B-cell Chronic Lymphocytic Leukemia Cases Research Articles

Identification of novel, clonally stable, somatic mutations targeting transcription factors PAX5 and NKX2-3, the epigenetic regulator LRIF1, and BRAF in a case of atypical B-cell chronic lymphocytic leukemia harboring a t(14;18)(q32;q21).

Diagnosis of B-cell chronic lymphocytic leukemia (B-CLL) is usually straightforward, involving clinical, immunophenotypic (Matutes score), and (immuno)genetic analyses (to refine patient prognosis for treatment). CLL cases with atypical presentation (e.g., Matutes ≤ 3) are also encountered, and for these diseases, biology and prognostic impact are less clear. Here we report the genomic characterization of a case of atypical B-CLL in a 70-yr-old male patient; B-CLL cells showed a Matutes score of 3, chromosomal translocation t(14;18)(q32;q21) (BCL2/IGH), mutated IGHV, deletion 17p, and mutations in BCL2, NOTCH1 (subclonal), and TP53 (subclonal). Quite strikingly, a novel PAX5 mutation that was predicted to be loss of function was also seen. Exome sequencing identified, in addition, a potentially actionable BRAF mutation, together with novel somatic mutations affecting the homeobox transcription factor NKX2-3, known to control B-lymphocyte development and homing, and the epigenetic regulator LRIF1, which is implicated in chromatin compaction and gene silencing. Neither NKX2-3 nor LRIF1 mutations, predicted to be loss of function, have previously been reported in B-CLL. Sequencing confirmed the presence of these mutations together with BCL2, NOTCH1, and BRAF mutations, with the t(14;18)(q32;q21) translocation, in the initial diagnostic sample obtained 12 yr prior. This is suggestive of a role for these novel mutations in B-CLL initiation and stable clonal evolution, including upon treatment withdrawal. This case extends the spectrum of atypical B-CLL with t(14;18)(q32;q21) and highlights the value of more global precision genomics for patient follow-up and treatment in these patients.

Flow Cytometric Analysis of ZAP-70 Protein Expression for B-Cell Chronic Lymphocytic Leukemia Prognostication: Usefulness and Limitations.

Objectives: The heterogenous clinical course in B-cell chronic lymphocytic leukemia (B-CLL) can be linked to several genetic and phenotypic characteristics of malignant B-cells. Prognostic analysis in B-CLL is routinely carried out to assist patient management; particularly to predict the time to initiate treatment. Increased ZAP-70 expression is a surrogate marker for unmutated immunoglobulin genes and inferior clinical outcomes which can be quantified to predict future outcomes in B-CLL patients. The study determined the ZAP-70 expression pattern using Z-index in Pakistani patients with B-CLL.Methods: A retrospective analysis of B-CLL cases diagnosed and confirmed on flow cytometry at Aga Khan University Hospital for the last six years which had also undergone ZAP-70 analysis were included. In all these cases, ZAP-70 expression was quantified by measuring mean fluorescence intensities (MFIs) of normal B-cells, T-cells, and CLL-cells (CD19 and CD5 double-positive population). ZAP-70 expression was divided into high, low, and negative categories based on Z-index calculation. Mann-Whitney U test was utilized to determine the significance of ZAP-70 variations in different age groups and genders. P-value <0.05 was considered significant.Results: A total of 120 patients of B-CLL had ZAP-70 analysis during the study period. The median age was 62 with an interquartile range of 35-87 and male to female ratio of 2:1. ZAP-70 expression was high in 18 (15%), low in 52 (43.3%) and negative in 50 (41.7%) cases. No significant difference in ZAP-70 expression with respect to the age or gender of the study population was identified using appropriate statistical calculations.Conclusions: This study showed only 15% of B-CLL cases showing high ZAP-70 expression, a surrogate biomarker for possible aggressive behavior which may necessitate therapeutic intervention and close surveillance.

Polyclonal B-cell lymphocytosis in English bulldogs.

