Cell Lymphocytosis Research Articles

Circulating B Cell Clones in Familial Waldenström Macroglobulinemia.

Abstract 2703 Background and Significance:Lymphoplasmacytic lymphoma (LPL) is an indolent but incurable B cell lymphoproliferative characterized by the clonal expansion of plasmacytoid lymphocytes in the lymph nodes and bone marrow. The majority of cases are associated with an IgM isotype paraprotein, and when present, is termed Waldenström macroglobulinemia (WM). WM shows familial aggregation suggesting an inherited risk for disease, and also co-aggregates with CLL in families. In our previous work, we have shown that clonal populations of peripherally circulating B cells can be identified in 18% of unaffected family members from CLL kindreds. Most of these clonal populations have a typical CLL immunophenotype and have been termed CLL-like monoclonal B cell lymphocytosis (MBL). Because CLL and WM have related gene expression profiles and appear to have shared genetic risk, we hypothesized that unaffected family members of WM kindreds would have detectable circulating clonal B cell populations. Further, we undertook systematic flow cytometric screening of familial WM and IgM MGUS cases to determine the prevalence, immunphenotype, and biologic characteristics of circulating malignant B cells. Methods:A diagnosis of LPL / WM or IgM MGUS was determined using standard WHO criteria. All patients and unaffected family members were ascertained at the National Cancer Institute and provided informed consent. Peripheral blood mononuclear cells were isolated using density centrifugation and viably frozen in DMSO containing media. We developed a two tube, nine color flow cytometric assay: the first tube allowed for detection of CLL-like clones based upon co-expression of CD5, CD20, and CD23; the second tube targeted WM populations based upon expression on CD19, CD20, CD25, CD38, and surface IgM. Cell populations were considered clonally restricted if the κ: λ was > 3.0 or < 0.3. Clonal populations were then isolated using fluorescence activated cell sorting (FACS). RNA and genomic DNA were extracted for genetic and genomic studies using phenol: chloroform purification. Results:A total of 155 individuals were analyzed: 54 WM / LPL, 17 IgM MGUS, 1 IgG MGUS, 1 IgA MGUS, 1 NHL, and 81 unaffected family members. Twenty of 54 WM patients had detectable peripherally circulating populations. Thirteen WM patients had no detectable B cells, of these, 11 patients had prior treatment. As such, among the 41 WM patients with a B cell compartment that was analyzable by flow cytometry, 49% (20 of 41) had peripherally circulating B cell clones detected. Four of these 20 cases showed two immunophenotypically distinct clonal B cell populations. The immunophenotype was somewhat heterogenenous: 18 cases expressed surface IgM, CD38 was variable but expressed in most cases, CD25 was not detected in any case, and 4 cases showed a CLL like (CD5+CD20dimCD23+) immunophenotype. Interestingly, we detected peripherally circulating B cell clones in 9 of 17 cases (53%) of IgM MGUS, a proportion nearly identical to that identified in WM / LPL. Three of 9 were “CLL-like” with co-expression of CD5 and CD23, while the majority of clones were CD5negIgM+CD38+. Among unaffected family members, we identified B cell clones in only 4 of 81 (5%). All 4 cases expressed CD5, and 3 showed a CLL-like phenotype, consistent with these individuals having MBL. Among all study subjects, 20 clonal B cell populations of > 104 B cells were FACS purified from 18 different cases: 12 WM, 4 IgM MGUS, 1 IgA MGUS, and 1 unaffected family member. Conclusions:Peripherally circulating B cell clones with an immunophenotype similar to that of LPL can be identified in approximately half of patients with WM / LPL. We observed for the first time that a similar proportion of patients with IgM MGUS have detectable clonal populations with an immunophenotype similar to that observed in WM patients. Genetic and genomic studies to determine the lineage of these populations are currently underway. The frequency of MBL among unaffected family members with WM is lower than that observed in CLL kindreds. CLL-like MBL can be detected at very low numbers because the cell population is immunophenotypically abnormal. The peripherally circulating clones identified in WM / LPL patients have an otherwise normal B cell immunophenotype and can only be detected by light chain restriction. This likely significantly limits the ability of flow cytometry to detect pre-clinical CD5neg IgM expressing clonal populations. Disclosures:No relevant conflicts of interest to declare.

