Low-count Monoclonal B-cell Lymphocytosis Research Articles

Progressive multifocal leukoencephalopathy as the presenting feature in a patient with occult low-count monoclonal B-cell lymphocytosis.

Low Count Monoclonal B-Cell Lymphocytosis (LC-MBL) is a relatively poorly understood entity which has been suggested to be very common in asymptomatic adults and possibly related to infectious complications despite not progressing to CLL. We describe the first case of Progressive Multifocal Leukoencephalopathy (PML) presenting in a 72-year-old man with LC-MBL but no other immunocompromising conditions. A diagnosis of PML was confirmed with classic MRI findings in association with a high CSF John Cunningham polyomavirus (JCV) viral load (4.09' 105 copies/mL). An extensive search for underlying immunocompromising conditions only demonstrated LC-MBL representing approximately 4% of total leukocytes (0.2' 109/L). This is the first report of PML in association with LC-MBL. Careful review of peripheral blood flow cytometry results is necessary to identify this disorder. Further study of the epidemiology and infectious complications of LC-MBL are warranted.

Risk of incident melanoma among individuals with low-count monoclonal B-cell lymphocytosis (MBL).

7577 Background: Prior research has consistently shown that patients with chronic lymphocytic leukemia (CLL) have an ~2-fold increased risk of developing melanoma compared to controls. MBL is a common condition affecting 8-10 million adults over the age of 40 years in the United States and is a precursor to CLL. Little is known about the association between MBL and melanoma. Herein, we evaluated the risk of incident melanoma in a large cohort of individuals screened for MBL. Methods: Participants from the Mayo Clinic Biobank who had no prior history of hematologic malignancy, were 40 years or older, were residents of counties surrounding Mayo Clinic, and who provided blood samples between 7/2009 and 4/2022 were included in the study. Peripheral blood mononuclear cells were screened for MBL using highly sensitive eight-color flow cytometry. Individuals with MBL were classified as CLL-like MBL (CD5+, CD20dim) or atypical MBL (CD5+, CD20+) based on immunophenotypes, and also classified as low-count MBL (LCMBL) or high-count MBL (HCMBL) based on whether the percent clonal B-cell count was < 85% or ≥85%, respectively. Diagnosis of melanoma was ascertained using ICD codes and review of the electronic health records. All individuals were followed from date of sample upon which MBL screening was performed to the earliest of incident melanoma, loss to follow-up, withdrawal from the study, or September 30, 2022. We used Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (CI), adjusting for age, sex, and history of melanoma in previous 5 years. Results: A total of 7,334 participants were screened for MBL, of which 1,151 (16%) individuals had MBL. Among those with MBL, 1,107 (96.2%) had CLL-like MBL and 1098 (95.4%) had LC MBL. The median age at entry into the MBL screening cohort was 67 years (range 40-101) and 37% were male; individuals with MBL were older (median age 72 vs 65 years) and male (49% vs 35%). After a median follow-up of 2.9 years (range 0-13.2), 121 of the 7,334 (1.6%) participants developed incident melanoma after the date of MBL screening. The 10-year cumulative incidence of melanoma among individuals with MBL was 7.8% (95% CI 4.5-13.5%) compared to 3.4% (2.7-4.4%) in those who screened negative for MBL. After adjusting for age, sex, and history of prior melanoma, we found a 1.81-fold increased risk of incident melanoma (95% CI 1.19-2.76) among individuals with MBL. The increased risk of melanoma persisted when the analysis was restricted to individuals with LCMBL (HR = 1.84; 95% CI 1.21-2.82). Conclusions: In the largest MBL screening cohort to date, we found that LCMBL, a common condition, was associated with an 84% elevated risk of melanoma, similar to the ~2-fold increased risk observed in patients with CLL. These data, together with our prior findings of a heightened risk of serious infections and lymphoid malignancies, suggest that individuals with LCMBL are at increased risk for adverse health outcomes.

Age- and Sex-Matched Normal Leukocyte Subset Ranges in the General Population Defined with the EuroFlow Lymphocyte Screening Tube (LST) for Monoclonal B-Cell Lymphocytosis (MBL) vs. Non-MBL Subjects.

Reference ranges of blood-circulating leukocyte populations by, e.g., age and sex, are required for monitoring immune-cell kinetics. Most previous reports in which flow cytometry has been used to define the reference ranges for leukocyte counts included a limited number of donors and/or cell populations and/or did not consider age and sex simultaneously. Moreover, other factors not previously considered in the definition of normal ranges, such as the presence of chronic-lymphocytic-leukemia (CLL)-like low-count monoclonal B-cell lymphocytosis (MBLlo), might also be associated with an altered distribution of leukocytes in blood in association with an immunodeficiency and increased risk of infection and cancer. Here, we established reference cell-count ranges for the major populations of leukocytes in blood of non-MBL and MBLlo adult Caucasians matched by age and sex using the EuroFlow Lymphocyte Screening Tube (LST). A total of 706 Caucasian adult donors—622 non-MBL and 84 MBLlo—were recruited from the general population. Among non-MBL donors, the total leukocyte, neutrophil, basophil dendritic cell and monocyte counts remained stable through adulthood, while the absolute numbers of T- and B-cell populations and plasma cells decreased with age. The number of eosinophils and NK-cell increased over time, with clear differences according to sex for certain age ranges. In MBLlo subjects, few differences in the absolute cell counts by age (vs. non-MBL) were observed, and MBLlo men and women showed similar trends to non-MBL subjects except for the B-cell count drop observed in >70 y-men, which was more pronounced in MBLlo vs. non-MBL controls. Building robust age- and sex-matched reference ranges for the most relevant immune-cell populations in the blood of non-MBL donors is essential to appropriately identify an altered immune status in different clinical settings and highlight the altered immune-cell profiles of MBLlo subjects.