BackgroundEnglish bulldogs disproportionally develop an expansion of small B‐cells, which has been interpreted as B‐cell chronic lymphocytic leukemia (BCLL). However, clonality testing in these cases has often not been supportive of neoplasia.HypothesisEnglish bulldogs have a syndrome of nonneoplastic B‐cell expansion.AnimalsEighty‐four English bulldogs with small‐sized CD21+ B‐cell lymphocytosis in the blood as determined by flow cytometry.MethodsThis is a retrospective study. We characterized this syndrome by assessing B‐cell clonality, clinical presentation, flow cytometric features, and immunoglobulin gammopathy patterns. We identified 84 cases with CD21+ lymphocytosis among 195 English bulldogs with blood samples submitted to the Colorado State University‐Clinical Immunology laboratory for immunophenotyping between 2010 and 2019. Flow cytometry features were compared to normal B‐cells and BCLL cases. PCR for antigen receptor rearrangements (PARR) by multiple immunoglobulin primers was performed to assess B‐cell clonality. A subset of cases with gammopathy were examined by protein electrophoresis, immunofixation, and immunoglobulin subclass ELISA quantification.ResultsSeventy percent (58/83) of cases had polyclonal or restricted polyclonal immunoglobulin gene rearrangements, suggesting nonmalignant B‐cell expansion. The median age of all dogs in the study was 6.8 years and 74% were male. The median (range) lymphocyte count was 22 400/μL (2000‐384 400/μL) and B‐cells had low expression of class II MHC and CD25. Splenomegaly or splenic masses were detected in 57% (26/46) of cases and lymphadenopathy in 11% (7/61). Seventy‐one percent (52/73) of cases had hyperglobulinemia and 77% (23/30) with globulin characterization had IgA ± IgM polyclonal or restricted polyclonal gammopathy patterns.Conclusions and Clinical ImportancePolyclonal B‐cell lymphocytosis in English bulldogs is characterized by low B‐cell class II MHC and CD25 expression, splenomegaly and hyperglobulinemia consisting of increased IgA ± IgM. We hypothesize that this syndrome has a genetic basis.

Expression of the CD5 Molecule in Chronic Lymphocytic Leukemia

Abstract CD5 is a T cell marker, aberrantly expressed in B cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL). Other chronic B cell malignancies, including hairy cell leukemia (HCL) and B cell prolymphocytic leukemia (B-PLL), are usually CD5-. In this study, CD5 expression was investigated in leukemia cells from 42 patients with chronic B cell lymphoproliferative disease. Immunophenotyping was accomplished by flow cytometry with a panel of monoclonal antibodies specific to chronic lymphoproliferative disease such as: CD3, CD4, CD5, CD7, CD8, CD10, CD14, CD16/56, CD19, CD22, CD23, HLADR, CD45, anti-immunoglobulin heavy chain (IgH), anti- kappa and lambda light chain. The hematological parameters were obtained using a hematological analyzer and cytomorphology in Leishman-stained blood films. Information about the patients such as age, sex and clinical data related to the disease was collected. Of the 42 patients, 22 were male and 20 female. Immunophenotyping showed that 35 cases were B-CLL, 3 B-PLL and HCL and one MCL. The lymphadenopathy and splenomegaly were present in 73.8% and 71.4% of cases, respectively. CD5 expression was present in all B-CLL and MCL cases. However, in HCL, and B-PLL, CD5 expression was negative in all cases. In conclusion, these data demonstrated that CD5 expression is useful in distinguishing B-CLL from other chronic B cell malignancies such as HCL and B-PLL, but not between B-CLL and MCL, where positivity may be present in both. Disclosures No relevant conflicts of interest to declare.

A Possible Risk Gene for B-cell Chronic Lymphocytic Leukemia: NRIP1

Background: B-cell chronic lymphocytic leukemia (B-CLL) is the most common type of leukemia in adults, with the underlying mechanisms remains unclear. The aim of this study is to investigate the novel genetic risk of B-CLL by systematically reviewing the published literatures and performing a meta-analysis.Methods: A comprehensive search of electronic databases was completed by using Illumina BioEngine. Twenty one B-CLL case/control bio-sets from four different studies were selected, including 195 B-CLL cases and 31 controls. The selected top B-CLL risk genes were further analyzed by integrating an online open source B-CLL genetic database. Pathway enrichment analysis (PEA) and network connectivity analysis (NCA) were conducted to identify potential functional association between target genes and B-CLL.Results: One novel gene (NRIP1) and two known genes (INPP5F and LEF1) were identified through the meta-analysis as top target genes for B-CLL. These genes play important roles within multiple B-CLL genetic pathways and tightly related to known B-CLL target genes. NCA results also revealed strong functional association between these genes and B-CLL.Conclusion: This study identified known as well as novel B-CLL target genes and their functional pathways that involved in the B-CLL pathogenesis. Our results may provide new insights into the understanding of the genetic mechanisms of B-CLL.

Opposite changes of regulatory T cell blood content may differentially contribute to atherosclerosis or lymphoproliferative disorders

Background. Chronic autoimmune inflammation in arterial wall may lead to atherosclerosis progression. Objective. The aim of this study was to investigate the association between Treg, Th17 and B1a cell blood frequencies as well as IgM autoantibodies to oxLDL and the abundance of carotid atherosclerosis. Material and methods. 18 patients with increased IMT (intima-media thickness) and 65 patients with different severity of carotid atherosclerotic plaques were included. Treg, Th17 and B1a cell blood frequencies were assessed via direct immunofluorescence staining and flow cytometry, oxLDL as well as IgM autoantibodies to oxLDL were measured with commercial kits. Results. We observed higher values of Treg in patients without carotid atherosclerosis. Patients with intact carotid arteries as compared to patients with mild atherosclerotic plaques had decreased Th1 levels. OxLDL IgM levels were higher in patients with intact carotid arteries. Patients who received statin treatment had higher levels of Treg. Immunophenotyping of B cells revealed two cases of monoclonal B-cell lymphocytosis and 1 case of B-CLL (B-cell chronic lymphocytic leukemia) in elderly patients with intact carotid arteries. Conclusion. We hypothesize that certain parameters of cell immunity may hamper atherosclerosis while protecting from lymphoproliferative disorders.