A Role for Interferon Regulatory Factor 4 in the Development of CLL

AbstractAbstract 714Chronic Lymphocytic Leukemia (CLL) representing 30% of all the adult leukemia is a heterogeneous B cell malignancy with complex clinical behavior and pathophysiology. Even though an increasing body of literature has helped in understanding the biology of CLL the precise molecular events that underlie the pathogenesis of this disease are still not fully understood. Recent Whole Genome Sequencing and Genome Wide Association Studies have linked different genetic loci to the origin and evolution of the disease. A Genome Wide Association Study (GWAS) by Di Bernardo et al identified Single Nucleotide Polymorphisms in the 3'UTR of Interferon Regulatory Factor 4 (IRF4) as a familial risk allele for CLL. These SNPs are associated with lower mRNA levels of IRF4 in individuals carrying the risk allele. Another study by Havelange V. et al identified heterozygous mutations in the DNA binding domain of IRF4 in patients with CLL. These studies have linked lower levels of IRF4 to the pathogenesis of CLL. Even though studies have linked low levels of IRF4 to the pathogenesis of CLL, whether low levels of IRF4 indeed contribute to CLL development remains to be determined.In rodent, CLL cells are presumably derived from B1 cells. Like CLL cells, B1 cells are B220+CD5+ and possess self-reactive B cell receptors (BCR). B1 cells normally reside in peritoneal and pleural cavities and constitute only about 5% of total B lymphocytes in mice. However, B1 cell population is dramatically elevated in the VH11 knock-in mice. VH11 family of variable IgH chain is expressed exclusively on B1 cells where it pairs with Vk9 to form BCR that recognizes phosphatidylcholine on senescent RBCs. We did not observe an overt increase of CLL development in IRF4 deficient mice. However, when we crossed the VH11 knock-in mice to an IRF4 deficient mice it resulted in a CLL like lymphoproliferative disorder with marked expansion of CD5+ B220low/int population in blood, spleen and other lymphoid organs. To monitor pathogenesis of CLL, we collected blood from saphenous vein of IRF4−/−VH11 and IRF4+/+VH11 littermate control mice monthly. A total of ten mice from each group were used for this study. The appearance of a monoclonal B220lowCD5+ population and its percentage among peripheral blood mononuclear cells (PBMC) were used for the initial CLL diagnosis. The diagnostic criterion for CLL in VH11 mice is the appearance of monoclonal B220lowCD5+ cells constituting over 20% of PBMCs. If the percentage of the B220lowCD5+ clone is under 20% of the PBMC, the diagnosis would be monoclonal B cell lymphocytosis (MBL), a disease which can progress into CLL. Interestingly, 6 out of 10 IRF4−/−VH11 mice developed CLL after just 5 months while the four remaining IRF4−/−VH11 mice developed MBL. In contrast, none of the IRF4+/+VH11 control mice developed CLL or MBL. Two of the mice developed aggressive CLL where leukemic cells infiltrated liver and other non-lymphoid organs. BrdU incorporation assay identified spleen as the primary site of CLL cells expansion although proliferating CLL cells were also identified in lymph nodes and bone marrow. We further isolated CLL cells from IRF4−/−VH11 mice and transplanted them into immunodeficient mice (Rag2−/−gamma−/−). Our result shows that the disease was transplantable as all the transplanted mice succumbed to CLL within 1 month post-transplantation. Treatment of IRF4−/−VH11 CLL cells with specific Syk and Btk inhibitors targeting BCR signaling showed that like human CLL, the leukemic cells were depended on active BCR signaling for their survival and proliferation.In summary, we provide evidence for a causal relationship between low levels of IRF4 and development of CLL. Further studies are needed to provide mechanistic insights into how IRF4 is involved in the development and progression of this disease. The fine dissection of this relationship will allow for development of new therapeutic strategies and identification of new prognostic markers for diagnosis and treatment of CLL. Disclosures:No relevant conflicts of interest to declare. Disclosures:No relevant conflicts of interest to declare.

Congenital B cell lymphocytosis explained by novel germline CARD11 mutations

Nuclear factor-κB (NF-κB) controls genes involved in normal lymphocyte functions, but constitutive NF-κB activation is often associated with B cell malignancy. Using high-throughput whole transcriptome sequencing, we investigated a unique family with hereditary polyclonal B cell lymphocytosis. We found a novel germline heterozygous missense mutation (E127G) in affected patients in the gene encoding CARD11, a scaffolding protein required for antigen receptor (AgR)-induced NF-κB activation in both B and T lymphocytes. We subsequently identified a second germline mutation (G116S) in an unrelated, phenotypically similar patient, confirming mutations in CARD11 drive disease. Like somatic, gain-of-function CARD11 mutations described in B cell lymphoma, these germline CARD11 mutants spontaneously aggregate and drive constitutive NF-κB activation. However, these CARD11 mutants rendered patient T cells less responsive to AgR-induced activation. By reexamining this rare genetic disorder first reported four decades ago, our findings provide new insight into why activating CARD11 mutations may induce B cell expansion and preferentially predispose to B cell malignancy without dramatically perturbing T cell homeostasis.

