responsible for the progression of MBL to CLL and determine an individual patient's disease course. Therefore, we applied exome sequencing to a cohort of MBL patients to understand the mutation profile of pre-emergent CLL and the determinants of progression from MBL to CLL.Thirty-six clinical MBL (CD19+ cell count: 0.5 – 5.0 x109/L) cases were ascertained through flow cytometric screening of a population based cohort of non-hematology patients identified via an absolute lymphocytosis on a routine complete blood count. Thirteen cases had a follow-up sample obtained at an average time of 4.8 years after initial study enrollment. All cases had a detectable CLL-phenotype clone but did not meet iwCLL criteria for a diagnosis of CLL. Samples with clinical follow-up were characterized as progressive or non-progressive based on whether the CLL phenotype clone expanded at the time of follow-up. CLL-phenotype cells and non-malignant monocytes were purified using FACS and DNA was extracted. We performed whole-exome sequencing using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13).A total of 86 exomes were sequenced for this study. Seven MBL cases were progressive and 29 MBL cases were non-progressive. Paired normal DNA was sequenced for all of these cases. Variants were filtered both through pair-wise comparisons of MBL and monocyte sequencing data and by prioritizing acquired mutations identified in know tumor suppressor or oncogenes. In all, we generated over 8 GB of sequencing data using high throughput sequencing on the Illumina platform. The number of mutations per exome were similar between progressive and non-progressive cases. Numerous non-synonymous variants in known tumor suppressor or oncogenes were identified in non-progressive cases including: NOTCH2, PIK3R1, SYK, MLL2, CDK12, CIITA, and CD79A. Non-synonymous variants identified exclusively in progressive cases included CREBBP and BRCA2.This study represents the first in-depth investigation of MBL exomes. Our data show that individual events in known oncogenes and tumor suppressor genes occur commonly in non-progressive MBL. Their presence does not indicate poor prognosis. These findings imply that sequential oncogenic mutations and combinations of specific genetic events are likely required for the progression of MBL to CLL. The knowledge of genetic variants that collectively contribute to the development of CLL would allow the development of risk stratification strategies for patients with MBL. Disclosures:No relevant conflicts of interest to declare.

Risk of Cancer in Patients with Clinical Monoclonal B-Cell Lymphocytosis (MBL): A Cohort Study of Newly Diagnosed Patients Compared to Controls.