Murine double minute-2 expression in B-cell chronic lymphocytic leukemia: correlation with apoptosis and disease outcome

Introduction B-cell chronic lymphocytic leukemia (B-CLL) is characterized by accumulation of quiescent monoclonal CD5+ B cells, which arise from undefined defects in apoptotic cell death. murine double minute-2 (MDM2) oncoprotein exists in an autoregulatory feedback loop with the tumor suppressor protein p53 and block p53-mediated transactivation and apoptosis. Aim of this work The aim of the study was to evaluate the overexpression of MDM2 in B-CLL patients and correlate it with apoptosis using annexin-V expression and disease outcome. Patients and methods This study was conducted on 55 patients including 45 patients with typical B-CLL; their ages ranged from 55 to 72 years, with a mean age of 58.55 ± 3.9 years. Ten age-matched and sex-matched apparently healthy individuals were included as the control group. The diagnosis of B-CLL was based on clinical, hematological, immunophenotypic, and cytogenetic criteria. Annexin-V and MDM2 oncoprotein expression was assessed by flow cytometric analysis. Results Our study revealed that MDM2 was overexpressed in 53% of B-CLL cases and it was significantly elevated in comparison with the control group (P < 0.001). There was an inverse correlation between MDM2 overexpression and apoptosis (P < 0.001). The mean level of MDM2 expression was significantly reduced after therapy compared with the pretreatment level in the responder group (P < 0.001), whereas there was no significant difference (P>0.05) in the nonresponder group. The expression of MDM2 significantly correlated with the percentage of CD38 expression, LDH level, and B2M level (P < 0.001, P < 0.001, and P < 0.001, respectively) and patients who had responded to treatment showed a significant increase in the level of annexin-V (P < 0.001). MDM2 expression, percentage of CD38 expression, and LDH and B2M levels were significantly higher in the high-risk patients compared with low-risk and intermediate-risk patients (P < 0.001, P < 0.01, P < 0.05, and P < 0.001, respectively).Conclusion MDM2 expression was inversely correlated with apoptosis, with decreased MDM2 expression in responder patients.

Clinico-pathological impact of cytogenetic subgroups in B-cell chronic lymphocytic leukemia: Experience from India

The present study of 238 B-cell Chronic Lymphocytic Leukemia (B-CLL) patients were undertaken to seek the prevalence and to evaluate clinico-pathological significance of recurrent genetic abnormalities such as del(13q14.3), trisomy 12, del(11q22.3) (ATM), TP53 deletion, del(6q21) and IgH translocation/deletion. We applied interphase - fluorescence in situ hybridization (FISH) on total 238 cases of B-CLL. Our study disclosed 69% of patients with genetic aberrations such as 13q deletion (63%), trisomy 12 (28%), 11q deletion (18%), 6q21 deletion (11%) with comparatively higher frequency of TP53 deletion (22%). Deletion 13q displayed as a most frequent sole abnormality. In group with coexistence of ≥2 aberrations, 13q deletion was a major clone indicating del(13q) as a primary event followed by 11q deletion, TP53 deletion, trisomy 12, 6q deletion as secondary progressive events. In comparison with del(13q), trisomy 12, group with coexistence of ≥2 aberrations associated with poor risk factors such as hyperleukocytosis, advanced stage, and multiple nodes involvement. In a separate study of 116 patients, analysis of IgH abnormalities revealed either partial deletion (24%) or translocation (5%) and were associated with del(13q), trisomy 12, TP53 and ATM deletion. Two of 7 cases had t(14;18), one case had t(8;14), and four cases had other variant IgH translocation t(?;14). Detail characterization and clinical impact are necessary to ensure that IgH translocation positive CLL is a distinct pathological entity. Our data suggests that CLL with various cytogenetic subsets, group with coexistence of ≥2 aberrations seems to be a complex cytogenetic subset, needs more attention to understand biological significance and to seek clinical impact for better management of disease.