Cellular origin and pathophysiology of chronic lymphocytic leukemia

The cellular origin of chronic lymphocytic leukemia (CLL) is still debated, although this information is critical to understanding its pathogenesis. Transcriptome analyses of CLL and the main normal B cell subsets from human blood and spleen revealed that immunoglobulin variable region (IgV) gene unmutated CLL derives from unmutated mature CD5(+) B cells and mutated CLL derives from a distinct, previously unrecognized CD5(+)CD27(+) post-germinal center B cell subset. Stereotyped V gene rearrangements are enriched among CD5(+) B cells, providing independent evidence for a CD5(+) B cell derivation of CLL. Notably, these CD5(+) B cell populations include oligoclonal expansions already found in young healthy adults, putatively representing an early phase in CLL development before the CLL precursor lesion monoclonal B cell lymphocytosis. Finally, we identified deregulated proteins, including EBF1 and KLF transcription factors, that were not detected in previous comparisons of CLL and conventional B cells.

Guidelines on the diagnosis, investigation and management of chronic lymphocytic leukaemia

Royal Bournemouth Hospital, Bournemouth, 2 Royal Marsden Hospital, London, 3 Southampton General Hospital, Southampton, Cardiff and Vale NHS Trust, Cardiff Cambridge University Hospitals NHS Foundation Trust, Cambridge UK; St. James's Institute of Oncology, Leeds, Christie Hospital NHS Trust, Manchester Royal Marsden Hospital London; Heart of England NHS Foundataion Trust, Birmingham; 10 Royal Liverpool University Hospital, Liverpool; Churchill Hospital, Headington, Oxford, Norfolk and Norwich University Hospital, Norwich

Signification clinique des expansions polyclonales lymphocytaires T CD8+/CD57+

Polyclonal CD8(+)/CD57(+)T cell lymphocytosis can be observed in various conditions such as chronic viral infections, autoimmune cytopenias, connective tissue diseases, chronic graft-versus-host disease and primary or secondary immune deficiencies. This population results from the chronic stimulation of CD8(+)/CD28(+)/CD57(-)lymphocytes by exogenous (mostly infection-related), autologous or allogeneic antigens. Paralleling chronic antigen stimulation, these CD8(+) T cells acquire a poor capacity to proliferate in standard conditions in relation with the loss of CD28, whereas CD57 antigen becomes expressed at their surface. CD8(+)/CD57(+)T cells represent activated cytotoxic T lymphocytes at a terminal stage of their differentiation with evidence of immunological senescence, which have usually lost their cytotoxic properties to become "regulatory" T cells. Patients with a CD8(+)/CD57(+)T cell lymphocytes expansion can display features of organ infiltration, as well as chronic idiopathic neutropenia. The search of this population has therefore a diagnostic value in clinical practice. A CD8(+)/CD57(+)T cell lymphocytes expansion must be suggested in patients with an organomegaly or organ(s) infiltration, particularly in patients infected by the human immunodeficiency virus or in the setting of allogeneic hematopoietic stem cell transplantation, as well as in patients with a neutropenia of unexplained origin. The identification of a CD8(+)/CD57(+)T cell lymphocytes expansion also has therapeutical consequences since patients with an organ infiltration or a neutropenia may respond remarkably to immunomodulatory therapies. The search of a CD8(+)/CD57(+) T cell expansion thus represents a useful and still poorly known diagnostic tool which clinical interest deserves further evaluation.

Congenital B cell lymphocytosis linked to a novel germline CARD11 mutation (109.13)

Abstract Nuclear Factor-kappa B (NF-κB) governs the transcription of many genes for normal lymphocyte functions, but constitutive NF-κB activation is most often associated with B cell malignancy. Using high-throughput whole transcriptome sequencing, we recently investigated a family with a unique, hereditary polyclonal B cell lymphocytosis. Here we report our discovery of a novel, germline heterozygous missense mutation (E127G) in affected patients in the gene encoding CARD11, a lymphocyte-specific scaffolding protein required for antigen receptor (AgR)-induced NF-κB activation in both B and T lymphocytes. Similar to somatic, gain-of-function CARD11 mutations described in diffuse large B cell lymphoma, E127G CARD11 spontaneously oligomerizes and activates NF-κB when upon ectopic expression in lymphoid cell lines. Primary lymphocytes from these patients also display evidence of CARD11 aggregation and constitutive NF-κB activity. In contrast to B cells, however, we observed that E127G CARD11 contributes to patient T cell hyporesponsiveness upon AgR stimulation, even though the proximal CARD11-associated signaling machinery is highly similar between B and T cells. Our findings suggest that an activating, germline CARD11 mutation can selectively induce B cell expansion and preferentially predispose to B cell malignancy without perturbing T cell homeostasis.

