Comparison of flow cytometric methods for the measurement of ZAP‐70 expression in a routine diagnostic laboratory

Chronic lymphocytic leukaemia (CLL) follows a variable clinical course with patient survival ranging from only a few years despite treatment, to several decades in patients who may never require clinical intervention. Determination of the mutational status of a patient's immunoglobulin heavy chain variable region (Ig V_H) gene has been used to provide prognostic information, but this assay is not available in most laboratories. The discovery of the expression of the protein tyrosine kinase zeta‐associated protein (ZAP)‐70 in V_H‐unmutated CLL cases led to its proposal as a surrogate marker for V_H status. This study investigated the measurement of ZAP‐70 expression in CLL using different flow cytometric protocols. Two different antibodies and two different staining methods were compared. The Caltag ZAP‐70 antibody and Fix & Perm kit were the easiest to use and were the most sensitive and specific combination, with 91% concordance between ZAP‐70 expression and V_H status. Three patients (9%) were discordant (two V_H mutated/ZAP‐70 positive, and one V_H unmutated/ZAP‐70 negative). No correlation existed between CD38 and either ZAP‐70 expression or V_H status. Measurement of ZAP‐70 expression using the Caltag antibody/kit combination provides a standardized flow cytometric method that could be introduced into a routine CLL immunophenotyping panel in a clinical diagnostic laboratory.     

Feasibility of an easily applicable method of ZAP-70 measurement in chronic lymphocytic leukemia in the routine flow cytometry setting: a methodological approach

Zeta-associated protein 70 (ZAP-70), determined by flow cytometry, has been advocated a surrogate marker of immunoglobulin (Ig)V_H unmutated status in B chronic lymphocytic leukemia (CLL). The aim of the current study was to test the applicability of an easy flow cytometry protocol for ZAP-70 measurement in CLL samples. Samples from 61 CLL patients and 44 normal subjects were analyzed using a commercial ZAP-70 monoclonal antibody (1E7.2 clone) conjugated with phycoerythrin (PE) and Alexa 488 fluorochromes. Modifications of the published methods led to the structure of a simplified in-house method of ZAP-70 measurement. A three-color approach was used with CD19, CD3 gating comparing with the isotype control provided by the same manufacturer. The cutoff levels for ZAP-70 positivity were defined from a receiver operator characteristic curve in relation to the IgV_H mutational status and from the ln normalized mean value +2 SD of normal controls. Using the 20% cutoff value for ZAP-70 positivity in CLL patients defined by the literature, ZAP-PE had a sensitivity of 55% and a specificity of 98% in predicting the IgV_H mutational status, whereas the corresponding values for ZAP-Alexa were 55% and 84%, respectively. Using the 7% cutoff value for CD38 positivity, the sensitivity was 55%, whereas the specificity was 76%. ZAP-70-positive patients showed a shorter time to disease progression in comparison with ZAP-70-negative patients (p<0.001). In conclusion, the 100% specific prediction of mutational status is accompanied by reduced sensitivity, thus limiting ZAP-70’s applicability either as a single marker or combined with CD38 for the assessment of the mutational status of CLL.     

Cryptochrome-1 expression: a new prognostic marker in B-cell chronic lymphocytic leukemia

Chronic lymphocytic leukemia is an adult-onset leukemia with a heterogeneous clinical behavior. When chronic lymphocytic leukemia cases were divided on the basis of IgV_H mutational status, widely differing clinical courses were revealed. Since IgV_H sequencing is difficult to perform in a routine diagnostic laboratory, finding a surrogate for IgV_H mutational status seems an important priority. In the present study, we proposed the use of Cryptochrome-1 as a new prognostic marker in early-stage chronic lymphocytic leukemia. Seventy patients (Binet stage A, without treatment) were included in the study. We correlated Cryptochrome-1 mRNA with well established prognostic markers such as IgV_H mutations, ZAP70, LPL or CD38 expression and chromosomal abnormalities. High Cryptochrome-1 expression correlated with IgV_H unmutated samples. In addition, Cryptochrome-1 was a valuable predictor of disease progression in early-stage chronic lymphocytic leukemia, therefore it can be introduced in clinical practice with the advantage of a simplified method of quantification.     