The Repertoire of Heavy Chain Immunoglobulin Genes in B-Cell Chronic Lymphocytic Leukemia in Russia and Belarus

Abstract 4579 Introduction.Mutation status of the heavy chain variable region genes is known as an important factor in long-term prognosis in B-cell chronic lymphocytic leukemia (B-CLL). A more detailed study of the gene sequences of immunoglobulin heavy chain (IgVH) led to the discovery of stereotyped antigen receptors (SAR) - receptors that have the same set of VH-, D- and JH- genes used. SARs have been found almost in a quarter of all B-CLL cases. Since this study there have been no data available concerning VH-gene usage and SARs in Russian and Belarusian B-CLL patients. Patients and methods.DNA or cDNA was amplified in 6 separated reactions using primers specific for VH-families, and a consensus JH primer [Campbell et al. 1992] or primer sets recommended by the BIOMED-2 [van Dongen et al. 2003]. PCR products were sequenced using family-specific primers and Big Dye Terminator v3.1 kit (Applied Biosystems). Sequences were analyzed with IgBlast (http://www.ncbi.nlm.nih.gov/igblast). 98% homology cutoff was used to discriminate between mutated and unmutated cases. Results.Total of 547 patients with B-CLL where analyzed; 192 patients with mutated IgVH-genes (35%) and 355 patients with unmutated ones (65%). We have identified 65 stereotyped receptors (SARs) in 198 of 491 Russian patients (40%). Twenty one SARs (confirmed) appeared in more than 3 patients (110 out of 198, 55%), 44 SARs (potential) found in 2 patients (88 out of 198 cases, 45%). The vast majority of confirmed SARs were found in the subgroup of patients without mutations in IgVH genes (95%). Among the potential SARs 15 pairs were from patients with and without mutations (34%), seven pairs from patients with mutations (16%) and 22 pairs from patients without IgVH mutations (50%). The most common SARs were: VH1–69/D3-3/JH6 (24 patients, 5%); VH1–69/D3–16/JH3 (8 patients); VH1–69/D2-2/JH6 (8 patients); VH1–69/D3–10/JH6 (6 patients). Among 56 Belarusian patients we have identified only 4 SARs, one confirmed SAR (4 cases) and 3 potential ones. Confirmed (VH1–69/D3-3/JH6) and one potential (VH1–69/D3–16/JH3) were also found in Russia while other two potential (VH1–69/D2–15/JH6 and VH2–5/D2-2/JH6) were not observed in Russia. Discussion.In Russia and Belarus, VH1–69 gene is found in 20% of all cases of B-CLL, and almost always (95%) in unmutated cases. This finding well correlates to the data obtained by [Kryachok et al. 2012] concerning high VH1–69 usage in Ukrainian patients. In other European countries, this gene is less common: about 14% of cases in Sweden, France and Spain, 11% in the UK and about 6–7% in Greece and Italy [Ghia et al. 2005; Tobin et al. 2004; Duke et al. 2003]. In all these countries, this gene is also prevalent in patients with unmutated VH-genes. Nordic countries are characterized by very frequent use of gene VH3–21 - 9%, while in Russia and the countries of central and southern Europe, it occurs at least 3 times less (about 3%), and was not found in Belarusian samples. This gene is also associated with poor prognosis of B-CLL, regardless of mutation status of IgVH genes. Interestingly, in Asia (China, Japan, Iraq) VH1–69 and VH3–21 genes are almost not observed [Nakamura et al. 1999; Farsangi et al. 2007; Lijuan Chen et al. 2008]. Narrowing of the repertoire of IgVH genes - specific feature of B-CLL indicatites that influence of antigen (at least in some cases) occurs during the development of the disease. Also, factors of genetic background as well as geographical environment could be important. It is possible that future treatment decisions will be based not only on the IgVH mutation status, but also on the characteristics of certain antigen receptor. Disclosures:No relevant conflicts of interest to declare.

A Comparative Analysis of Cutaneous Marginal Zone Lymphoma and Cutaneous Chronic Lymphocytic Leukemia

The morphologic distinction between cutaneous marginal zone lymphoma (CMZL) and secondary cutaneous involvement by B-cell chronic lymphocytic leukemia (B-CLL) can be difficult. Both entities can show very similar architectural patterns of involvement in the skin and not uncommonly, the skin can be the first site of presentation of B-CLL in the elderly. We reviewed biopsies of 13 patients with cutaneous B-CLL and 14 patients with CMZL to compare their histologic and immunohistochemical features. CMZL and cutaneous B-CLL both predominantly exhibited a nodular pattern of skin involvement (9 of 13 B-CLL, 9 of 14 CMZL) with a minority of cases demonstrating a diffuse pattern (4 of 13 B-CLL, 4 of 14 CMZL). Although reactive germinal centers (12 of 14 cases) and plasma cells (10 of 14 cases) were seen more often in CMZL, plasma cells were also observed in cases of B-CLL (4 of 13). The lesional cells of B-CLL expressed CD79, CD5, CD23, and CD43, although CMZL did not express CD5 or CD43. Although we noted light chain restriction in 13 of 14 cases of CMZL cases, we also observed light chain restriction in 4 of 13 cases of B-CLL. Our results indicate that CMZL and B-CLL can be morphologically similar and both may show light chain restriction. Complete immunophenotyping is necessary to ensure that all cases are correctly classified.