High frequency of low-count monoclonal B-cell lymphocytosis in hospitalized COVID-19 patients

High frequency of low-count monoclonal B-cell lymphocytosis in hospitalized COVID-19 patients

Abstract 5256: Incidence of squamous cell carcinoma (SCC) in a large screening cohort of monoclonal B-cell lymphocytosis (MBL)

Abstract MBL is a common pre-malignant condition characterized by circulating clonal B-cells with an absolute B-cell count <5x109/L and no lymphadenopathy, organomegaly, or cytopenias. MBL is the precursor to CLL. The incidence of cutaneous SCC in CLL is significantly higher compared to controls. The incidence of SCC in MBLs has not yet been determined. Study participants from the Mayo Clinic Biobank who had no prior history of hematologic malignancy, were 40 years or older, and were Olmsted County residents completed a health questionnaire and provided blood samples between 7/2009 and 12/2020. Stored peripheral blood mononuclear cells were screened for MBL using flow cytometry. We defined three MBL immunophenotypes: CLL-like MBL (CD5+, CD20dim), atypical MBL (CD5+, CD20+), and non-CLL-like MBL (CD5-, CD20+). MBL individuals were also classified by cell count into low-count MBL (LC-MBL) and high-count MBL (HC-MBL), with HC-MBL having a percent clonal B-cell count ≥85%. Data on newly diagnosed SCC was abstracted from the medical records, and prior history of skin cancer before sample collection was ascertained from patient questionnaires. Individuals were followed from sample date to the earliest of SCC, death, loss to follow-up, progression, or 12/31/2020. Cumulative SCC incidence was adjusted for competing risk of death. Cox regression was used to calculate hazard ratios (HR) and 95% confidence intervals (CIs) adjusted for age and sex. A total of 5,470 participants were screened for MBL and included 949 (17%) with LC-MBL, 63 (1%) with HC-MBL, and 4,458 (81%) controls (negative for MBL). Individuals with HC-MBL (median age 75 years) or LC-MBL (median age 73 years) were significantly older (P<0.001) than controls (median age 66 years). There were 41 (65%) males among HC-MBL, 447 (47%) among LC-MBL, and 1,551 (35%) among controls. Prior skin cancer history was highest among HC-MBLs (N=17, 27%) or LC-MBLs (N=236, 25%) compared to controls (N=767, 17%). After a median follow-up of 18 months (range 0-138), 154 of the 5,470 individuals were identified to have incident SCC following MBL screening. At least one SCC was observed in 3 individuals with HC-MBL, 33 individuals with LC-MBL, and 118 controls. The 5- and 10-year cumulative incidence of SCC in individuals with MBLs was 7% and 16%, respectively; control estimates were 4% and 8%, respectively. However, after adjusting for age and sex, we observed no evidence of an association between MBL and risk of incident SCC (HR=0.95, CI=0.65-1.40, P=0.80), nor when we stratified individuals by age or by sex (all P>0.05). In the largest MBL screening cohort to date, individuals with MBL do not have an increased risk of incident SCC compared to controls. In contrast to individuals with CLL, these individuals with screening MBL do not need increased dermatologic examination for skin cancer, which is important given the high prevalence of MBL (18% of the population above age 40). Citation Format: Ahmed Nadeem Ansari, Sara J. Achenbach, Sameer A. Parikh, Geffen Kleinstern, Aaron D. Norman, Kari G. Rabe, Connie E. Lesnick, Timothy G. Call, Janet E. Olson, James R. Cerhan, Neil E. Kay, Celine M. Vachon, Esteban Braggio, Curtis A. Hanson, Tait D. Shanafelt, Christian L. Baum, Susan L. Slager. Incidence of squamous cell carcinoma (SCC) in a large screening cohort of monoclonal B-cell lymphocytosis (MBL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5256.