Modern and conventional prognostic markers of chronic lymphocytic leukaemia in the everyday haematological practice

Objectives: The impact of modern prognostic markers on clinical course of chronic lymphocytic leukaemia (CLL) in everyday practice has been not yet well defined, especially in large series of patients. Therefore, the goal of this study was to assess the influence of conventional as well as modern prognostic factors on overall survival (OS) and time to therapy (TTT) of patients with CLL. Methods: We retrospectively analysed data of all patients consecutively entered into the databases of five large academic centres in the Czech Republic. The total of 1300 patients was included in the analysis. Results and conclusion: Through the use of uniparametric analysis, it was determined that gender, clinical stage Rai II–IV, unmutated IgV_H status, deletion 17p (for both 5% and 20% cut‐off), deletion 11q, ZAP‐70 positivity and high expression of CD38 had significant negative influence on OS. TTT was significantly influenced by gender, Rai stage, IgV_H status, deletion 11q, deletion 17p, deletion 13q and CD38 expression. Multiparametric analysis revealed that OS was significantly influenced by gender, age, IgV_H status and deletion 17p. If only patients who died of CLL were included, gender, age, Rai stage, IgV_H status and deletion 17p had significant influence on OS. Based on our results, the examination of biological prognostic markers can give an insight into the possible disease evolution in daily clinical practice. Biological prognostic markers are, however, not ready (maybe except deletion 17p in younger patients) to be used for guidance of therapy at least outside of clinical trials.     

A proliferation-inducing ligand (APRIL) serum levels predict time to first treatment in patients affected by B-cell chronic lymphocytic leukemia

Purpose: A proliferation‐inducing ligand (APRIL), a tumor necrosis factor superfamily member involved in B‐lymphocytes differentiation and survival, plays a role in protecting B‐Cell Chronic lymphocytic leukemia (B‐CLL) cells from apoptosis. Having observed that APRIL serum (sAPRIL) levels were higher in B‐CLL patients with CLL at diagnosis as compared to healthy donors (14.61 ± 32.65 vs. 4.19 ± 3.42 ng/mL; P < 0.001), we tested the correlation existing in these patients between sAPRIL, clinical–biological parameters and disease progression. Experimental design: sAPRIL levels were measured by ELISA in 130 patients with B‐CLL at diagnosis and in 25 healthy donors. Results: sAPRIL levels did not correlate with gender, age, clinical stage, blood cell counts, β2‐microglobulin (β2M) levels, ZAP‐70 and CD38 expression. Using median sAPRIL natural logarithm (ln) as cutoff, we distinguished two groups of patients (APRIL_LOW and APRIL_HIGH) who were comparable with regard to clinical–biological parameters and overall survival, but different with regard to time to the first treatment (TTFT; P = 0.035). According to univariate analysis, high lymphocyte count, high β2M, Binet stage B–C, ZAP‐70 expression and ln(sAPRIL) above median were associated with earlier TTFT. Advanced clinical stage, high β2M, ZAP‐70 expression and ln(sAPRIL) above median remained independently predictive of shorter TTFT at multivariate analysis. Moreover, sAPRIL increased its prognostic significance when patients were stratified according to independent favorable clinical–biological characteristics (low β2M, stage A and lack of ZAP‐70 expression). Conclusions: sAPRIL is a novel indicator of shorter TTFT in B‐CLL and a predictor of progression especially in patients otherwise considered at low risk according to validated prognostic factors.     

Significant change in ZAP‐70 expression during the course of chronic lymphocytic leukemia

Introduction: It is widely accepted that expression of ZAP‐70 in chronic lymphocytic leukemia (CLL) remains stable in time. However, data supporting this notion are surprisingly scarce. Therefore, we assessed expression of ZAP‐70 in serial samples taken during the course of the disease. Patients and Methods: We studied 44 patients with CLL diagnosed according to NCI‐WG criteria (34 men, 10 women, median age 62, range, 36–81). A total of 104 samples were examined; all patients had at least two measurements. Median interval between the first and the second sample was 13 months (range, 2–36). ZAP‐70 expression was detected by flow cytometry using phycoerythrin‐conjugated monoclonal antibody clone 1E7.2 and negative isotype control. Twenty percent of positive cells were considered as the threshold of positivity. Results: Significant change in ZAP‐70 expression (i.e. from positivity to negativity and vice versa) was detected in 15/44 patients (34%). Interestingly, 7/8 patients whose ZAP‐70 expression converted to positivity had unmutated IgVH genes. In addition, the conversion was accompanied by clinical progression or relapse in all but one patient. On the other hand, 5/7 patients with loss of ZAP‐70 had stable clinical course. One patient became ZAP‐70‐negative during treatment with prednisone for autoimmune hemolytic anemia. Conclusions: In contrast to commonly accepted opinion, significant change in ZAP‐70 expression in time was detected in a substantial proportion of our patients with CLL. While the conversion to ZAP‐70 negativity was found predominantly in patients with stable disease, change to positivity was typical in patients with unmutated IgVH genes at the time of progression or relapse. Based on our pilot results, repeated assessment of ZAP‐70 expression might be especially useful at the time of progression or relapse in patients who were initially ZAP‐70‐negative.     