Abstract LB-342: Matrix metalloproteinase-9 is a novel pathogenic factor in B-cell chronic lymphocytic leukemia

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation in peripheral blood of CD5+ B lymphocytes, which ‘ progressively infiltrate lymphoid tissues. Several molecules, including matrix metalloproteinase-9 (MMP-9), are involved in these processes and could represent therapeutic targets. We previously showed that MMP-9 plays an essential role in B-CLL cell migration through basement membranes or endothelial cells (Redondo-Muñoz et al., Blood 108: 3143, 2006). We also recently demonstrated that alpha4beta1 integrin and a 190 kDa CD44v isoform are receptors for MMP-9 in B-CLL but not in normal B cells and that the MMP-9 hemopexin domain is essential for these interactions. Binding of MMP-9 to B-CLL cells inhibited cell migration and this required MMP-9 proteolytic activity (Redondo-Muñoz et al., Blood 112: 169, 2008). To further define the role of surface-bound MMP-9 in B-CLL we have employed several techniques including western blot, zymography, immunofluorescence, immunoprecipitation, siRNA transfection, cell adhesion/soluble binding assays, apoptosis, and cell migration assays. B-CLL cells cultured on pro- or mature MMP-9, proMMP-9 hemopexin domain or proMMP-9 catalytically-inactive mutant, showed decreased spontaneous apoptosis. This was due to induction of a survival pathway involving Lyn activation, STAT3 phosphorylation and Mcl-1 upregulation and did not require catalytic activity. This pathway was induced in all B-CLL cases and was active in B-CLL lymphoid tissues. Our results highlight the role of MMP-9 in B-CLL pathogenesis and indicate that MMP-9 hemopexin domain could constitute a therapeutic target in this malignancy. Supported by grants PI060400 and SAF2009-07035 from the Ministerio de Ciencia e Innovación and by the Fundación de Investigación Médica Mutua Madrileña. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-342.

KIR Genes and Their HLA-C Ligands in B-Cell Chronic Lymphocytic Leukemia in a Polish Population.

Abstract 4402 IntroductionDysfunction in cellular and humoral immunity entails an increased risk of B-cell chronic lymphocytic leukemia (B-CLL). It has been suggested that innate immunity, especially natural killers cells play key role in antitumor cytotoxicity regulated by interaction between killer immunoglobulin-like receptors (KIRs) on NK cells and some T-cell subpopulations and their HLA-class I ligands on target cells. KIRs receptors have been divided into two functional groups: with long cytoplasmic tails (DL) - inhibitory and with short cytoplasmic tails (DS) - activating. Two broad haplotypes of KIR genes have been defined: the A haplotype characterized by presence of single activating KIR gene (2DS4) and the B haloptype characterized by two or more activating KIR genes (2DS1, 2DS2, 2DS3, 2DS5 and 3DS1).The 2DL2, 2DL3 and 2DS2 KIRs bind to HLA-C1 allotype (Asp80), while 2DL1 and 2DS1 KIRs bind to HLA-C2 allotype (Lys80). Due to lack of representative data in literature the study was undertaken to determine the association between polymorphism of KIR genes and HLA-C allotypes and susceptibility to B-CLL. Patients and methodsThe following KIR genes were typed: 2DL1, 2DL2, 2DL3, 3DL1, 2DS1, 2DS2, 2DS3, 2DS4fl, 2DS4del, 2DS5, 3DS1, in 200 B-CLL patients and 199 healthy individuals using PCR-SSP method. The HLA-C1 and HLA-C2 were determined using Olerup SSP typing kit in 187 B-CLL patients and 104 controls. ResultsIndividual KIR gene content was similar in B-CLL cases and healthy controls although patients with KIR2DS3 gene were more common among cases compared to controls (36% vs. 27%, p=0.06) (Table1). The frequency of AA haplotype did not differ between studied groups (29% vs. 28%). Moreover no differences were found between incidence of HLA-C allotypes (HLA-C1: 74.3% vs. 76.0%, HLA-C2: 67.4% vs.70.2%), genotypes (C1/C1: 32.6% vs 29.8%, C1/C2: 41.7% vs 46,2% and C2/C2: 25.7% vs 24.1) and KIR genes in combination with genes for their HLA-C ligands in B-CLL patients and controls (Table 2).Table 1Frequency of KIR genes in B-CLL patients and controls.2DL12DL22DL32DS12DS22DS32DS4del2DS4fl2DS53DL13DS1B-CLL (n=200)1951061767510854166565318670[%]97.553.088.037.554.027.083.028.026.593.035.0Controls (n=199)1971061808111171162586118178[%]99.053.390.540.755.835.781.429.130.791.039.2Table.2Frequency of the KIR-HLA gene combinations in B-CLL patients and controls.B-CLL (n = 187)Controls (n = 104)N%N%2DL1+/C2+12265.247370.192DL2/3+/C1+13974.337975.962DS1+/C2+4926.202423.082DS2+/C1+7137.974038.462DL1+/C2-6032.083028.802DL2/3+/C1-4825.692524.042DS1+/C2-2211.761817.312DS2+/C1-1910.371110.58 ConclusionsThe data obtain in this study imply that there may be not direct association between KIR and HLA-C gene content in the genome and the presence of B-CLL. Disclosures:No relevant conflicts of interest to declare.