High Frequency of Low-Count Monoclonal B-Cell Lymphocytosis in Hospitalized COVID-19 Patients

High Frequency of Low-Count Monoclonal B-Cell Lymphocytosis in Hospitalized COVID-19 Patients

Prevalence and Overall Survival of Low Count Monoclonal B-Cell Lymphocytosis (LC-MBL): A Screening Study of 8,297 Individuals from the Mayo Clinic Biobank

Prevalence and Overall Survival of Low Count Monoclonal B-Cell Lymphocytosis (LC-MBL): A Screening Study of 8,297 Individuals from the Mayo Clinic Biobank

Low-Count Monoclonal B-Cell Lymphocytosis Persists after 7 Years of Follow-up and Confers a Higher Risk of Death

Monoclonal B-cell lymphocytosis (MBL) is characterized by the presence of low numbers of circulating clonal B cells that usually display highly-similar phenotypic features to chronic lymphocytic leukemia (CLL) cells. This entity is divided into high-count (MBLhi) and low-count (MBLlo), depending on the number of peripheral blood (PB) clonal B cells (higher vs. lower than 0.5x109/L, respectively). Whereas MBLhi is frequently detected in laboratory routine analyses and precedes CLL, MBLlo is mainly detected in population-based screening studies, making its biological and clinical significance elusive. Of note, prevalence of MBLlo is significantly higher than that of MBLhi and CLL, and increases with age, with a prevalence >20% among individuals older than 70y.Here, we re-evaluated 65/91 MBLlo subjects (54 CLL-like and 11 non-CLL-like) followed-up for >5 years (median of 7 years), using high-sensitivity flow cytometry. Cytogenetic alterations related to CLL (i.e. del13q14, trisomy 12, del11(q22-q23) and del17p13) and other chronic B-cell tumors were studied by iFISH in purified MBL clonal B cells.Overall, the clone size significantly increased (median of 2-3-fold) in 69% of MBLlo cases, but only one subject progressed to MBLhi. In parallel, the frequency of cytogenetic alterations increased over time (32% vs. 61% of cases at baseline and at follow-up, respectively). The absolute number of the major T- and NK-cell populations was also increased, but only among CLL-like cases with increased clone size. To prevent a potential age-related bias and to further confirm these findings, we compared the number of PB normal lymphocyte subsets in CLL-like MBLlo subjects vs . a large series of age- and gender-matched non-MBL healthy donors.Although progression to CLL was not observed, overall survival of MBLlo subjects was significantly reduced vs. non-MBL controls studied in parallel (P=0.03) plus the general population from the same region (P≤0.001), particularly among females (P=0.01); infection and cancer, were the main causes of death in MBLlo. In summary, despite mid-term progression from MBLlo to MBLhi and CLL appears to be unlikely, MBLlo clones persist at increased numbers usually carrying more genetic alterations, in association with higher-risk of death in comparison with healthy individuals, particularly among females. DisclosuresNo relevant conflicts of interest to declare.

A Tissue Counterpart to Monoclonal B-Cell Lymphocytosis.

B-cell clones discovered in tissue biopsies, without overt lymphoma, may represent a tissue counterpart to peripheral blood monoclonal B-cell lymphocytosis (MBL), herein termed tMBL. To characterize the clinicopathologic features of tMBL. During a 10-year period, we retrospectively identified non-bone marrow/peripheral blood cases with monotypic B cells detected by tissue-based flow cytometry but without an identifiable lymphomatous infiltrate on routine histopathology. We excluded cases with prior diagnosis of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma or MBL. Fifty-four cases were identified (35 lymph node, 3 splenic, and 16 soft tissue/viscera). Forty-six cases were CLL-type, 2 were atypical CLL, and 6 were non-CLL. tMBL was detectable by immunohistochemistry in 14 cases (26%, all CLL-type). Concurrent blood flow cytometry, available in 10 cases, showed 4 with low-count MBL (3 CLL-type, 1 with non-CLL-type), 5 with high-count MBL (all CLL-type), and 1 case negative for clonal population. With median follow-up of 51 months, 2 patients had progression of disease (CLL, 68.7 months; and diffuse large B-cell lymphoma, 5.9 months). Patients with immunohistochemistry-detectable tMBL had increased monoclonal B cells per total lymphocyte events (P = .01), morphologic evidence of bone marrow involvement (P = .04), higher white blood cell count (P = .02), and increased absolute lymphocyte count (P = .02). tMBL spans an immunophenotypic spectrum similar to MBL, is detectable by immunohistochemistry in a minority of cases (often CLL immunophenotype), and is likely systemic in most cases. Development of overt lymphoma is uncommon but may occur, warranting clinical follow-up.

Natural history of monoclonal B-cell lymphocytosis among relatives in CLL families

Chronic lymphocytic lymphoma (CLL) has one of the highest familial risks among cancers. Monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, has a higher prevalence (13%-18%) in families with 2 or more members with CLL compared with the general population (5%-12%). Although, the rate of progression to CLL for high-count MBLs (clonal B-cell count ≥500/µL) is ∼1% to 5%/y, no low-count MBLs have been reported to progress to date. We report the incidence and natural history of MBL in relatives from CLL families. In 310 CLL families, we screened 1045 relatives for MBL using highly sensitive flow cytometry and prospectively followed 449 of them. MBL incidence was directly age- and sex-adjusted to the 2010 US population. CLL cumulative incidence was estimated using Kaplan-Meier survival curves. At baseline, the prevalence of MBL was 22% (235/1045 relatives). After a median follow-up of 8.1 years among 449 relatives, 12 individuals progressed to CLL with a 5-year cumulative incidence of 1.8%. When considering just the 139 relatives with low-count MBL, the 5-year cumulative incidence increased to 5.7%. Finally, 264 had no MBL at baseline, of whom 60 individuals subsequently developed MBL (2 high-count and 58 low-count MBLs) with an age- and sex-adjusted incidence of 3.5% after a median of 6 years of follow-up. In a screening cohort of relatives from CLL families, we reported progression from normal-count to low-count MBL to high-count MBL to CLL, demonstrating that low-count MBL precedes progression to CLL. We estimated a 1.1% annual rate of progression from low-count MBL, which is in excess of that in the general population.