The clinical and biological features of a series of immunophenotypic variant of B‐CLL

Objectives: To describe the clinical and biological features of a series of immunophenotypic variant of B‐CLL (v‐CLL) characterised by intermediate RMH score, in the absence of t(11;14)(q13;q32) in FISH analysis in comparison with a series of typical CLL. Methods: We studied the clinical and biological features of 63 cases of v‐CLL and 130 cases of CLL. Results: We observed significant differences in terms of age <70 yr (P < 0.001), lymphocytosis <20 × 10⁹/L (P < 0.001), lymphocyte doubling time ≤12 months (P = 0.02), high serum β2‐microglobulin levels (P < 0.001) and splenomegaly (P = 0.002); CD38, CD49d, CD1c were more expressed in v‐CLL, CD43 in CLL (P < 0.001). IgV_H mutation and trisomy 12 were more frequent in v‐CLL group (P = 0.001; P < 0.001); del13q14 in CLL (P = 0.008). Gene expression profiling of nine v‐CLL and 60 CLL indicated that the atypical group presented a specific molecular pattern. After a median follow‐up of respectively, 55 (4–196) and 60 months (6–180), 25/42 patients with v‐CLL (48%) and 55/93 patients with CLL (59%) were treated. Time to treatment was significantly shorter in IgV_H‐mutated v‐CLL vs. mutated CLL (P = 0.006). The median overall survival was worse in v‐CLL‐mutated cases (P = 0.062). Conclusion: v‐CLL should be identified and dealt with separately from classic CLL. In particular, the prognostic markers that are routinely used to characterise classical B‐CLL should not be interpreted as having the same meaning.     

Clinical utility of molecular and flow cytometric markers in chronic lymphocytic leukaemia

Background: Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous disease. While immunoglobulin variable region heavy chain (IgVH) mutational status remains the ‘gold standard’ in molecular prognostication, a range of additional markers is increasingly being used in clinical trials. As awareness of trial data increases, requests to determine these prognostic markers for new CLL patients are becoming more prevalent in Australia. Aim: To explore the clinical utility of currently available prognostic markers for CLL in an Australian cohort. Methods: IgVH mutational status and gene usage was determined and compared with other reported immunophenotypic markers, cytogenetics and clinical outcome as defined by treatment‐free survival (TFS), lymphocyte doubling time and clinical stage in a cohort of 65 CLL patients. Results: An unmutated IgVH gene, high expression of CD38, ZAP‐70, CD25, CD49d, CD54 or low expression of CD49c was associated with shorter TFS indicating an adverse clinical prognosis in our cohort. High expression of each of CD38, ZAP‐70, CD49d and CD54 was significantly associated with an unmutated IgVH gene; however, associations were not absolute. IgVH and CD25 expression retained their significance in multivariate analysis. Concordant CD25^high/IgVH unmutated CLL patients had the shortest median TFS interval (40 months) in our cohort. Conclusions: Molecular and immunophenotypic markers remain useful as adjuncts to clinical prognostication; however, as single parameters they are unable to dictate the timing of therapeutic intervention. The combined use of CD25 and IgVH mutational status may be clinically relevant to CLL prognostication while also providing insight into the biological pathways involved in disease progression.     