MicroRNA expression in chronic lymphocytic leukaemia

MicroRNA expression in chronic lymphocytic leukaemia

Value of Additional CD19 Lymphocyte Counts for the Detection of B-CLL in a Primary Health Care Laboratory

Background: B-cell chronic lymphocytic leukaemia (B-CLL) is the most common type of lymphoma in the Western world and is often diagnosed by coincidence in patients with an increased absolute lymphocyte count (ALC). Although immunophenotyping is advised for all patients with an increased ALC of unknown aetiology, referral for immunophenotyping in all patients with increased ALC will result in high costs and inconvenience for patients, since a considerable number of patients with increased ALC has no B-CLL or other malignancy. On the other hand, early haematological consultation at diagnosis is becoming more important, since initiation of treatment of B-CLL is changing from a symptom-based strategy towards a strategy based on biological markers. In this context, an additional CD19 count might be a valuable laboratory test to reduce unnecessarily referrals and to facilitate early haematological consultation of patients with B-CLL.Aim: To investigate the value of an additional CD19 count in detecting B-CLL in patients with increased ALC in a primary care laboratory.Methods: In the period from March 2001 until October 2006, a CD 19 lymphocyte count was performed in all patients, living in a previously defined geographic area, aged ≥ 40 years with an abnormal White Blood Cell count (WBC), defined as absolute lymphocyte count (ALC) ≥ 6.0x109 cells/L, relative lymphocyte count (RLC) ≥ 60% or atypical lymphocytes (AL) ≥ 2+. WBC's were requested by general practitioners for any reason not further specified. Patients with positive EBV or CMV serology were excluded. CD19 lymphocyte counts were classified as negative (<1.0x109/L) or positive (≥1.0x109/L). In case of a positive test, referral to a haematologist was advised. A geographic area in which the contingency areas of SALTRO and the referral hospitals overlapped was based on postal codes in order to link patients in whom a CD19 lymphocyte count was performed to newly diagnosed B-CLL patients in the hospitals. In October 2007, all cases of B-CLL in this area were identified by a stepwise search-strategy with a survey among general practitioners, including two postal questionnaires and a telephone reminder and by consultation of three different databases. All data were collected anonymously and CD19 lymphocyte count cases and B-CLL cases were cross-referenced by postal code and date of birth.Results: A CD19 lymphocyte count was performed in 543 patients of 199,108 (0.3%) patients in whom a WBC was performed; 229 (42%) were positive and 314 (58%) were negative. In total, 120 patients with a B-CLL were identified; 119 in the positive test group en 1 in the negative group. This resulted in a sensitivity and Positive Predictive Value (PPV) of 99% (95%CI 97.9–99.7) and 52% (95%CI 47.6–56.2) respectively and specificity and Negative Predictive Value (NPV) of 74% (95%CI 70.1–77.7) and 100% (95%CI 98.7–100) respectively. This resulted in a Number Needed to Screen (NNS) of 1.9. The AUC for CD19 count and ALC, 0.92 (95% CI 0.90–0.94), was significantly higher than the AUC for ALC only; 0.85 (95% CI 0.81–0.88). The PPV of ALC only, 34% (118/348) was significantly lower compared to the PPV of the CD19 count and ALC combined; 52% (119/229).Conclusion: CD19 count is a valuable laboratory test in detecting B-CLL in a primary care setting with a high sensitivity and PPV. The CD19 count is superior to ALC only. Introduction of a CD19 count in a primary health care laboratory has additional value in the selection of patients with increased ALC for referral to haematologists. The value of early detection strategies for B-CLL, such as CD19 counts in a primary health laboratory, will depend on the outcome of ongoing studies of treatment, based on new biological markers indicative of a poor prognosis, in early stages of B-CLL.

Impact of trisomy 12, del(13q), del(17p), and del(11q) on the immunophenotype, DNA ploidy status, and proliferative rate of leukemic B‐cells in chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (B-CLL) is a well-defined clinical entity with heterogeneous molecular and cytogenetic features. Here, we analyze the impact of trisomy 12, del(13q), del(17p), and del(11q) as determined by interphase fluorescence in situ hybridization analysis of purified neoplastic B-CLL cells on their immunophenotype, DNA ploidy status and proliferative rate.Overall, 111 of 180 (62%) B-CLL cases studied displayed one (50%) or more (12%) genetic abnormalities, del(13q) (35%) being more frequently detected than trisomy 12 (23%) followed by del(11q) (9%) and del(17p) (8%). Trisomy 12 was associated with a higher frequency of DNA aneuploidy, stronger expression of CD19, CD20, CD22, CD24, CD27, CD79b, CD38, and sIg and lower reactivity for CD43 with respect to cytogenetically nonaltered cases. In turn, cases with del(13q) displayed greater reactivity for CD20, FMC7, CD27, CD22, CD5, and bcl2, while del(11q) was associated with brighter expression of CD38, FMC7, CD25, and sIg. Hierarchical clustering analysis of the immunophenotype of B-CLL cases with cytogenetic abnormalities allowed the identification of three different groups of patients with increasing frequencies of trisomy 12, del(11q), and del(13q). Remarkably, none of the cytogenetic abnormalities analyzed except coexistence of 13q- and 17p- had a clear impact on the proliferative index of B-CLL cells.

