Evidence of Pre-Diagnostic Monoclonality in Blood Obtained up to 6.4 Years Preceding Diagnosis of Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3082-3082
Author(s):  
Ola Landgren ◽  
Maher X. Albitar ◽  
Wanlong X. Ma ◽  
Fatima R. Abbasi ◽  
Richard B. Hayes ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Gene Family ◽  
B Cell ◽  
Suppressor Gene ◽  
Blood Draw ◽  
Color Flow ◽  
Mutation Status ◽  
Diagnostic Samples ◽  
Vh Gene ◽  
Clinical Records

Abstract Background: Monoclonal B-cell lymphocytosis (MBL) is reported in about 3% of the general adult population 65 yrs or older. Risk of progression from MBL to CLL requiring treatment has been estimated to about 1% per yr. For the first time we established the occurrence of preceding MBL using prospectively collected pre-diagnostic samples from CLL pts. Also, we tested the hypothesis that indolent (IgVH mutated) CLL occurs after MBL while aggressive (IgVH unmutated) CLL has an alternative route that develops rapidly or bypasses MBL. Methods: Using the population-based nationwide PLCO (Prostate, Lung, Colorectal and Ovarian) cancer screening trial (n>144,500 cancer-free adults), we identified 46 CLL pts with available baseline cryopreserved whole blood obtained 3 to 77 months prior to CLL diagnosis. Clinical information was retrieved from screening questionnaires and medical records. 10 healthy adult controls were included. 6-color flow cytometry (CD45/CD19/CD5/CD10/kappa/lambda) and reverse transcription/polymerase chain reaction (RT/PCR) for IgH gene were used to determine evidence of expression of monoclonal IgH gene family and to establish the VH gene mutation status. Direct sequencing without subcloning (for clonal cases) and with subcloning (for non-clonal cases) was used to determine mutation status. We sequenced exons 5, 6, 7, 8, and 9 of the p53 suppressor gene for mutations. Results: After exclusion of one subject with absent viable cells, we analyzed pre-diagnostic blood specimen from 45 CLL pts. CLL diagnosis was based on clinical criteria and confirmed by review of clinical records. Immunophenotyping data were available for 39 (87%) pts. The Rai CLL stage distribution was: 0 (n=26), 1 (n=9), 2 (n=2), 3 (n=1), missing/unclear (n=7). The mean time between pre-diagnostic blood draw and subsequent CLL diagnoses was 3 yrs. Using flow cytometry, sequencing, and PCR, we demonstrated evidence of monoclonality in 43/45 (96%) pre-diagnostic samples from CLL pts. Among individuals whose blood was obtained <3 yrs vs. >3 yrs prior CLL diagnosis, the proportion of pre-diagnostic monoclonality was 28/28 (100%) vs. 15/17 (88%), respectively; the proportion of IgVH mutated pre-diagnostic clones was the same in both groups: 23/28 (82%) vs. 14/17 (82%). The most frequently expressed VH gene family was VH3 (40%) followed by VH4 (29%), VH1 (9%), VH5 (9%), VH2 (7%), and VH6 (2%), with no expression of VH7 gene families. We found 2 pts with mutations in the p53 suppressor gene; one in exon7/M237K, and one in exon 6, P22 that is silent and has been reported as in some tumors and a polymorphism. One sample with unmutated IgVH appeared to have a deletion at p53 in both alleles. Immunophenotyping analyses for pre-diagnostic clones showed kappa n=27, lambda n=8, n=4 biclonal, and missing/indeterminate n=6 (kappa:lambda ratio >3:1). Comparisons of pre-diagnostic and diagnostic kappa/lambda status in the same persons showed 100% concordance. All negative controls lacked evidence of monoclonality. Conclusions: In this first prospective study examining pre-diagnostic blood obtained 3 to 77 months prior to CLL diagnosis, we found a monoclonal B-cell population in virtually all the persons; 82% were IgVH mutated. Our findings suggest that CLL is commonly preceded by the precursor condition MBL.

scholarly journals VH gene analysis of hairy cell leukemia reveals a homogeneous mutation status and suggests its marginal zone B-cell origin

Leukemia ◽  
2004 ◽  
Vol 18 (10) ◽  
pp. 1729-1732 ◽  
Author(s):  
V Vanhentenrijk ◽  
A Tierens ◽  
I Wlodarska ◽  
G Verhoef ◽  
C D Wolf-Peeters
Keyword(s):  
B Cell ◽  
Hairy Cell Leukemia ◽  
Marginal Zone ◽  
Gene Analysis ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Mutation Status ◽  
Vh Gene

Comparative study of VH gene family usage by newbornxid and non-xid mice, newborn NZB and adult NZB mice, and by splenic and peritoneal cavity B cell compartments