ZAP‐70 expression is associated with increased risk of autoimmune cytopenias in CLL patients

Autoimmune cytopenias (AIC) are frequent in chronic lymphocytic leukemia (CLL) patients, but risk factors and prognostic relevance of these events are controversial. Data about the influence on AIC of biological prognostic markers, as ZAP‐70, are scanty. We retrospectively evaluated AIC in 290 CLL patients tested for ZAP‐70 expression by immunohistochemistry on bone marrow biopsy at presentation. They were 185 men, median age 63 years, 77.9% Binet stage A, 17.6% B and 4.5% C. AIC occurred in 46 patients (16%): 31 autoimmune hemolytic anemias, 10 autoimmune thrombocytopenias, four Evans syndromes, and one pure red cell aplasia. Of the 46 cases of AIC, 37 (80%) occurred in ZAP‐70 positive patients and nine (20%) in ZAP‐70 negatives. ZAP‐70 expression [Hazard Ratio (HR) = 7.42; 95% confidence interval (CI): 2.49–22.05] and age >65 years (HR = 5.41; 95% CI: 1.67–17.49) resulted independent risk factors for AIC. Among the 136 patients evaluated both for ZAP‐70 expression and IGHV status, the occurrence of AIC was higher in ZAP‐70 positive/IGHV unmutated cases (35%) than in patients ZAP‐70 negative/IGHV mutated (6%) or discordant for the two parameters (4%; P < 0.0001). In ZAP‐70 positive patients, occurrence of AIC negatively influenced survival (HR = 1.75; 95% CI: 1.06–2.86). The high risk of developing AIC in ZAP‐70 positive CLL, particularly when IGHV unmutated, should be considered in the clinical management. Am. J. Hematol. 2010. © 2010 Wiley‐Liss, Inc.     

Quantification of ZAP-70 expression in chronic lymphocytic leukemia: T/B-cell ratio of mean fluorescence intensity provides stronger prognostic value than percentage of positive cells

Expression of ZAP-70 measured by flow cytometry belongs to the most powerful prognostic parameters in chronic lymphocytic leukemia (CLL). However, many technical factors such as setting of the positivity threshold may significantly influence results.. Quantification using mean fluorescent intensity (MFI) may eliminate the subjective error which is inevitable in the isotype control method. The aim of the present project was therefore to assess the prognostic significance of ZAP-70 using three different methods. Between 2005 and 2010 we measured ZAP-70 expression in 157 patients with CLL (108 males, 49 females, median age 60 years [range, 31-82]; low/intermediate/high Rai risk in 41/48/11%). Expression of ZAP-70 was determined by flow cytometry using phycoerythrin (PE)-conjugated monoclonal antibody, clone 1E7.2. Evaluation was performed by 1) percentage of positive cells compared to isotype control (cut-off 20%), 2) MFI ratio of T-cells/CLL cells (cut-off 3.0); 3) MFI ratio of ZAP-70/isotype control on CLL cells (cut-off 2.5). MFI method with T-cells/CLL cells ratio was the best in the identification of patients with unfavourable outcome: ZAP-70 positive patients had significantly shorter time to treatment (TTT, median 24 vs. 55 months, p=0.0001) and overall survival (OS, median 97 vs 174 months, p=0.0074). The differences in TTT a OS were not significant with the use of isotype percentage and MFI isotype methods. Combined analysis of ZAP-70 with CD38 expression or IgVH mutation status lead to identification of a subgroup with the longest TTT and OS (ZAP-70 and CD38 negative, p<0.0001 and p=0.012; ZAP-70 negative and mutated IgVH genes, p<0.0001 and p=0.0019). In conclusion, our results suggest that measurement of ZAP-70 expression in CLL by MFI using T-cells/CLL cells ratio might be the optimal method for accurate prediction of clinical course. Combined analysis of ZAP-70 with CD38 or IgVH mutation status further refined individual patient′s prognosis.     

FCRL2 mRNA expression is inversely associated with clinical progression in chronic lymphocytic leukemia

Fc receptor‐like 2 (FCRL2) is highly expressed on B‐cell chronic lymphocytic leukemia (B‐CLL) cells and could possibly influence disease pathogenesis. Therefore, we investigated FCRL2 mRNA expression in a large cohort with 152 CLL patients in order to assess its role in risk prediction in B‐CLL. FCRL2 mRNA expression was found to be expressed at considerably higher levels in peripheral blood mononuclear cells (PBMC) of B‐CLL patients compared to controls (range 1.35‐ to 210‐fold upregulation; P < 0.0001) and cells of other hematological diseases. Patients with high FCRL2 expression (according to ROC‐analysis) had a significantly longer treatment‐free survival (TFS) and overall survival (OS) than patients with low FCRL2 expression (median TFS: 119 vs. 34 months, P < 0.0001; median OS: 321 months vs. not reached, P = 0.009). Univariate comparisons found that FCRL2 expression was weakly associated with IGHV mutation status (P = 0.05), CD38 status (P < 0.0001) and ZAP‐70 status (P < 0.0001). Furthermore, we show that the combination of FCRL2 with ZAP70‐, CD38‐ or IGHV‐status could further significantly refine the prognostic information provided by either of the factors alone in TFS and OS. In multivariate analysis low FCRL2 expression was a significant independent prognostic factor (HR 2.4; P = 0.005). Here we demonstrate that the level of FCRL2 expression is correlated with prognosis in B‐CLL.     