The presence of high‐risk chromosome aberrations in chronic lymphocytic leukaemia does not correlate with centrosome aberrations

Chromosome aberrations are frequently found in B-cell chronic lymphocytic leukaemia (B-CLL), and specific chromosome aberrations identify poor prognostic subgroups. Almost all the aberrations identified in B-CLL involve loci where genes with a role in the regulation of centrosome duplication have been mapped. Centrosome aberrations have been described as a possible cause of numerical chromosome abnormalities in both solid and haematological tumours. However, little is known about the possible role of centrosome aberrations in B-CLL. To investigate whether centrosome aberrations do occur in B-CLL and correlate with cytogenetically defined prognostic subgroups, we examined a set of 64 B-CLL samples by immunofluorescent staining. B-CLL cases differed significantly from controls in the mean frequency of cells with centrosome aberrations, while no difference was found between subgroups with or without specific chromosome aberrations. Our results indicated that although centrosome aberrations were a common feature in B-CLL, they did not represent a reliable prognostic marker.

Regulation and Function of NOTCH2 in B-CLL Cells.

B-cell chronic lymphocytic leukemia (B-CLL) represents a clonal expansion of self-reactive CD5+ B-lymphocytes which seems to be resistant to apoptosis in vivo. One of the characteristics of B-CLL lymphocytes is the high expression of the B-cell differentiation/activation marker CD23 which we recently identified as a target gene for NOTCH2 signaling. NOTCH2 is implicated in the development/homeostasis of murine CD5+ (B-1a) B-cells, suggesting a function for human NOTCH2 in B-CLL leukemogenesis. Here we show that peripheral B-CLL lymphocytes overexpress a transcriptionally active form of NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 expression, and cytogenetics). Although the majority of unstimulated B-CLL samples downregulate their N2IC activity in vitro, DNA-bound N2IC complexes could be maintained by the protein kinase C (PKC) activator TPA (12-O-tetradecanoylphorbol 13-acetate) accompanied by an upregulation of the NOTCH2 target gene CD23 and increased B-CLL cell viability. These effects are sensitive to the PKC-δ selective inhibitor Rottlerin. In 80% of B-CLL cases, NOTCH2 signaling was found to be resistant to the γ-secretase inhibitors (GSI's) Dapt and compound E, indicative for the expression of truncated forms of NOTCH2 which do not require γ-secretase for processing and function. Inhibition of NOTCH2 either by Dapt in GSI sensitive B-CLL cases or, more specifically, by RNA interference downregulates CD23 expression on the mRNA and protein level and sensitizes B-CLL cells for apoptosis. Since self-reactive B-cells are normally eliminated either by chronic (anergy) or apoptotic (negative selection) B-cell receptor (BCR) signaling, we asked whether NOTCH2 modulates B-cell fate decisions triggered by the BCR. For this reason, we stably transduced the human B-cell line BL41 with constitutive active forms of NOTCH2 and found that NOTCH2 inhibits BCR mediated apoptosis induced by surface-IgM cross-linking. In summary, the data demonstrate that NOTCH2 signaling is deregulated in B-CLL cells and might be critically involved in the PKC-dependent maintenance of their malignant phenotype.

MiRNA Expression Profile of B-SLL Consistent with Normal Memory B Cells:BIC/miR–155 Specific Location in Proliferation Center.