Clinical Monoclonal B Cell Lymphocytosis Is a Strong Favorable Prognosticator Independent From Biological and Clinical Parameters,

Abstract 3887Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic monoclonal expansion of <5.0 × 109/L circulating CLL (chronic lymphocytic leukemia)-phenotype B-cells. CLL-like MBL, observed in the clinical setting and defined as clinical MBL (cMBL), demonstrated a substantial risk of leukemic progression to symptomatic disease requiring treatment that can be predicted by biological prognosticators (Rawstron, 2008; Rossi, 2009). Based on this background, the aims of our study were: (i) to define clinical features at diagnosis and a biological profile of cMBL and Rai 0 subsets; (ii) to determine treatment free survival (TFS) within cMBL in comparison with Rai 0 CLL; (iii) to identify clinical and biological variables at diagnosis that predict the risk of evolution from MBL to CLL requiring treatment; and finally (iiii) to validate cMBL as an independent prognostic factor. For this purpose, we investigated 516 patients, median age 66 years (range 33–89), 288 males and 228 females. According to the IWCLL 2008 guidelines, 109 cases were diagnosed as cMBL and 407 cases as CLL (Rai 0: 106; I-II: 290; III-IV: 11). Cases presented for initial evaluation at our Institution between June 1989 and June 2009. The database was updated for the analysis in January 2011. The median follow up was 6.5 years (range 1–20). Among cMBL, median absolute lymphocyte count was 7.3 × 109/L (range 3.2–12.6 × 109/L) and median CLL-phenotype lymphocyte count was 3.5 × 109/L (range 0.6–4.9 × 109/L). cMBL were characterized by both lower beta2-microglobulin (beta2-M) and soluble CD23 (sCD23) levels (P=0.008 and P=0.0005) than Rai 0 CLL. Moreover cMBL showed a more favorable genetic profile compared to Rai 0 CLL: more specifically, cMBL showed both a significant higher prevalence of normal karyotype (45/72 [63%]] vs 30/75 [40%]) and a lower incidence of del11q/del17p (5/72[7%] vs 11/75[15%] (P=0.020). IgVH mutational status as well as ZAP-70 and CD38 percentages did not differ significantly between these two groups. Lymphocyte doubling time was <12 months in 6/109 (5.5%) cMBL cases compared to 19/106 (18%) Rai 0 CLL (P=0.005). With regard to clinical outcome, only 12/109 (11%) cMBL patients required treatment, while 35/106 (33%) Rai 0 CLL underwent chemotherapy (P=0.00012). Noteworthy, also the time to treatment was significantly longer in cMBL patients (71.5 months vs 53.5 months, P=0.03). The slower disease kinetics was consistent with a longer TFS in cMBL compared to Rai 0 CLL (72% vs 42% at 14 years, P=0.00001, Figure). Interestingly, while, within Rai 0 CLL subset, biological prognosticators such as IgVH status (P=0.01), ZAP-70 (P=0.0002) and cytogenetics (P=0.02) as well as markers of tumor burden such as beta2-M (P=0.002) and sCD23 (P=0.0006) were confirmed to predict TFS, within cMBL none of these factors was able to predict progression free survival (PFS) and/or TFS, although some biological prognosticators (ZAP-70 and IgVH) were represented in similar percentages between these two groups. It might be hypothesized that some other factors related to the immune function (e.g., phenotype and function of T-lymphocyte subsets [G.D.P., unpublished observation]) could be able to explain the different time to progression of the cMBL patients. Finally, multivariate analysis, combining cMBL and Rai 0 CLL, documented that having a diagnosis of cMBL is an independent protective factor against the risk of treatment (P=0.011), together with a normal karyotype (P=0.020) and a lower ZAP-70 expression (P=0.0001). In conclusion, cMBL represents a biological and clinical distinct entity from Rai 0 CLL with a more favorable outcome, displaying some degree of heterogeneity in the risk of treatment requirement, probably due to both humoral and cellular immune dysfunctions. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The detection of minimal residual disease status in chronic lymphocytic leukemia