Polygenic Risk Score and Risk of Chronic Lymphocytic Leukemia, Monoclonal B-Cell Lymphocytosis (MBL), and MBL Subtypes

Background MBL is a precursor to chronic lymphocytic leukemia (CLL) and is subclassified into low-count (LC) MBL (absolute B-cell count<0.5x109/L) and high-count (HC) MBL (absolute B-cell count between 0.5 and 5x109/L). We previously reported that a polygenic risk score (PRS) based on a weighted average of 41 CLL-susceptibility variants was associated with risk of both MBL and CLL among a cohort of individuals from CLL families. Here we evaluate this PRS in an independent cohort of MBL and CLL individuals of European ancestry (EA), all of whom were ascertained agnostic to CLL family-history status. We also evaluate the PRS by MBL subtype (LC/HC), and in African American (AA) CLL cases and controls. Methods We genotyped 535 EA MBLs (139 HC-MBL, 396 LC-MBLs), 735 CLLs (640 EA, 95 AA), and 2,866 controls (2,631 EA, 235 AA) from the Mayo Clinic CLL Resource, Duke University, and Weill Cornell Medical College. We computed the CLL-PRS for each individual and used logistic regression to estimate odds ratios (OR) and 95% confidence intervals, adjusting for age and sex. To assess discriminatory accuracy, we computed the c-statistic. Among EA individuals, we calculated a trend test among LC-MBL, HC-MBL, and CLL risk using the P-value for heterogeneity from a polytomous logistic regression analysis. Moreover, we plotted a boxplot for the PRS among controls, LC-MBL, HC-MBL, and EA CLL, as well as for AA CLL cases and controls, and tested the statistical difference using the Kruskal Wallis test and Mann-Whitney test, respectively. Results We found a significant association of PRS with overall MBL risk (OR=1.87, P=1.1x10-28) with good discrimination (c-statistic=0.72). Significant associations were also found for LC-MBL (OR=1.75, P=7.5x10-19, c-statistic=0.72), HC-MBL (OR=2.22, P=1.4x10-17, c-statistic=0.74), and CLL of EA (OR=2.60, P=1.2x10-62, c-statistic=0.78), with a significant difference among these cohorts (Figure 1.A) and a significant positive trend across these cohorts (Pheterogeneity=8.4x10-6). Although we observed a 33% increased risk of CLL in AA (c-statistic=0.57), the PRS was borderline significant (P=0.07, Figure 1.B). Conclusion The CLL-PRS is a strong prediction-tool for risk of CLL and MBL among individuals of EA. Future studies are needed to improve the PRS for AAs including performing GWAS of AA in order to identify CLL-susceptibility SNPs that are more representative within known CLL loci and to discover novel CLL loci that are unique for AAs. Disclosures Parikh: GlaxoSmithKline: Honoraria; Janssen: Honoraria, Research Funding; Ascentage Pharma: Research Funding; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Verastem Oncology: Honoraria. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Brander:Genentech: Consultancy, Honoraria, Other, Research Funding; Juno/Celgene/BMS: Other, Research Funding; MEI Pharma: Other, Research Funding; Ascentage: Other, Research Funding; ArQule: Consultancy, Other, Research Funding; NCCN: Other; Teva: Consultancy, Honoraria; Tolero: Research Funding; AstraZeneca: Consultancy, Honoraria, Other, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Other, Research Funding; Pfizer: Consultancy, Other; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Novartis: Consultancy, Other; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Tolero: Research Funding; Teva: Consultancy, Honoraria; DTRM: Other, Research Funding; BeiGene: Other, Research Funding; Novartis: Consultancy, Other; NCCN: Other; Verastem: Consultancy, Honoraria, Other, Research Funding. Cerhan:NanoString: Research Funding; BMS/Celgene: Research Funding. Kay:Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; MEI Pharma: Research Funding; Abbvie: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees. Furman:Acerta: Consultancy; AstraZeneca: Consultancy, Research Funding; Beigene: Consultancy; Abbvie: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy, Research Funding; Verastem: Consultancy; Incyte: Consultancy; Genentech: Consultancy; Janssen: Consultancy, Speakers Bureau; Loxo Oncology: Consultancy; Oncotarget: Consultancy. Shanafelt:Mayo Clinic: Patents & Royalties: and other intellectual property; Genentech, Pharmacyclics LLC, an AbbVie Company, AbbVie, GlaxoSmithKline, and Merck: 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