AbstractAbstract 2893 Introduction:Monoclonal B-Cell Lymphocytosis (MBL) is a common asymptomatic condition characterized by a clonal B-cell population detected in the blood or bone marrow. The prevalence of MBL is approximately 3–5% in adults over age 40 but rises with age. MBL is a precursor state to CLL and other types of indolent non-Hodgkin lymphoma (NHL). Only a small subset of patients with MBL comes to clinical attention, typically during evaluation for low grade lymphocytosis (designated now as clinical MBL). The rate of progression to CLL requiring treatment among individuals with clinical MBL is ∼1%/yr, and the vast majority of patients with MBL will never develop a B-cell malignancy. Recent data indicate that individuals with MBL are at increased risk of infection similar to CLL. It is unknown whether individuals with MBL are at risk for other adverse outcomes associated with CLL, such as increased risk of non-hematologic cancers (NHC). We investigated the incidence of NHC among patients with clinical MBL and compared this to a cohort of general medicine patients and a cohort of CLL patients. Methods:Mayo Clinic Rochester (MCR) is the primary center for hematologic care in a region including southeastern Minnesota, northern Iowa, and western Wisconsin. No other hematology specialty centers are available within a 50 mile radius of MCR. To explore the risk of NHC in a local, community dwelling cohort of individuals with clinically-identified, CLL-phenotype MBL, we used the MCR CLL database to identify all individuals with newly diagnosed MBL seen in the MCR Hematology Division between January 1, 1999 and December 31, 2009. Analysis was limited to 154 MBL cases residing within 50 miles of MCR. The comparison cohort consisted of 596 adult general medicine patients, with no prior cancer, residing within 50 miles of MCR, who were seen for a general medical exam and enrolled as controls in a case-control study for NHL between April 25, 2004 and December 31, 2009. Rates of NHC in the MBL cohort were also compared to a cohort of 174 patients with newly diagnosed CLL seen in the same time interval as the MBL cohort and also living within 50 miles of MCR. NHC were identified using the Mayo Clinic Cancer Tumor Registry as well as review of medical records in all 3 cohorts. Patients with a NHC diagnosis >3 months prior to the diagnosis date of MBL/CLL were excluded from analysis while those with MBL/CLL and a NHC within 3 months of one another were considered to have a concurrent diagnosis. Results:There were 125 patients in the MBL cohort, 153 in the CLL cohort, and 596 in the control cohort after excluding patients with prior cancers. The MBL and CLL patients tended to be older (median age MBL=69, CLL=68, Control=58; p<0.0001) and more likely to be male (% male MBL=61, CLL=67, Control=46; p<0.0001) than the controls.After a median follow-up of 4.2 years (range 0–11), 20 (16%) individuals with MBL developed NHC as compared to 22 (4%) of the controls (Hazard Ratio (HR) MBL relative to control=5.0; 95% confidence interval (CI)=(2.6, 9.4; p<0.0001). Twenty CLL patients (13%) developed NHC, supporting a similar rate to that of MBL patients (HR MBL relative to CLL=1.3; 95% CI=(0.6, 2.5; p=0.49;See Figure). Specifically, the MBL cohort had a higher incidence of cancer of the breast, lung, and nervous system than that of the control cohort. The higher risk of NHC among individuals with CLL and MBL persisted even when patients with “concurrent diagnosis” of CLL/MBL and NHC were excluded (p=0.006) and when patients treated for progressive CLL were censored at the date of treatment (p=0.001)Finally, we performed a pooled multivariable analysis of all patients (n=874) adjusting for age, sex, major co-morbidities, presence of MBL, and diagnosis of CLL. Age (HR each year older=1.03; 95% CI 1.01–1.06; p=0.003), major comorbidities (HR=1.81; 95% CI 1.07–3.00; p=0.02), CLL (HR=2.10; 95% CI 1.01–4.01; p=0.04), and MBL (2.47; 95% CI 1.30–4.69; p=0.02) were independent risk factors for NHC. The results were similar when the effects of age was modeled in a non-linear (e.g. logarithmic) manner. Conclusions:In this cohort study, patients with newly diagnosed clinical MBL had a 2.5-fold increased risk of developing NHC relative to the control cohort. MBL was an independent risk factor for development of NHC after adjusting for age, sex, and other comorbidities. This finding adds to the growing evidence that clinical MBL has a distinct clinical phenotype. [Display omitted] Disclosures:Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Infectious Complications Among Individuals with Monoclonal B-Cell Lymphocytosis (MBL): A Prospective Case-Control Study of Newly Diagnosed Patients,