1988 ◽  
Vol 18 (12) ◽  
pp. 1979-1983 ◽  
Author(s):  
Guillaume Dighiero ◽  
Annick Lim ◽  
Marie-Pierre Lembezat ◽  
Azad Kaushik ◽  
Luis Andrade ◽  
...  
Keyword(s):  
Comparative Study ◽  
Gene Family ◽  
B Cell ◽  
Peritoneal Cavity ◽  
Cell Compartments ◽  
Vh Gene

Distinctive IgVH Gene Segments Usage and Mutation Status in Chinese Patients with Chronic Lymphocytic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4708-4708
Author(s):  
Lijuan Chen ◽  
Yaping Zhang ◽  
Wenjuuan Zheng ◽  
Jianyong Li ◽  
Changgeng Ruan
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Gene Family ◽  
Gene Families ◽  
Lymphocytic Leukemia ◽  
Chinese Patients ◽  
Chain Gene ◽  
Mutation Status ◽  
Significant Difference ◽  
Variable Heavy ◽  
Vh Gene

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the relentless accumulation of monoclonal B cells with the appearance of small mature lymphocytes and a characteristic CD5 and CD19 co-expression immunophenotype. The incidence of CLL in Asian countries is lower than that in the Western ones, where CLL is the most common leukemia. To evaluate the frequency and mutation status of immunoglobulin (Ig) variable heavy chain gene (IgVH) expression in Chinese patients with CLL. We investigated IgVH gene segments usage and mutation status by multiplex RT-PCR in 52 CLL patients, and analyzed the relationship between IgVH somatic mutation status and the expression of CD38, ZAP-70 and CLLU1. 38 patients had mutated IgVH, and 14 had unmutated IgVH. The most frequently expressed VH gene family was found to be VH3 (46.2%) followed by VH4 (40.4%), VH1 (5.8%), VH2 (5.8%) and VH7 (1.9%), with no expression of VH5 and VH6 gene families. VH1-69 and VH3-21 which commonly overused in Western CLL weren’t detected in our cohort. The frequency of IgVH gene families indicates significant difference in Chinese CLL patients compared with Western patients, suggesting involvement of ethnic and/or environmental factors in CLL disease initiation. IgVH gene mutation status was significantly associated with the expression of CD38 and CLLU1. The expression of them may be simple and reliable surrogates for the identification of IgVH mutations. VH gene family usage and mutation status VH family n Mutated VH gene Unmutated VH gene VH1 3 3 0 VH2 3 2 1 VH3 24 19 5 VH4 21 16 5 VH5 0 0 0 VH6 0 0 0 VH7 1 0 1

scholarly journals 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

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 831-831
Author(s):  
Tait D. Shanafelt ◽  
Neil E. Kay ◽  
Curtis A. Hanson ◽  
Sameer A. Parikh ◽  
Sara J Achenbach ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Mayo Clinic ◽  
Research Funding ◽  
Olmsted County ◽  
Sample Collection ◽  
Serious Infection ◽  
Clinical Records ◽  
The Relationship

Abstract 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&gt;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&lt;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. Figure 1 Figure 1. Disclosures Shanafelt: 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.

scholarly journals Differential expression of VH gene families in peripheral B cell repertoires of newborn or adult immunoglobulin H chain congenic mice.

1992 ◽  
Vol 175 (6) ◽  
pp. 1449-1456 ◽  
Author(s):  
A C Viale ◽  
A Coutinho ◽  
A A Freitas
Keyword(s):  
Gene Family ◽  
B Cell ◽  
Expression Pattern ◽  
Gene Families ◽  
Cell Repertoire ◽  
Adult Mice ◽  
Igh Locus ◽  
B Cell Repertoire ◽  
Vh Gene ◽  
Old Mice

The pattern of VH gene family expression in the primary B cell repertoire of the mouse is strain dependent. In C57Bl/6 mice, the VH J558 family is expressed by more than 45% of the cells, while the expression of VH 7183, VH Q52, and VH 36-60 families together does not exceed 20%. In BALB/c mice, relative expression of VH J558 is lower than 35%, while the sum of the other three families reaches 25%. To assess which genetic loci control strain-specific VH gene family expression, we studied VH gene family usage in splenic B cell repertoires of different congenic strains of mice. Changes in major histocompatibility complex or immunoglobulin (Ig) K light chain genes did not modify VH gene family expression in adult mice. Differences at the IgH locus, however, modified VH gene family usage. In 1-d-old mice, the strain-specific VH gene family expression pattern is determined by the IgH haplotype. In adult mice, the VH gene family expression pattern of resting B cells is independent of the IgH locus and follows the genetic background of the congenic strain, while it is determined by the IgH haplotype among Ig-secreting spleen cells. In F1(B6 x BALB/c) mice, each of the two spleen B cell populations, sorted on the basis of mu heavy chain allotype expression, shows an independent VH gene family expression pattern, determined by the IgH locus. The implications of these results in the control of VH gene family expression, and in the selection of peripheral B cell repertoires are discussed.