Association between the Risk of Low/High-Count Monoclonal B-Cell Lymphocytosis (MBL) and the Chronic Lymphocytic Leukemia (CLL) Polygenic Risk Score (PRS)

BackgroundMBL is a precursor state to CLL, with a prevalence of ~5-8% in the general population and 15-18% in CLL families. To date, 41 single nucleotide polymorphisms (SNPs) have been found to be associated with CLL. We previously showed that a PRS of the weighted average of the number of risk alleles of these SNPs is associated with CLL risk using cases and controls from the International Lymphoma Epidemiology (InterLymph) Consortium. We validated this score in an independent sample of CLL cases and controls from the Genetic Epidemiology of CLL (GEC) Consortium, a cohort of families with ≥2 members with CLL. In the CLL families we also reported an association between the PRS and MBL risk, where 93% of MBLs were low-count MBL. Here we evaluate the PRS in an independent sample ascertained from the Mayo Clinic Biobank.MethodsThe Mayo Clinic Biobank is a large-scale bio-repository of adult patients assembled to provide a wide array of health-related research studies. Biobank participants (N=2530) were screened for MBL using a highly-sensitive, 8-color (CD38, CD45, Kappa, Lambda, CD19, CD23, CD5 and CD20) flow-cytometry assay on stored cryopreserved peripheral blood mononucleotide cells. Low-count and high-count MBL were defined as those individuals who had <85% and ≥85 clonal B-cells out of total B-cells, respectively. Individuals without MBL (“controls”) were frequency matched to MBLs based on age and sex, after excluding non-Caucasian and non-interpretable flow results. Participants were genotyped using the Illumina OmniExpress array. Standard genotyping quality-control metrics were applied. We computed the PRS as previously published and categorized it by quintiles based on values previously used with InterLymph controls. The middle quintile served as the reference category. We used logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CI), adjusting for age at consent, sex, and socioeconomic status. In addition, we compared the median PRS by low-count MBL and high count MBL using the Mann-Whitney U test.ResultsWe identified 276 MBLs overall and 2109 controls; 97% (N=267) of MBLs had low-count MBL and only 3% (N=9) had high-count MBL. We then selected 844 controls frequency matched to the MBL cases on age and sex. Family history of leukemia/lymphoma was 11.3% among MBLs overall and 8.8% among controls. The median PRS was 7.88 and 7.46 (Figure) among MBLs overall and controls, respectively. The continuous PRS had a 1.75-fold increased risk for MBL overall (CI:1.5-2.1, P=2.65x10-12). Compared to the middle quintile, the highest quintile had 2.2-fold increased risk for MBL overall (CI:1.5-3.3, P=1.46x10-4) and the lowest quintile had 0.6-fold decreased risk (CI:0.4-0.96, P=0.03). The median PRS for low-count and high count MBL were 7.86 and 8.04, respectively. The continuous PRS had a 1.75-fold increased risk for low-count MBL (CI:1.5-2.1, P=4.81x10-12) and a 1.89-fold increased risk for high-count MBL (CI:0.8-4.2, P=0.13), however, the effect is not statistically significant between high-count and low-count MBL (P=0.89).ConclusionIn this independent sample, we validated our previous findings that the CLL PRS is associated with MBL risk. Although the sample size of high-count MBL was limited (N=9), there is evidence of a weak trend towards a higher PRS compared to low-count MBL and controls Larger sample size of high-count MBL is warranted in order to stratify MBL risk by low-count and high-count MBL. These results may help identify individuals at higher risk of MBL including low-count and high-count MBL, beyond the risk associated with age and family history of CLL.Figure: Polygenic risk score distribution by control and MBL status DisclosuresParikh:Janssen: Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding. Cerhan:Nanostring: Research Funding; Celgene: Research Funding; Jannsen: Other: Scientific Advisory Board. Kay:Janssen: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Shanafelt:Genentech: Research Funding; Pharmacyclics: Research Funding; GlaxoSmithKline: Research Funding; Jansen: Research Funding.

Low-count monoclonal B-cell lymphocytosis persists after seven years of follow up and is associated with a poorer outcome

Low-count monoclonal B-cell lymphocytosis is defined by the presence of very low numbers of circulating clonal B cells, usually phenotypically similar to chronic lymphocytic leukemia cells, whose biological and clinical significance remains elusive. Herein, we re-evaluated 65/91 low-count monoclonal B-cell lymphocytosis cases (54 chronic lymphocytic leukemia-like and 11 non-chronic lymphocytic leukemia-like) followed-up for a median of seven years, using high-sensitivity flow cytometry and interphase fluorescence in situ hybridization. Overall, the clone size significantly increased in 69% of low-count monoclonal B-cell lymphocytosis cases, but only one subject progressed to high-count monoclonal B-cell lymphocytosis. In parallel, the frequency of cytogenetic alterations increased over time (32% vs. 61% of cases, respectively). The absolute number of the major T-cell and natural killer cell populations also increased, but only among chronic lymphocytic leukemia-like cases with increased clone size vs. age- and sex-matched controls. Although progression to chronic lymphocytic leukemia was not observed, the overall survival of low-count monoclonal B-cell lymphocytosis individuals was significantly reduced vs. non-monoclonal B-cell lymphocytosis controls (P=0.03) plus the general population from the same region (P≤0.001), particularly among females (P=0.01); infection and cancer were the main causes of death in low-count monoclonal B-cell lymphocytosis. In summary, despite the fact that mid-term progression from low-count monoclonal B-cell lymphocytosis to high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia appears to be unlikely, these clones persist at increased numbers, usually carrying more genetic alterations, and might thus be a marker of an impaired immune system indirectly associated with a poorer outcome, particularly among females.