Introduction: B cell Chronic Lymphocytic Leukemia (B-CLL) is characterized by the accumulation of nonproliferating mature-appearing lymphocytes in the blood, marrow, lymph nodes, and spleen. B-CLL behaves like leukemia and is found mostly in the blood. The expression of ZAP-70 and mutation status of the immunoglobulin heavy-chain gene (IgH) can serve as prognostic markers in B-CLL. ZAP-70 positive cases usually present with unmutated IgH genes and have a bad prognosis, whereas ZAP-70 negative cases mostly present with mutated IgH genes and have good prognosis. Gene expression studies of B-CLL indicated that the profile of IgH mutated and unmutated cases are similar to normal memory B cells. Several studies showed that miRNAs play important roles in pathogenesis of B-CLL and some miRNAs correlate with the prognosis in B-CLL. B-SLL is considered to be the same disease entity as CLL, but this variant is found mostly in bone marrow and the lymphatic system. The most aggressive type of B-SLL is characterized by neoplastic cells that are more responsive to B-cell receptor signaling and are characterized by proliferation centers (PCs), a potentially important site of neoplastic cell stimulation. Until now, only a few reports have been published about the ZAP-70 expression and IgH mutation status and no data are available about the microRNA expression profile.Methods: 33 B-SLL cases were retrieved from the pathology files. ZAP-70 expression was analyzed by using immunohistochemistry. IgH mutation status was determined using PCR followed by direct sequencing. Cases with homology of ≥98% with germline sequences were considered as unmutated and cases with homologies <98% as mutated. Levels of 15 miRNAs were determined by qRT-PCR using U6 as a housekeeping gene in 28 B-SLL cases with good quality RNA. As a control we also analyzed miRNA levels in normal naïve, GC and memory B cell subsets. RNA in-situ hybridization (ISH) was used to localize the most abundantly expressed miRNAs in B-SLL tissues.Results: 16 B-CLL cases were ZAP70+ with the vast majority of tumor cells staining positive and 17 were negative. Of the ZAP70+ cases 10 carried unmutated and 3 mutated IgH genes and 3 cases failed due to bad quality DNA. 14/17 ZAP-70- cases carried mutated IgH genes and 3 cases failed. miR-150, miR-21, miR-16, miR-92 and miR-155 were expressed at high levels in all B-SLL cases independent of the ZAP70 and IgH mutation status. The miRNA expression pattern in B-SLL was very similar to normal memory B cells. RNA-ISH for BIC, the primary transcript of miR-155, demonstrated the most abundant expression in the proliferation centers of B-SLL cases.Conclusion: In B-SLL there is significant correlation between ZAP-70 expression and IgH mutation status similar to B-CLL cases. miRNA expression levels in B-SLL did not correlate with ZAP-70 or IgH status. The overall expression profile is very similar to normal memory B cells. BIC/miR-155 expression is observed specifically in the proliferation center of B-SLL tissues.

IgVH Mutational Status and Clonality Analysis of Richter's Transformation

Approximately 5% of B-cell chronic lymphocytic leukemia (B-CLL) patients develop a secondary aggressive lymphoma, usually of diffuse large B-cell type (DLBCL), termed Richter's transformation (RT). Rarely, classic Hodgkin lymphoma (HL) is observed. Published small series suggest that tumor cells in DLBCL and HL can be clonally identical to the B-CLL clone or arise as an independent, secondary lymphoma. We describe the morphology, immunophenotype, and clinical features of 34 classic RT patients with DLBCL, 6 cases of B-CLL with HL, and 8 cases with scattered CD30-positive Hodgkin and Reed-Sternberg (HRS)-like cells. The clonal relationship of the 2 components was analyzed using sequencing analysis of immunoglobulin heavy chain variable region (IgVH) genes. In classic RT, 18/23 B-CLL cases (78%) showed clonal progression to DLBCL with identical IgVH sequences in both lymphoma components, whereas in 5 cases (22%) the DLBCL was clonally unrelated. Among clonally related RT samples, 73% carried unmutated IgVH genes, whereas 4/5 unrelated cases were mutated. Immunophenotypically, most cases of DLBCL irrespective of clonal relatedness showed significant differences in phenotype compared with the B-CLL, with common loss of CD5 and CD23. Using immuno-laser capture microdissection, sequencing of the IgVH CDR3 region of isolated HRS cells showed that 2/2 cases with HL were clonally unrelated, whereas they were clonally identical in 1/2 cases of B-CLL with scattered HRS-like cells. HRS or HRS-like cells in all 3 unrelated cases showed evidence of Epstein-Barr virus infection. Of interest, 5/6 cases of B-CLL with HL, and 5/6 cases of B-CLL with HRS cells showed mutated IgVH genes.

Analysis of 11q22–q23 deletion target genes in B-cell chronic lymphocytic leukaemia: Evidence for a pathogenic role of NPAT, CUL5, and PPP2R1B

Deletion of 11q22–q23 is associated with an aggressive course of B-cell chronic lymphocytic leukaemia (B-CLL). Since only in a subset of these cases biallelic inactivation of ATM was observed, we sought to identify other disease-associated genes within 11q22–q23 by analysing NPAT (cell-cycle regulation), CUL5 (ubiquitin-dependent apoptosis regulation) and PPP2R1B (component of the cell-cycle and apoptosis regulating PP2A) for point mutations and their expression in B-CLL by single-strand conformation polymorphism/sequence analysis of the transcripts and real-time polymerase chain reaction. Though none of the genes were affected by deleterious mutations, we observed a significant down-regulation of NPAT in B-CLL versus CD19+ B cells and of CUL5 in 11q deletion versus non-deletion B-CLL samples and measured reduced PPP2R1B transcript levels in a subset of B-CLL cases. Alternative splicing of PPP2R1B transcripts (skipping of exons 2/3, 3, 9) was associated with a reduced activity of protein phosphatase 2A. Together, these results implicate deregulation of the cell-cycle and apoptosis regulators NPAT, CUL5 and PPP2R1B and a role for these genes in the pathogenesis of B-CLL.