Prognostic relevance of serum levels and cellular expression of adiponectin in B-cell chronic lymphocytic leukemia

The correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukaemia (CLL) [i.e., mutational status of the immunoglobulin heavy chain variable region (IgV_H), ZAP-70- and CD38-expression] and adiponectin serum concentration was evaluated in a cohort of 69 previously untreated Binet stage A CLL patients. Adiponectin levels inversely correlated with absolute peripheral blood lymphocyte count (r = −0.254; P = 0.03), CD38-positive CLL cells (r = −0.294; P = 0.04) and ZAP-70 (r = −0.285; P = 0.03). The univariate Cox proportional hazard model demonstrated that, in addition with lower serum levels of adiponectin (P = 0.01), the unmutated IgV_H condition (P = 0.002) and ZAP-70-positivity (P = 0.02) were associated with a shorter time to first treatment (TFT). However, in multivariate analysis only ZAP-70 positivity emerged as predictor of the TFT (P = 0.008). The levels of adiponectin in CLL were evaluated in 60 patients from an independent cohort investigated by gene expression profiling. Adiponectin gene expression was invariably low suggesting a limited (if any) role of leukemic cells in the production of circulating adiponectin levels. In contrast, both adiponectin receptor 1 (AdipoR1) and AdipoR2 mRNA were highly expressed by CLL cells with a degree of inter-patient variability. Our results, although preliminary, lend support to the idea that adiponectin secretion by bone marrow adipocytes might represent a possible promising drug target in the field of hematology.     

Baff serum level predicts time to first treatment in early chronic lymphocytic leukemia

We analyzed the correlation between well‐established biological parameters of prognostic relevance in B‐cell chronic lymphocytic leukemia (CLL) [i.e. mutational status of the immunoglobulin heavy chain variable region (IgVH), ZAP‐70 and CD38 expression] and serum levels of B cell–activating factor (BAFF of the TNF family) by evaluating the impact of these variables on the time to first treatment (TFT) in a series of 169 previously untreated CLL patients in Binet stage A. Higher levels of BAFF were more frequently associated with female gender (P = 0.02), younger age (P = 0.01), Rai stage 0 (P = 0.002), higher platelet count (P = 0.005), mutated IgVH disease (P = 0.002), higher occurrence of normal cytogenetic profile or presence of 13q deletion (P = 0.02), low ZAP‐70‐ (P = 0.003), and CD38‐expression (P = 0.02). Maximally selected log‐rank statistic plot identified a serum BAFF concentration of 0.313 ng/mL as the best cut‐off (P < 0.0001). This threshold recognized two subsets of patients with different TFT (P < 0.0001). Because in multivariate analysis soluble BAFF [Hazard ratio (HR), 8.23; confidence Interval (CI) 95%,3.0–22.6, P < 0.0001] and mutational status of IgVH (HR = 2.60; CI 95% 1.10–6.14, P = 0.03) maintained the discriminating power their combined effect on clinical outcome was assessed. When three groups were considered: ( 1 ) low‐risk (n = 93), patients with concordant IgVH_mut and higher soluble BAFF; ( 2 ) intermediate‐risk (n = 50), patients with IgVH_mut and low BAFF levels or IgVH_unmut and soluble higher BAFF;( 3 ) high‐risk (n = 26), patients with concordant IgVH _unmut and low soluble BAFF, the 2‐yr TFTs were, respectively, 95%, 85%, and 41% (P < 0.0001). In conclusion, our results indicate that in early B‐cell CLL, the biological profile including among other parameters soluble BAFF may provide a useful insight into the complex interrelationship of prognostic variables.     