Highly similar genomic landscapes in monoclonal B-cell lymphocytosis and ultra-stable chronic lymphocytic leukemia with low frequency of driver mutations

Despite the recent discovery of recurrent driver mutations in chronic lymphocytic leukemia, the genetic factors involved in disease onset remain largely unknown. To address this issue, we performed whole-genome sequencing in 11 individuals with monoclonal B- cell lymphocytosis, both of the low-count and high-count subtypes, and 5 patients with ultra-stable chronic lymphocytic leukemia (>10 years without progression from initial diagnosis). All three entities were indistinguishable at the genomic level exhibiting low genomic complexity and similar types of somatic mutations. Exonic mutations were not frequently identified in putative chronic lymphocytic leukemia driver genes in all settings, including low-count monoclonal B-cell lymphocytosis. To corroborate these findings, we also performed deep sequencing in 11 known frequently mutated genes in an extended cohort of 28 monoclonal B-cell lymphocytosis/chronic lymphocytic leukemia cases. Interestingly, shared mutations were detected between clonal B cells and paired polymorphonuclear cells, strengthening the notion that at least a fraction of somatic mutations may occur before disease onset, likely at the hematopoietic stem cell level. Finally, we identified previously unreported non-coding variants targeting pathways relevant to B-cell and chronic lymphocytic leukemia development, likely associated with the acquisition of the characteristic neoplastic phenotype typical of both monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia.

Prevalence of Low Count (LC) Monoclonal B Cell Lymphocytosis (MBL) and Serious Infections in a Population-Based Cohort of U.S. Adults Participating in a Large Bio-Repository

Background: There is little prospective, longitudinal data on the health outcomes of low count MBL in the U.S. adult population. We screened for MBL in asymptomatic adults with normal blood counts using highly sensitive flow-cytometry in a clinic-based cohort of participants from the Mayo Clinic Biobank and explored the relationship between MBL and risk of hospitalization with infection.Methods: The Mayo Clinic Biobank is a large scale biorepository of 50,000 adults (age>18) seen in primary care-based clinics. All consented participants completed a health history questionnaire and provided blood samples. For the present study, we focused on the study participants from Olmsted County, Minnesota, 40 years of age or older who had stored PBMC. We used these PBMC to screen for MBL using an 8 color (CD38, CD45, Kappa, Lambda, CD19, CD23, CD5 and CD20) flow cytometry assay validated to consistently detect clonal B-cell events to the 0.005% level (1/20,000 events). The sensitivity of this approach of collecting 500,000 events from isolated PBMC is 2-3-fold higher than collecting 500,000 events from whole blood due to the increased concentration of lymphocytes in these samples.Infections associated with hospitalization were considered serious or life-threatening infections. Every hospitalization at both Mayo Clinic and Olmsted Medical Center hospitals in Olmsted County, MN following the sample collection was reviewed to document hospitalization with infection. Data on hospitalization with infection was abstracted from clinical records using our previously published approach (Moriera, Leukemia 27:136). This approach allows medical record validation and review of detailed clinical and treatment information related to infection. For this analysis, follow-up began at date of sample collection and ended at the earliest of death, migration from Olmsted County, or 12/31/2016. The individual abstracting information on hospitalization for infection was blinded to the results of MBL screening.Chi-square tests were used to assess associations between MBL status and demographic characteristics. Cox regression models were used to determine association with risk of hospitalization with infection accounting for the competing risk of death using Fine Gray models.Results: We screened for MBL in 1045 Olmsted County residents age 40 and older. Of these, 984 (94%) had interpretable results. 119 of these 984 (12%) had a clonal B-cell population detected including 106 (11%) with CLL phenotype (CD5, CD19, CD20 [dim], CD23, kappa or lambda light chain restriction [dim]).This analysis focused on the 106 individuals with CLL-like MBL clones (mean age 69 years, 55% male) and the 865 individuals with normal immunophenotype (mean age 61 years, 40% male).The presence of CLL phenotype MBL was strongly associated with age. MBL was detected in 3% of patients age 40-49 years, 7% of those 50-59, 10% of those 60-69, 20% of those 70-79, and 27% of those age 80+ (p<0.001). MBL was also more common among men (women 8%; men 14%; p=0.003).After a median follow-up of 7 years from biobank enrollment, 76 of the 971 participants were hospitalized with infection in Olmsted County at least once. The estimated cumulative incidence of infections at 7 years was 19% for LC MBL and 9% for those without MBL (Figure). The most frequent site of infections for which these individuals were first hospitalized were pneumonia (14 individuals; 18%), cellulitis (20 individuals; 26%), urinary tract infection (21 individuals; 26%), and blood stream infection (6 individuals; 8%). An association between LC MBL and hospitalization with infection was observed with the unadjusted HR for hospitalization with infection among the 106 Olmsted County residents with LC MBL relative to the 865 Olmsted County residents with normal immunophenotype is 2.41 (95%CI: 1.39-4.19; p=0.002). After adjusting for age and sex, the HR is 1.65 (95% CI: 0.95-2.88; p=0.08).Conclusions:We report one of the first studies to systematically explore the relationship between LC MBL and the risk of serious infection. It appears that individuals with LC MBL may be at increased risk for serious infection. If this observation is confirmed, it would suggest that 5-10% of adults over age 40 (6-12 million U.S. adults) have a largely unstudied condition with potentially serious health implications. [Display omitted] DisclosuresShanafelt:Hospira: Research Funding; AbbVie: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Jannsen: Research Funding; GlaxoSmithKline: Research Funding; Genentech: Research Funding. Kay:Gilead: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Tolero Corporation: Research Funding. Parikh:Pharmacyclics: Research Funding; AstraZeneca: Honoraria; Pharmacyclics: Honoraria.