Abstract 3903▪▪This icon denotes a clinically relevant abstract INTRODUCTION:Monoclonal B-Cell Lymphocytosis is an asymptomatic hematologic condition characterized by the presence of small populations of clonal B-cells in the blood or bone marrow occurring in 3–5% of the general population over age 40. Although MBL is considered the precursor state to CLL and other sub-types of indolent non-Hodgkin Lymphoma, most individuals with MBL will not develop a B-cell malignancy. It is unknown, however, whether individuals with MBL may be at risk for other adverse outcomes associated with CLL, such as increased risk of infection. To explore this aspect, we evaluated the risk of hospitalization for infection among patients with clinically recognized MBL and compared this to a cohort of general medicine patients and a cohort of CLL patients. METHODS:The Mayo Clinic Rochester (MCR), a tertiary referral center in rural southeastern Minnesota, is also the primary hematology center for southeastern Minnesota, northern Iowa, and western Wisconsin. There are no metropolitan areas or hematology specialty centers within a 50 mile radius. To explore the natural history of infection in a local, community dwelling cohort of individuals with clinically identified, CLL-phenotype MBL, we used the Mayo Clinic CLL database to identify all patients with newly diagnosed MBL seen in the MCR Hematology Division between January 1999 and December 2009. Analysis was limited to MBL cases who resided within 50 miles of MCR. The comparison cohort consisted of 689 adult patients also resided within 50 miles of MCR who were seen for a general medical examination between September 2002 and December 2009 and who were enrolled as controls in a case-control study of non-Hodgkin lymphoma. Rates of infection among individuals with MBL were also compared to a cohort of 174 patients with newly diagnosed CLL diagnosed in the same time interval and who resided within 50 miles of MCR. All hospitalizations in these 3 groups were audited to document hospitalization with infection including cross-referenced with the electronic Mayo Clinic infection database, which contains all culture results obtained from hospitalized patients at MCR. Serologies and viral assays (e.g. CMV viral load) were also reviewed. Patients whose cultures were negative but who were given a clinical diagnosis of infection (e.g. pneumonia) and who were treated with a full course of antibiotics were considered to have a culture negative infection. Among the cases, only hospitalizations after CLL or MBL diagnosis were considered. RESULTS:In May 2010, there were 524 individuals in the Mayo Clinic MBL/CLL Database with the diagnosis of CLL-phenotype MBL made between January 1999 – December 2009. Of these, 154 (29%) patients resided within a 50 mile radius of Mayo Clinic and were considered a local, community-based cohort of MBL patients. Median age at MBL diagnosis for these 154 patients was 70 years (range 45–94).After median follow-up of 4.2 years (range 0.3–10.6 years), 25 (16%) individuals with MBL were hospitalized with infection as compared to 20/689 (3%) individuals in the control population (odds ratio MBL relative to control=6.5; p<0.001) and 32/174 (18%) in the cohort of patients with newly diagnosed CLL (OR MBL relative to CLL=0.9; p=0.61) (Figure 1). The 25 MBL patients hospitalized for infection were hospitalized a total of 39 times (median 1 hospitalization for infection per individual). A specific infectious organism was identified by cultures or serologies in 8/39 (21%) hospitalizations among individuals with MBL, 10/27 (37%) hospitalizations for infection among controls, and 60 (63%) of the 89 hospitalizations for infection in CLL cases.Finally we performed a pooled multivariable analysis of all patients (n=1017) adjusting for age, sex, presence of MBL, diagnosis of CLL, and whether patients with CLL or MBL received treatment for CLL during follow-up. All factors were independently associated with risk of hospitalization for infection [Age (per year, OR=1.02, p=0.02), sex (male, OR=2.6, p=0.001), presence of MBL (OR=3.3, p<0.001), diagnosis of CLL (OR=3.0, p=0.002), and treatment for CLL (OR=3.3, p<0.001)]. CONCLUSIONS:In this cohort study, patients with newly diagnosed clinical-MBL had a 6.5 fold risk of hospitalization for infection relative to the control cohort. MBL was an independent risk factor for hospitalization for infection after controlling for age, sex, and treatment. [Display omitted] Disclosures:Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding. Kay:Biothera: Research Funding; Clegene: Research Funding; Cephalon: Research Funding; Genentech: Research Funding; Glaxo Smith Kline: Research Funding; Hospira: Research Funding; Novartis: Research Funding; Supergen: Research Funding; Calistoga: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Emergent Biosolutions (Formerly Trubion): Membership on an entity's Board of Directors or advisory committees.

Circulating Regulatory T Cells in “Clinical” Monoclonal B-Cell Lymphocytosis

Regulatory T-cells (Tregs) constitute a small subset of cells involved in antitumour immunity and are generally increased in patients with chronic lymphocytic leukemia (CLL). No data is available on Tregs in monoclonal B-cell lymphocytosis (MBL), a disease entity characterized by less than 5000/microL circulating clonal B-cells in absence of other features of lymphoproliferative disorders. We used multicolour flow cytometry to evaluate the number of circulating Tregs in 56 patients with &#x0022;clinical&#x0022; MBL, 74 patients with previously untreated CLL and 40 healthy subjects. MBL patients showed a lower absolute number of Tregs, compared to CLL patients, but slightly higher than controls. Moreover, the absolute cell number of Tregs directly correlated both with more advanced Rai/Binet clinical stages and peripheral blood B-cell lymphocytosis. Of note, the absolute number of Tregs was found lower in MBL patients than in CLL patients staged as 0/A Rai/Binet. The study showed that Treg increase gradually from normal subjects to &#x0022;clinical&#x0022; MBL patients and are significantly higher in CLL patients as compared to MBL patients. Moreover, a significant direct relationship was found between higher Treg values and a higher tumor burden expressed by B-lymphocytosis or more advanced clinical stages. In light of this data, MBL seems to be a preliminary phase preceding CLL. The progressive increase of Treg numbers might contribute both to the clinical evolution of MBL to overt CLL and to CLL progression.