In this issue of Clinical Cytometry, Durrieu et al. on behalf of GEIL-GOELAMS present evidence that circulating levels of CD19+ CD5+ chronic lymphocytic leukemia (CLL)-like cells can be used to define a threshold for the detection of minimal residual disease (MRD) in CLL (this issue: page 346). This study was performed in 16 different hematology laboratories or services, and the data is analyzed centrally. The panel consisted of five tubes of four reagents each with CD19 and CD5 being present in each tube. They determined that the level of two CLL-like immunophenotypes CD19+/CD5+/CD43int/CD79blo and CD19+/ CD5+/CD81lo/CD22lo were less than 4 × 10−4 per WBC in unwashed, lyzed normal whole blood. Levels greater than this found in post treatment CLL samples were taken to signify the presence of MRD. The author further state that they are unable to determine monoclonality using light chain restriction due to their polyclonal nature in normal blood donors and the presence of cytophilic antibodies being present in the unwashed, lyzed method. And that bright CD5+ B cells are often lambda positive, and polyclonal. I have several preferences (biases) in this matter, and they include the use of CD20 rather than CD19 or preferably both CD20 and CD19 in combination to define B cells and the clone, demonstration of monoclonality or monotypia, and that reports be based on the absolute cell-count (WBC or absolute lymphocyte count ALC) and not just the percentage < or > 10−4. If the WBC is used, it should be used in conjunction with the ALC and even a B cell absolute count. I sympathize with the problem of cytophilic antibodies but in monoclonal B cell lymphocytosis studies, values of 5–50 light chain restricted B cells per microliter can be routinely detected (1). I was also surprised to see that the lower range of 1–3% CD5+ B cells seems to be the norm, whereas values of 10–25% seem to be more the rule. The European Research Initiative on CLL (ERIC) has published a standardized four-color approach to MRD detection in CLL (2). Both the authors of that study and this study find a WBC threshold of 10−4 below which is considered MRD negativity. The ERIC study allows for the primary role of kappa lambda tube for historical purposes but favors the combination of CD20/CD38/CD19/CD5 over the combinations of CD43/CD79b and CD81/CD22. They further point out the need for the evaluation of gate contamination, number of total events collected (500,000), and operator training. Certain precautions are required for differentiating between blood and marrow involvement in the setting of antibody therapies. However, due to the correlation of MRD between blood and marrow, blood should be the first site tested for MRD. The complete absence of B cells (B cell aplasia) might also be taken as a clinical measure of MRD. If ascertainment of marrow involvement in this setting were considered important, a bone marrow study would be indicated. Conversely, the timing of the blood MRD analysis could be postponed. The German CLL Study Group have also standardized three four tube panels of four-color reagents using a larger volume of blood, and where appropriate 2 million leucocytes were collected (3). This paper also shows that the overall ability of MRD flow is to detect and quantify residual CLL cells and is not affected by the addition of rituximab to FC chemotherapy (3). In a 2005 editorial, Montserrat (4) called for standardization and revision of 1996 NCI response guidelines to include MRD negative status as a goal. In 2008, Hallek et al. (5) revised the NCI guidelines to include not only a definition of MRD but also encouraged that the MRD detection become part of future CLL clinical trials because MRD negative status has prognostic impact. The GCLLSC, ERIC, and GEIL studies have all demonstrated that multicolor flow cytometry can meet the needs of a robust MRD assay. All three groups used a four-color, five tube panel and a sequential gating strategy. Newer clinical flow cytometers now allow for the use of 6–8 colors. The Salamanca group has developed an automated method for doing MRD detection by merging files on the same patient with the addition normal donor virtual 1:1 dilution as a control (6). Knowledge of the original CLL immunophenotype would be useful. One could envision an integration of the GCLLSC, ERIC, and GEIL methods combining CD19/CD20/CD5/CD38/CD79b/ CD22/CD41/CD81 and CD19/CD20/CD5/CD45/CD3/CD14/kappa/lambda. Del Poeta et al. (7) concluded that the addition of rituximab consolidation and maintenance therapy prolonged response duration significantly in patients with MRD + CLL. MRD detection is indicated in the investigation, care, and treatment of the CLL patient. Clinical trials will be required to answer the question of overall survival in CLL. Regardless, CLL remains a model disease for the appropriate treatment of the elderly with progressive disease.

Surprise! HSC Are Aberrant in Chronic Lymphocytic Leukemia

In this issue of Cancer Cell, Kikushige etal. report the surprising finding that, in human chronic lymphocytic leukemia (CLL), hematopoietic stem cells (HSC) aberrantly generate clonal B cells with CLL-like phenotypes, which implicate HSC in the pathogenesis of this mature lymphoid malignancy and has major implications for CLL therapies.