Host virus and pneumococcus-specific immune responses in high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia: implications for disease progression.

Patients diagnosed with chronic lymphocytic leukemia (CLL) display a high incidence of infections due to an associated immunodeficiency that includes hypogammaglobulinemia. A higher risk of infections has also been recently reported for high-count monoclonal B-cell lymphocytosis, while no information is available in low-count monoclonal B-cell lymphocytosis. Here, we evaluated the status of the humoral immune system in patients with chronic lymphocytic leukemia (n=58), as well as in low- (n=71) and high- (n=29) count monoclonal B-cell lymphocytosis versus healthy donors (n=91). Total free plasma immunoglobulin titers and specific levels of antibodies against cytomegalovirus, Epstein-Barr virus, influenza and S.pneumoniae were measured by nephelometry and ELISA-based techniques, respectively. Overall, our results show that both CLL and high-count monoclonal B-cell lymphocytosis patients, but not low-count monoclonal B-cell lymphocytosis subjects, present with relatively high levels of antibodies specific for the latent viruses investigated, associated with progressively lower levels of S.pneumoniae-specific immunoglobulins. These findings probably reflect asymptomatic chronic reactivation of humoral immune responses against host viruses associated with expanded virus-specific antibody levels and progressively decreased protection against other micro-organisms, denoting a severe humoral immunodeficiency state not reflected by the overall plasma immunoglobulin levels. Alternatively, these results could reflect a potential role of ubiquitous viruses in the pathogenesis of the disease. Further analyses are necessary to establish the relevance of such asymptomatic humoral immune responses against host viruses in the expansion of the tumor B-cell clone and progression from monoclonal B-cell lymphocytosis to CLL.

Telomere length analysis in monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia Binet A.

Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic clinical entity characterized by the proliferation of monoclonal B cells not meeting the diagnosis criteria for chronic lymphocytic leukemia (CLL). MBL may precede the development of CLL, but the molecular mechanisms responsible for disease progression and evolution are not completely known. Telomeres are usually short in CLL and their attrition may contribute to disease evolution. Here, we determined the telomere lengths of CD5+CD19+ cells in MBL, CLL, and healthy volunteers. Twenty-one CLL patients, 11 subjects with high-count MBL, and 6 with low-count MBL were enrolled. Two hundred and sixty-one healthy volunteers aged 0 to 88 years were studied as controls. After diagnosis confirmation, a flow cytometry CD19+CD5+-based cell sorting was performed for the study groups. Telomere length was determined by qPCR. Telomere length was similar in the 3 study groups but shorter in these groups compared to normal age-matched subjects that had been enrolled in a previous study from our group. These findings suggest that telomere shortening is an early event in CLL leukemogenesis.

Monoclonal B-cell lymphocytosis: from literature to laboratory practice

Monoclonal B-cell lymphocytosis (MBL) is defined as an asymptomatic condition characterized by the presence of less than 5,000 monoclonal B-cells per microliter and the absence of clinical signs or symptoms of a B-cell lymphoproliferative disorder. Most MBL cases involve B cells presenting an identical phenotype to CLL (CLL-like MBL) with a Catovsky-Matutes score of 3 to 5 and share the same chromosomal abnormalities than CLL. Depending on the absolute B cell count, one may distinguish low-count CLL-like MBL (<500 B cells/μL) which have no evidence of progression, no reduction in overall survival, no increase in infection risk and do not require any specific follow-up. Patients with clinical CLL-like MBL (>500 B cells/μL) have a 1% to 2% per year risk of progression to CLL requiring therapy, a higher risk of infectious complications and mortality implicating an annual follow-up by hematologist. MBL may also express other less common phenotypes and are named atypical MBL in case of CD5 antigen expression (Catovsky-Matutes score: 1-2) and non-CLL-like MBL for CD5 negative cases (Catovsky-Matutes score: 0-2). Their poorer prognosis implicates imaging studies, bone marrow biopsy and cytogenetic analysis in addition to physical examination in order to rule out non-hodgkinien lymphoma, and require a more frequent follow-up. This review focuses on key concepts in the classification, diagnosis, monitoring and biology of MBL in laboratory practice.