Prognostic importance of soluble CD23 in B‐cell chronic lymphocytic leukemia

Soluble CD23 (sCD23) was proposed as a marker of disease activity and as an important prognostic parameter in B‐cell chronic lymphocytic leukemia (B‐CLL). In this study, prognostic significance of sCD23 in B‐CLL was examined according to its temporal relationship with the known clinical parameters of the disease, CD38 and ZAP‐70. Serum sCD23 levels of 36 B‐CLL patients, followed up in our clinic between 1999 and 2005, and 15 healthy subjects were measured with enzyme‐linked immunosorbent assay. The mean serum sCD23 level of the B‐CLL patients (210.72 ± 193.67 and 6–600 U/ml) was significantly higher than the control group (18.20 ± 14.30 and 6–50 U/ml). Seventy‐eight percent of the B‐CLL patients with lymphocyte doubling time (LDT) <12 months and 24% of patients with LDT >12 months had high sCD23 levels (P = 0.008). Meanwhile, 81% of the patients with diffuse bone marrow infiltration and 33% of patients with nondiffuse infiltration had high levels of serum sCD23 (P = 0.029). A significant difference was found between B‐CLL patients with Binet stages A and C (P = 0.009). Peripheral blood flow cytometry of the patients revealed a significant CD38 expression in patients with high serum sCD23 levels (P = 0.002). Similarly, an increased bone marrow zeta‐chain associated protein kinase‐70 (ZAP‐70) expression was seen in patients with high serum sCD23 levels (P = 0.009, correlation co‐efficient was 0.714). Cumulative and the progression free survivals of the patients with low serum sCD23 levels were 60.1 ± 5.7 months [95% confidence interval (CI); 49.0–71.2] and 51.1 ± 6.6 months (95% CI; 38.0–64.1), respectively. However, they were 43.8 ± 6.5 months (95% CI; 31.0–56.6) and 26.5 ± 6.4 months (95% CI; 14.0–39.1) in patients with high levels. Serum sCD23 is increased in B‐CLL patients and can be used in the clinical follow‐up of the disease in prediction of the tumor mass and prognosis.     

Defining the prognosis of early stage chronic lymphocytic leukaemia patients

Approximately 70% of chronic lymphocytic leukaemia (CLL) patients present with early stage disease, therefore defining which patients will progress and require treatment is a major clinical challenge. Here, we present the largest study of prognostic markers ever carried out in Binet stage A patients (n = 1154) with a median follow‐up of 8 years. We assessed the prognostic impact of lymphocyte doubling time (LDT), immunoglobulin gene (IGHV) mutation status, CD38 expression, ZAP‐70 expression and fluorescence in situ hybridization (FISH) cytogenetics with regards to time to first treatment (TTFT) and overall survival (OS). Univariate analysis revealed LDT as the most prognostic parameter for TTFT, with IGHV mutation status most prognostic for OS. CD38 expression, ZAP‐70 expression and FISH were also prognostic variables; combinations of these markers increased prognostic power in concordant cases. Multivariate analysis revealed that only LDT, IGHV mutation status, CD38 and age at diagnosis were independent prognostic variables for TTFT and OS. Therefore, IGHV mutation status and CD38 expression have independent prognostic value in early stage CLL and should be performed as part of the routine diagnostic workup. ZAP‐70 expression and FISH were not independent prognostic markers in early stage disease and can be omitted at diagnosis but FISH analysis should be undertaken at disease progression to direct treatment strategy.     

Total body computed tomography scan in the initial work‐up of Binet stage A chronic lymphocytic leukemia patients: Results of the prospective,multicenter O‐CLL1‐GISL study

Total body computed tomography (TB‐CT) scan is not mandatory in the diagnostic/staging algorithm of chronic lymphocytic leukemia (CLL). The aim of this study was to determine the value and prognostic significance of TB‐CT scan in early stage CLL patients. Baseline TB‐CT scan was performed in 240 Binet stage A CLL patients (179 Rai low‐ and 61 Rai intermediate‐risk) included in a prospective multicenter observational study ( clinicaltrial.gov ID:NCT00917549). The cohort included 69 clinical monoclonal B lymphocytosis (cMBLs). Patients were restaged considering only radiological data. Following TB‐CT scans, 20% of cases reclassified as radiologic Binet (r‐Binet) stage B. r‐Binet B patients showed a higher incidence of unfavorable cytogenetic abnormalities (P = 0.027), as well as a shorter PFS (P = 0.001). At multivariate analysis, r‐Binet stage [HR = 2.48; P = 0.004] and IGHV mutational status [HR = 3.01; P = 0.002] retained an independent predictive value for PFS. Among 179 Rai low‐risk cases, 100 were redefined as r‐Rai intermediate‐risk based upon TB‐CT scan data, showing a higher rate of cases with higher ZAP‐70 (P = 0.033) and CD38 expression (P = 0.029) and β2‐microglobulin levels (P < 0.0001), as well as a shorter PFS than those with r‐Rai low‐risk (P = 0.008). r‐Rai stage [HR = 2.78; P = 0.046] and IGHV mutational status [HR = 4.25; P = 0.009] retained a significant predictive value for PFS at multivariate analysis. Forty‐two percent of cMBL patients were reclassified as r‐small lymphocytic lymphomas (r‐SLLs) by TB‐CT scan. TB‐CT scan appears to provide relevant information in early stage CLL related to the potential and the timing of patients to progress towards the more advanced disease stages. Am. J. Hematol. 88:539–544, 2013. © 2013 Wiley Periodicals, Inc.     