New insights into the pathobiology of chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a varying clinical outcome; however, the pathogenic mechanisms involved in disease development have remained largely unknown. In recent years, novel biomarkers, such as certain recurrent genomic alterations and the immunoglobulin heavy variable gene mutational status, have significantly improved the subdivision of the disease along with the prognostic assessment of individual patients. Advanced molecular studies have also revealed important genetic/epigenetic events and potential susceptibility loci for CLL, as well as implicating antigens in CLL development. Furthermore, the presence of monoclonal B cell lymphocytosis (MBL) has been demonstrated to precede CLL and appears to be a pre-leukemic condition. In this review, we will not only focus on recent developments made in the fields of genetics and immunogenetics in CLL, but also provide a brief overview of MBL, since we believe that advancements in these areas will have a major impact on our understanding of CLL pathobiology.

Amplification of 6p Associated with Familial CLL

AbstractAbstract 2432Chronic lymphocytic leukemia (CLL) is one of the most familial of all cancers but the genetic basis of this heritability remains poorly characterized. Families with very strong inheritance of CLL have been described in the literature, and recently the occurrence of CLL in one such family was associated with a polymorphism in the DAPK gene. Here we report the genomic characterization of a family in which CLL appears to be inherited in a Mendelian autosomal dominant manner. Within this family, five of eleven siblings of the first generation were affected, and one of those affected siblings had five children, of whom three were also affected (the second generation). The children of the second generation are currently aged 20–30 and hence too young to know whether they will develop CLL. We performed high-density single-nucleotide polymorphism (SNP) array analysis and gene expression profiling on tumor and germline DNA from four of the offspring of the second generation, as well as six of their children. Analysis of the SNP array data revealed a significant germline amplification of 6p, spanning 0–720 Mb and encompassing a known copy number variant (CNV) region but significantly larger than the CNV region. This amplification was found in both affected individuals with samples available from the second generation, and was transmitted by each of them to one of their two children in the third generation. This amplification was absent from the two unaffected members of the second generation, their children, or any of the other 189 individuals with CLL who were analyzed in our high-density SNP array dataset. None of the unaffected individuals with or without the amplification had evidence of monoclonal B cell lymphocytosis (MBL) by highly sensitive flow cytometry. These unaffected individuals also lacked any PCR-detectable oligoclonal or monoclonal immunoglobulin heavy chain gene rearrangement suggestive of MBL. The region of amplification contains four protein-coding genes: EXOC2, DUSP22, HUS1B and IRF4. We sequenced the coding regions of these four genes and the 5' and 3' UTRs of IRF4 in all family members, but found no somatic mutations in this family. All four genes were also sequenced in 92 other familial CLLs, identifying no somatic mutations. We then analyzed our gene expression profiling data to assess whether any genes in this region were altered in the affected individuals with the amplification. This analysis revealed a significant 1.74X increase in IRF4 expression in the CLLs with the amplification compared to those without (q value < 0.001). By Western blotting, we confirmed that IRF4 protein was increased approximately two-fold in amplified compared to non-amplified samples. These data suggest that the amplification may target IRF4, which has been previously implicated in CLL by a genome wide association study that identified a tag SNP in its 3' UTR as a CLL risk allele. Further analysis of our SNP data demonstrated allele specific amplification in this region, and mass-spectrometric genotyping confirmed enrichment of the CLL risk allele in the individuals with amplification. We conclude that amplification of IRF4 carrying the risk allele for CLL appears likely to be the culprit predisposing to CLL in this family. Disclosures:No relevant conflicts of interest to declare.

Hematopoietic Stem Cells Are Primarily Involved In Pathogenesis of Chronic Lymphocytic Leukemia.