Next-Generation IgVH Sequencing of Monoclonal B-Cell Lymphocytosis Reveals Clonal Heterogeneity, Ongoing Mutation, and Repertoire Differences Relative to Chronic Lymphocytic Leukemia

Background: Chronic lymphocytic leukemia (CLL) usually develops from asymptomatic monoclonal expansions of CD5 positive B-cells termed monoclonal B-cell lymphocytosis (MBL), present in the peripheral blood (PB) of approximately 5% of otherwise healthy older individuals. Although MBL only occasionally progresses to CLL, cases that do progress typically have higher MBL cell counts in the 1500-4000/µL range. Although antigen selection appears to play a central role in the development CLL, it is unclear whether this occurs at an early MBL stage or primarily during the progression of MBL to CLL. One prior study has reported clonal heterogeneity in MBL finding it in 4 of 6 low count MBL cases from familial CLL kindreds using a single cell PCR technique (Leukemia 2010,24:133-140). In this study, we assessed the VH repertoire and degree of clonal heterogeneity in sporadic MBL cases using next-generation sequencing (NGS) of the rearranged immunoglobulin heavy chain (IgH) locus.Methods: The 35 cases selected for sequencing represented residual, cryopreserved material from PB specimens submitted to ARUP for clinical phenotyping studies. All contained polytypic CD5 negative B-cells in addition to MBL/CLL phenotype cells, and had 2 or more vials for analysis. The majority (80%) had counts of MBL cells below 1000/µL (mean 294/, range 795-30 cells/µL). FACS purification of MBL cells (CD20+CD5+) and CD5 negative B-cells was performed on all samples. The IgH repertoire from the unsorted and two sorted populations was determined by NGS using the LymphoSIGHT method.Results: Five cases could not be analyzed due to insufficient numbers of MBL cells. Clonal VDJ rearrangements or clonotypes were identified in the remaining 30 based on their high frequency within the B-cell repertoire of the unsorted sample, and having a higher frequency in the sorted MBL cells relative to the sorted CD5 negative B-cells. Functional clonotypes were identified in 29 of these 30 cases. Interestingly, 5 cases had 2 functional unrelated clonotypes using different D and/or J segments that also employed different V segments. Of the 5 cases with 2 unrelated clonotypes, 3 had MBL cell counts below 1000/µL (32, 275, and 865) and 2 above (1640, 2600). Moreover, 1 of the clones in the case with 865 cells/µL represented only 25% of the MBL cells or 220 cells/µL, while 1 clone in the case with 2600 MBL cells/µL represented 18% of the MBL cells or 470 cells/µL. By flow cytometry, the CD5+ CD20+ cells in 2 of the cases with 2 functional clonotypes showed polytypic kappa/lambda expression (ratios near 1), 2 cases had uniform dim monotypic kappa expression, and 1 case showed 90% dim kappa and 10% dim lambda expression. The most frequently used VH segments were V4-34 in 6/34 or 18% of functional clonotypes, followed by V3-23 (11%), and V3-21 (9%). The V1-69 segment was used by only 1/34 (3%) functional clonotypes. The VH segments in 72% of cases with functional clonotypes were mutated (homology to germline < 98%), with 6 cases showing clear evidence of ongoing mutation by having 2 or more related clones.Conclusions: We demonstrate that MBL exhibits considerable clonal heterogeneity, with 2 distinct unrelated clones identified in 17% of 30 analyzed cases. Finding 2 distinct clones cannot be explained by a lack of allelic exclusion or the presence of 1 cell with 2 productive IgH rearrangements since each clone had different frequencies within the sorted MBL cell repertoire. This is further supported by finding the ratios of the two MBL clones in 2 cases being different in the unsorted compared to the MBL sorted cells. Clonal heterogeneity appears to occur at an early stage since the majority of clones (6/10) had cell counts below 500 cells/µL. We also found that clonal heterogeneity of MBL may not be detectable by flow cytometry or may appear as polytypic CD5+CD20+ B-cells. To our knowledge, this represents the first report of clonal heterogeneity in sporadic MBL. Our identification of infrequent use of V1-69 (1/34) supports prior studies indicating the VH repertoire of MBL is different than CLL which frequently employs V1-69. Finding evidence of ongoing VH mutation suggests antigen selection may occur in early MBL. Overall, our findings are consistent with recent observations (Cancer Cell 2011, 20;246-259) suggesting that hematopoietic stem cells from CLL patients can generate mono-or oligoclonal MBL phenotype cells that can then be selected through antigen binding for expansion. DisclosuresFaham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.