Igbeta(CD79b) mRNA expression in chronic lymphocytic leukaemia cells correlates with immunoglobulin heavy chain gene mutational status but does not serve as an independent predictor of clinical severity

The etiology of chronic lymphocytic leukemia (CLL) is poorly understood and its course is highly variable. Somatic hypermutation (SHM) of the immunoglobulin heavy chain (IgV(H)) gene and ZAP70 protein expression have been reported as prognostic indicators. However, these assays are not widely available and their concordance is imperfect. Thus a need exists to identify additional molecular determinants of CLL. The Igbeta (CD79b) subunit of the B cell antigen receptor is essential for B lymphocyte function. Defects in Igbeta expression are implicated in CLL pathogenesis. We have analyzed Igbeta mRNA expression in CLL cells in 40 consecutive patient samples. About 75% of the samples showed the expected decrease of Igbeta surface staining. Igbeta mRNA levels covered a wider range, did not correlate with Igbeta surface staining, but clearly distinguished the normal and CLL lymphocyte populations. Remarkably, Igbeta mRNA levels correlated strongly with SHM; Igbeta mRNA levels in CLL cells were significantly higher in patients with an unmutated IgV(H) gene when compared with those in whom IgV(H) was hypermutated (P = 0.008). In contrast, no correlation was observed between Igbeta mRNA levels and ZAP70 expression. Multiple parameters abstracted from chart reviews were used to estimate severity of CLL in each case. While severity correlated strongly with ZAP70 staining, and to a lesser extent with SHM status, there was no correlation with Igbeta mRNA levels. These data establish a strong linkage between Igbeta mRNA expression and SHM in CLL and highlight the complex relationships between biochemical parameters and clinical status in this disease.     

Monoclonal B‐cell lymphocytosis is closely related to chronic lymphocytic leukaemia and may be better classified as early‐stage CLL

The World Health Organization classification uses a cut‐off point of 5·0 × 10⁹/l cells with a chronic lymphocytic leukaemia (CLL)‐phenotype in peripheral blood to discriminate between monoclonal B‐lymphocytosis (MBL) and B‐CLL. This study analysed 298 MBL patients by multi‐parameter flow cytometry, chromosome banding analysis (CBA)/fluorescence in situ hybridization (FISH), and IGHV mutation status and compared them with 356 CLL patients. In MBL, CBA more frequently revealed a normal karyotype and FISH identified less frequently del(6q), del(13q) (as sole alterations), and del(17)(p13). Within the MBL cohort, a shorter time to treatment (TTT) was found for ZAP‐70‐positivity, 14q32/IGH‐translocations (CBA), del(11)(q22·3) (FISH) and unmutated IGHV status. Higher CD38 and ZAP‐70 expression, del(11)(q22·3) (FISH), trisomy 12 (FISH), and 14q32/IGH‐translocations (CBA) were correlated with a shorter TTT in the combined cohort (MBL + CLL); a sole del(13)(q14) (FISH) correlated with longer TTT. Regarding overall survival, unmutated IGHV status and ‘other’ alterations (CBA) had an adverse impact. There was no correlation between the concentration of CLL‐cells and TTT or overall survival. Multivariate analysis confirmed a negative impact on TTT for del(11)(q22·3)/ATM, trisomy 12 (both by FISH), and 14q32/IGH‐translocations by CBA. These data emphasize a close relationship between MBL and CLL regarding clinically relevant parameters and provide no evidence to strictly separate these entities by a distinct threshold of clonal B‐cells.