AbstractAbstract 4604Chronic lymphocytic leukemia (CLL) is characterized by consistent expansion of B cells in peripheral lymphoid organs. CLL B cells express CD5 antigen, and have clonal rearrangement of immunoglobulin heavy chain (IGH) genes with the restricted usage of VH genes. CLL has thus been believed to represent retention or proliferation of abnormal B cell clones presumably with anti-apoptotic potential, or with deregulated response to auto-antigens. In this study, we extensively search for CLL-initiating cells by utilizing the xenogeneic transplantation system, in which human hematopoietic stem cells (HSCs) can normally develop multi-lineage cells including polyclonal B cells. In the xenotransplantation system, neither human (h)CD34-hCD19+ circulating B cells, nor hCD34+hCD38+ bone marrow (BM) progenitor populations from CLL patients engrafted after injection of >106 cells. We then transplanted hCD34+hCD38- BM HSC population from 12 CLL patients. Mice transplanted with as few as 3×103 cells of the hCD34+hCD38- BM population from CLL patients exhibited multi-lineage human hematopoietic reconstitution. Surprisingly, monoclonal or oligoclonal hCD5+ mature CLL-like B cells were reconstituted in the mice transplanted with HSCs from 11 out of 12 CLL patients. IGH gene rearrangement analyses demonstrated that these clonal abnormal B cells derived from hCD34+hCD38- HSCs of CLL patients (CLL-HSCs) were completely independent from the original leukemia. Interestingly, these clonal B cells showed highly biased usage of VH genes for their IGH rearrangement like primary CLL cells. We then checked the chromosomal abnormalities including del13q14 and del11q22 in CLL-HSCs and the clonal B cells reconstituted in the recipient mice. In 3 cases examined, both of them were negative for the chromosomal abnormalities detected in the original CLL cells, suggesting that the chromosomal abnormalities were acquired as a 2nd leukemogeneic events during CLL progression in the patients.The fact that CD34+CD38- HSCs from CLL patients but not those from normal individuals reconstitute monoclonal B cell lymphocytosis (MBL)-like human hematopoiesis in our xenograft model strongly suggests that some genetic abnormalities for mature monoclonal B cell development are acquired already at the HSC level in CLL patients. HSCs in CLL patients are multipotent, but once they commit to the B cell lineage, they use preferentially the VH1, VH3 and VH4 regions for IGH recombination and MBL-like clonal B cell expansion occur. Our hypothesis is that such B cell clones may already be abnormal in that they clonally expand in response, for example, to auto-antigens (xeno-antigens in mice), and these preleukemic B cell clone may possibly sequentially receive additional genetic events including chromosomal abnormalities during development of CLL and finally progress to clinical CLL.Although this xenograft model may not recapitulate full picture of CLL progression, our data clearly show that primary leukemogenic event occurs at the multipotent HSC stage in human CLL. Disclosures:No relevant conflicts of interest to declare.

Paraneoplastic T cell lymphocytosis associated with a thymoma in a dog

A four-year-old male neutered Australian shepherd dog was diagnosed with a thymoma and concurrent mature T cell lymphocytosis. The lymphocytosis consisted of a mixed population of T cells expressing either CD4 or CD8 or neither marker, and the result of polymerase chain reaction for antigen receptor rearrangement was negative. The peripheral lymphocytosis resolved within 24 hours following thoracotomy and thymectomy. Similar cases have been reported in man, but the aetiology of the increased circulating lymphocytes remains unclear. Although peripheral lymphocytosis is an uncommon paraneoplastic syndrome associated with thymomas, thymoma should be considered as a differential when the increased lymphocytes consist of a mixed population of T cells.

Scleritis and multiple systemic autoimmune manifestations in chronic natural killer cell lymphocytosis associated with elevated TCRα/β+CD3+CD4−CD8− double-negative T cells

Background/aimsChronic natural killer lymphocytosis (CNKL) has been associated with systemic autoimmunity; however, its association with scleritis or ocular autoimmunity has not been characterised. The natural killer (NK) cell function and...

Monoclonal B cell lymphocytosis in hepatitis C virus infected individuals

Monoclonal B cell lymphocytosis (MBL) is a preclinical condition characterized by an expansion of clonal B cells in the absence of B lymphocytosis (BALC < 5 × 10(9)/L) in the peripheral blood, without clinical signs, suggestive of a lymphoproliferative disorder. B cell clonal expansions are also associated with hepatitis C virus (HCV) infection and they can evolve into lymphoproliferative disorders such as mixed cryoglobulinemia and non-Hodgkin lymphomas (NHL). The relationship between MBL and HCV infection has not been established yet. By five-colour flow cytometry, we analyzed 123 HCV positive subjects with diagnosis of chronic hepatitis (94) or cirrhosis (29); 16 of those with cirrhosis had a diagnosis of hepatocellular carcinoma. MBL were identified in 35/123 (28.5%), at significantly higher frequency than in the general population. Sixteen/thirty-five were atypical-chronic lymphocytic leukemia (CLL) MBL (CD5(+), CD20(bright)), 13/35 were CLL-like MBL (CD5(bright), CD20(dim)), and 6/35 were CD5(-) MBL. Twenty-four/ninety-four (25.5%) patients affected by chronic hepatitis had MBL, whereas 11/29 (37.9%) patients with cirrhosis showed a B cell clone. A biased usage of IGHVgenes similar to HCV-associated NHL was evident. All three types of MBL can be identified in HCV-infected individuals at a higher frequency than in the general population, and their presence appears to correlate with a more advanced disease stage. The phenotypic heterogeneity is reminiscent of the diversity of NHL arising in the context of HCV infection. The persistence of HCV may be responsible for the dysregulation of the immune system and in particular of the B cell compartment.