IL-10 Contributes to the Development of Immunosuppressive CD14+HLA-DRlow/- monocytes in B-Cell Non-Hodgkin’s Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2979-2979
Author(s):  
Bing Xiu ◽  
Yi Lin ◽  
Deanna Grote ◽  
Steven Ziesmer ◽  
Michael Gustafson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Poor Prognosis ◽  
Underlying Mechanism ◽  
Monocyte Count ◽  
Newly Diagnosed ◽  
Color Flow ◽  
Healthy Donors

Abstract Background - The phenotype and biological role of monocytes in B-cell non-Hodgkin lymphoma (NHL) is not fully understood, however, an increased absolute monocyte count in the peripheral blood of lymphoma patients is associated with a poor prognosis. We have previously reported that monocytes from patients with relapsed B-cell NHL displayed an immunosuppressive CD14+HLA-DRlow/- phenotype that correlated with a poor prognosis. However, the underlying mechanism by which CD14+HLA-DRlow/- monocytes develop in patients with B-cell NHL is unknown. The goal of this study was to determine whether cytokines are responsible for the increased number and phenotype of CD14+HLA-DRlow/-monocytes present in lymphoma patients. Methods – Whole blood from patients with newly diagnosed, untreated B-cell NHL (n=20) and healthy donors (n=20) was stained with a panel of antibodies and analyzed by 10-color flow cytometry. Cytokine levels in serum and culture supernatants were measured by Luminex and ELISA, respectively. Polarization of monocytes from healthy donors was performed by incubating CD14+ cells with specific cytokines or supernatants from B-cell NHL cell lines for 24 hours. Activation and proliferation of CD4+T cells cocultured with IL-10-pretreated monocytes were measured by flow cytometry. Results – By flow cytometry, we observed that the absolute number of peripheral monocytes was increased in newly-diagnosed lymphoma patients compared to healthy donors. A significant proportion of these monocytes displayed an immunosuppressive CD14+HLA-DRlow/- phenotype and the numbers of CD14+HLA-DRlow/- cells were significantly higher in lymphoma patients than in healthy donors. To identify the underlying mechanism by which CD14+HLA-DRlow/- monocytes develop thereby leading to increased numbers of CD14+HLA-DRlow/- monocytes in B-cell NHL, we tested a panel of cytokines and found that IL-10 played a key role in the process. Firstly, treatment of monocytes with IL-10 in vitro resulted in the generation of CD14+HLA-DRlow/- cells. Second, monocytes co-cultured with IL-10 producing lymphoma B-cells, or treated with supernatants from lymphoma cell cultures, developed a CD14+HLA-DRlow/- phenotype. Thirdly, IL-10 levels were increased in the serum of DLBCL (p<0.0001) and FL patients (p=0.010) compared to healthy controls, and serum IL-10 levels correlated with increased numbers of peripheral monocytes (p=0.02). Finally, IL-10-pretreated CD14+HLA-DRlow/- monocytes were significantly immunosuppressive and inhibited the proliferation and activation of CD4+T cells in co-culture assays. Conclusions- Taken together, our results suggest that IL-10 signaling contributes to the increased numbers of CD14+HLA-DRlow/- monocytes in B-cell NHL. Strategies to inhibit IL-10 production may therefore have therapeutic potential in B-cell NHL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comparison of the fetal and adult functional B cell repertoires by analysis of VH gene family expression.

1988 ◽  
Vol 168 (2) ◽  
pp. 589-603 ◽  
Author(s):  
H D Jeong ◽  
J M Teale
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
B Cells ◽  
Gene Family ◽  
B Cell ◽  
Cell Lineage ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
Vh Gene

The functional B cell repertoire in BALB/c mice was assessed at various stages in ontogeny. This was done by analyzing VH gene family expression using the sensitive technique of in situ hybridization. The B cell repertoire was probed with the mitogen, LPS, and the antigen DNP. DNP was chosen because B cells responsive to this hapten appear very early in ontogeny. The APCs that developed after stimulation with LPS or DNP were analyzed for VH gene expression by in situ hybridization of individual cells using radiolabeled VH gene family probes. The results indicated that VH gene expression in fetal B cells after stimulation was distinct from adult B cells in that there was a biased expression of D proximal families. The results indicated that this bias was associated with developmental age and not a given differentiation stage in the B cell lineage. In addition, stimulation of fetal B cells with DNP resulted in a large increase in expression of member(s) of VH 36-60, suggesting that the early appearance of DNP-responsive B cells is not strictly correlated with preferential rearrangement of D proximal families, VH 7183 and VH Q52. However, the results suggested that a large proportion of pre-B cells that preferentially rearrange D proximal families early in ontogeny become part of the functional developing repertoire.