Monoclonal B cell lymphocytosis: Clinical and population perspectives

Monoclonal B Cell Lymphocytosis (MBL) refers to clones of CLL-like cells that exhibit CLL characteristics that fall short of the numbers required for CLL diagnosis. Data from large CLL kindreds document increased prevalence of MBL suggesting a genetic contribution to its etiology. The molecular features that favor progression of MBL to CLL are poorly understood but an elevated B-cell count is a risk factor for progression. An important consideration when evaluating volunteers from CLL families who are willing to donate bone marrow is that MBL be ruled out since the MBL donor clone could result in a second CLL in the recipient. Further studies of MBL are needed to identify the molecular features and how they evolve during progression.

Long‐term follow‐up of monoclonal B‐cell lymphocytosis detected in environmental health studies

Four individuals in whom Monoclonal B cell Lymphocytosis (MBL) had been previously detected were evaluated for the fourth time after 15-18 years since initial testing. All four were environmental health study participants without hematologic malignancies who had elevated absolute B cell counts at initial testing. The current laboratory evaluation included complete blood counts, lymphocyte immunophenotypes, immunoglobulin heavy-chain variable (IGHV) gene mutation status, and serum tests for monoclonal immunoglobulins and free light chains. Results from this evaluation were compared with those from the three previous evaluations. Clinical status was assessed by reviewing medical records. B-cell clones with phenotypic characteristics of the original MBL clone were detected in three of the four individuals. Since the last evaluation in 2003, one participant who had a clinical diagnosis of Waldenstrom's Macroglobulinemia had developed a diffuse large cell lymphoma and was treated. Another participant continued to show a decline in lymphocyte and B cell counts, reaching clinical lymphocytopenia and B cell lymphopenia. The MBL clone was still detectable. The remaining two participants had stable blood counts and MBL phenotypes. Neither had been diagnosed with a hematologic malignancy. However, molecular analysis revealed clonal changes in both: one showed a marked decline in the percentage of somatically-mutated B cells, and the other showed a clonal transition from IGHV3-13 to IGHV4-34. A diversity of clonal evolution was observed in these MBL cases. These observations suggest that long-term follow-up studies using standardized MBL subcategories are essential to understanding B-cell pathobiology and optimizing clinical management.

Commentary: Comparison of current flow cytometry methods for monoclonal B cell lymphocytosis detection

Monoclonal B cell lymphocytosis (MBL) is now recognized as the B-lymphocyte analogue of a monoclonal gammopathy of unknown significance. MBL can be the precursor of chronic lymphocytic leukemia or associated with non-Hodgkin's lymphoma. It may be associated with an autoimmune abnormality or be related to aging (immunosenescence). The combination of available new fluorochrome-conjugated monoclonal antibody reagents, multilaser instrumentation, and improved software tools have led to a new level of multicolor analysis of MBL. Presently, several centers, including the University of Salamanca (Spain), Duke University (Durham, NC), Mayo Clinic (Rochester, MN), and the National Cancer Institute (Bethesda, MD) in conjunction with the Genetics and Epidemiology of Familial chronic lymphocytic leukemia Consortium, the Food and Drug Administration (Bethesda, MD), and the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry (Atlanta, GA) in collaboration with Saint Luke's Hospital (Kansas City, MO), the Università Vita-Salute San Raffaele in Milan (Italy), and Leeds Teaching Hospital (UK) are all actively conducting studies on MBL. This commentary is an updated summary of the current methods used in these centers. It is important to note the diversity of use in reagents, instruments, and methods of analysis. Despite this diversity, there is a consensus in what constitutes the diagnosis of MBL and its subtypes. There is also an emerging consensus on what the next investigative steps should be.

The DLEU2/ miR-15a/ 16-1 Cluster Controls B Cell Proliferation and Its Deletion Leads to Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a malignancy of B cells of unknown etiology. Deletions of the chromosomal region 13q14 are commonly associated with CLL, with monoclonal B cell lymphocytosis (MBL), which occasionally precedes CLL, and with aggressive lymphoma, suggesting that this region contains a tumor-suppressor gene. Here, we demonstrate that deletion in mice of the 13q14-minimal deleted region (MDR), which encodes the DLEU2/miR-15a/16-1 cluster, causes development of indolent B cell-autonomous, clonal lymphoproliferative disorders, recapitulating the spectrum of CLL-associated phenotypes observed in humans. miR-15a/16-1-deletion accelerates the proliferation of both human and mouse B cells by modulating the expression of genes controlling cell-cycle progression. These results define the role of 13q14 deletions in the pathogenesis of CLL.