scholarly journals THU0040 PROTEINASE 3-REACTIVE B CELL RECONSTITUTION AFTER TREATMENT WITH RITUXIMAB FOR ANCA-ASSOCIATED VASCULITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 233.1-233
Author(s):  
A. Berti ◽  
S. Hillion ◽  
A. Hummel ◽  
E. Carmona ◽  
T. Peikert ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Proteinase 3 ◽  
Research Support ◽  
Color Flow ◽  
Anca Associated Vasculitis ◽  
Cell Repopulation

Background:Proteinase 3 (PR3)-reactive B cells are present in PR3-ANCA-associated vasculitis (AAV) at levels higher than healthy controls.Objectives:To evaluate the dynamics of the PR3-reactive B cell repopulation in patients with PR3-AAV after treatment with rituximab, and to analyze possible associations between these immunological changes and long-lasting remissions.Methods:We analyzed all available frozen peripheral blood mononuclear cells (n=148) from 23 randomly-selected PR3-AAV patients who participated in the RAVE trial and achieved complete remission (BVAS=0, prednisone=0) after treatment with rituximab.We measured PR3-reactive B cells and the relative subsets by a multi-color flow cytometry panel including CD19, IgD, CD27, CD38, CD24, and a biotinylated PR3 revealed by fluorescent streptavidin. The clinical data of the trial were correlated with flow-cytometry data.Results:10/23 (43%) patients relapsed during the follow up, 8/10 relapses were severe. At baseline, clinical features, PR3-ANCA levels, % of total PR3-reactive B cells and PR3-reactive B cell subsets were similar between relapsers and non-relapsers. All patients were followed until the end of the trial, for a mean of 44 months (25-75%IQR 31-54), without difference in follow-up time between relapsers and non-relapsers (p=0.98).The majority of patients had B cell repopulation at 12 (range 12-24) months after rituximab. At the time of B cell repopulation, transitional (CD19+CD24+CD38+) and naïve (CD19+CD27+IgD-) B cells were higher compared to baseline, while total plasmablasts (PB) were unchanged, and mature B cells significantly decreased in both relapsers and non relapsers. PR3-reactive B cells reappeared in all the patients, and the % of PR3-reactive of B cells were higher at the B cell repopulation visit compared to baseline (5.82% vs 4.25%, p<0.05), while total B cells were lower (66/μL vs 151/μL, p<0.01), regardless of future relapse.Within PR3-reactive B cells, only the % of PB (CD19+CD27+CD38+PR3+) were higher in relapsers vs. non-relapsers (median [25-75%IQR]; 1.95% [1.315-3.845] vs 0.84% [0.05-1.66], p=0.022) and severe relapsers vs non-severe relapsers (2.165% [1.66-4.315] vs 0.84% [0.1-1.74], p=0.015). Time-to-relapse and time-to severe-relapse were significantly shorter in patients with circulating PR3-PB higher than the median value of the cohort (1.6%) during B cell reconstitution (Figure 1A-B).Conclusion:In PR3-AAV, during B cell reconstitution after rituximab, the total fraction of PR3-B cells increases, due to the expansion of the transitional and naïve B cell compartments. Circulating PR3-PB within PR3-B cells are enriched in the peripheral blood of relapsing and severely relapsing patients compared to non-relapsing patients. Higher levels of PR3-PB after rituximab during B cell reappearance significantly increased the risk of subsequent relapse and severe relapse.References:[1]Cornec D, Berti A, Hummel A, et al. J Autoimmun. 2017Disclosure of Interests:Alvise Berti: None declared, Sophie Hillion: None declared, Amber Hummel: None declared, Eva Carmona: None declared, Tobias Peikert: None declared, Carol Langford: None declared, Peter A. Merkel: None declared, Paul Monach: None declared, Philip Seo: None declared, Robert Spiera Grant/research support from: Roche-Genetech, GSK, Boehringer Ingelheim, Chemocentryx, Corbus, Forbius, Sanofi, Inflarx, Consultant of: Roche-Genetech, GSK, CSL Behring, Sanofi, Janssen, Chemocentryx, Forbius, Mistubishi Tanabe, E. William St. Clair: None declared, Fernando Fervenza: None declared, Kristina Harris: None declared, John H. Stone Grant/research support from: Roche, Consultant of: Roche, Jacques-Olivier Pers: None declared, Ulrich Specks: None declared, Divi Cornec: None declared

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