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.     

MFI ratio estimation of ZAP-70 in B-CLL by flow cytometry can be improved by considering the isotype-matched antibody signal

Introduction: The IgV_H mutational status of B‐cell chronic lymphocytic leukemia (B‐CLL) is of prognostic value. Expression of ZAP‐70 in B‐CLL is a surrogate marker for IgV_H unmutated (UM). As determination of IgV_H mutational status involves a methodology currently unavailable for most clinical laboratories, it is important to have available a reliable technique for ZAP‐70 estimation in B‐CLL. Flow cytometry (FC) is a convenient technique for this purpose. However, there is still no adequate way for data analysis, which would prevent the assignment of false positive or negative expression. Methods: We have modified the currently most accepted technique, which uses the ratio of the mean fluorescent index (MFI) of B‐CLL to T cells. The MFI for parallel antibody isotype staining is subtracted from the ZAP‐70 MFI of both B‐CLL and T cells. We validated this technique comparing the results obtained for ZAP‐70 expression by FC with those obtained with quantitative PCR for the same patients. Results: We applied the technique in a series of 53 patients. With this modification, a better correlation between ZAP‐70 expression and IgV_H UM was obtained. Conclusions: Thus, the MFI ratio B‐CLL/T cell corrected by isotype is a reliable analysis technique to estimate ZAP‐70 expression in B‐CLL.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

LDOC1 mRNA is differentially expressed in chronic lymphocytic leukemia and predicts overall survival in untreated patients

We previously identified LDOC1 as one of the most significantly differentially expressed genes in untreated chronic lymphocytic leukemia (CLL) patients with respect to the somatic mutation status of the immunoglobulin heavy-chain variable region genes. However, little is known about the normal function of LDOC1, its contribution to the pathophysiology of CLL, or its prognostic significance. In this study, we have investigated LDOC1 mRNA expression in a large cohort of untreated CLL patients, as well as in normal peripheral blood B-cell (NBC) subsets and primary B-cell lymphoma samples. We have confirmed that LDOC1 is dramatically down-regulated in mutated CLL cases compared with unmutated cases, and have identified a new splice variant, LDOC1S. We show that LDOC1 is expressed in NBC subsets (naive > memory), suggesting that it may play a role in normal B-cell development. It is also expressed in primary B-cell lymphoma samples, in which its expression is associated with somatic mutation status. In CLL, we show that high levels of LDOC1 correlate with biomarkers of poor prognosis, including cytogenetic markers, unmutated somatic mutation status, and ZAP70 expression. Finally, we demonstrate that LDOC1 mRNA expression is an excellent predictor of overall survival in untreated CLL patients.     

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.     

NOTCH1 mutations in CLL associated with trisomy 12

Two recent studies reported whole-genome sequencing of chronic lymphocytic leukemia (CLL) samples and found repeated mutations in the XPO1 and NOTCH1 genes. XPO1 was found mutated in 2.4% of cases, while NOTCH1 was found mutated in 12.2% or 15.1% of CLL samples. Here we report the results of sequencing of XPO1 and NOTCH1 in 186 CLL cases. Our results confirmed frequency of XPO1 mutations. However, we found only 5 NOTCH1 mutations in 127 IGVH unmutated/ZAP70(+) CLL samples (4%), and one mutation was found in IGVH mutated/ZAP70(-) CLL for a total percentage of 1.5%. Because 4 of 6 mutated samples also showed trisomy 12, we sequenced NOTCH1 in an additional 77 cases with trisomy 12 CLLs, including 47 IGVH unmutated/ZAP70(+) cases. Importantly, we found 41.9% NOTCH1 mutation frequency in aggressive trisomy 12 CLL cases. Our data suggest that activation of NOTCH1 plays a critical role in IGVH unmutated/ZAP70(+) trisomy 12 CLL.     

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.     

A unique proteomic profile on surface IgM ligation in unmutated chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course with 2 extreme subsets: indolent, ZAP70(-) and mutated immunoglobulin heavy chain gene (M-CLL); and aggressive, ZAP70(+) and unmutated immunoglobulin heavy chain (UM-CLL). Given the long-term suspicion of antigenic stimulation as a primum movens in the disease, the role of the B-cell receptor has been extensively studied in various experimental settings; albeit scarcely in a comparative dynamic proteomic approach. Here we use a quantitative 2-dimensional fluorescence difference gel electrophoresis technology to compare 48 proteomic profiles of the 2 CLL subsets before and after anti-IgM ligation. Differentially expressed proteins were subsequently identified by mass spectrometry. We show that unstimulated M- and UM-CLL cells display distinct proteomic profiles. Furthermore, anti-IgM stimulation induces a specific proteomic response, more pronounced in the more aggressive CLL. Statistical analyses demonstrate several significant protein variations according to stimulation conditions. Finally, we identify an intermediate form of M-CLL cells, with an indolent profile (ZAP70(-)) but sharing aggressive proteomic profiles alike UM-CLL cells. Collectively, this first quantitative and dynamic proteome analysis of CLL further dissects the complex molecular pathway after B-cell receptor stimulation and depicts distinct proteomic profiles, which could lead to novel molecular stratification of the disease.     

Distinct gene expression patterns in chronic lymphocytic leukemia defined by usage of specific VH genes

The mutation status and usage of specific VH genes such as V3-21 and V1-69 are potentially independent pathogenic and prognostic factors in chronic lymphocytic leukemia (CLL). To investigate the role of antigenic stimulation, we analyzed the expression of genes involved in B-cell receptor (BCR) signaling/activation, cell cycle, and apoptosis control in CLL using these specific VH genes compared to VH mutated (VH-MUT) and VH unmutated (VH-UM) CLL not using these VH genes. V3-21 cases showed characteristic expression differences compared to VH-MUT (up: ZAP70 [or ZAP-70]; down: CCND2, P27) and VH-UM (down: PI3K, CCND2, P27, CDK4, BAX) involving several BCR-related genes. Similarly, there was a marked difference between VH unmutated cases using the V1-69 gene and VH-UM (up: FOS; down: BLNK, SYK, CDK4, TP53). Therefore, usage of specific VH genes appears to have a strong influence on the gene expression pattern pointing to antigen recognition and ongoing BCR stimulation as a pathogenic factor in these CLL subgroups.     

Somatic hypermutation and V(H) gene usage in mantle cell lymphoma

Abstract: Mantle cell lymphoma (MCL) is considered to derive from naïve, pregerminal center (GC) CD5⁺ B‐cells. However, the cell of origin has been questioned in recent studies that showed somatic hypermutations in the immunoglobulin (Ig) variable heavy chain (V_H) genes in subsets of MCL. To clarify this issue, we analyzed the IgV_H genes for the presence of somatic hypermutations in 51 MCL cases. Twenty percent of the MCL cases displayed somatically mutated V_H genes (defined as >2% mutated), whereas 80% showed unmutated V_H genes. This finding suggests that MCL is a genetically heterogeneous disease, with the majority of cases originating from unmutated pre‐GC B‐cells and a subset deriving from more mature B‐cells which have been exposed to the GC environment and have undergone somatic hypermutation. A biased V_H gene usage has been demonstrated in several B‐cell malignancies; however, this has not yet been investigated in MCL, although V_H4‐34 overusage has been indicated by small studies. Interestingly, we found a restricted usage of three individual V_H genes in our MCL material; V_H4‐34 (22%), V_H3‐21 (16%) and V_H5‐51 (12%). This novel finding of preferential V_H gene usage in half of the MCL cases may suggest an antigen driven process occurring in B‐cells expressing specific V_H genes, thus implicating that Ig specificity could be involved in mantle cell lymphoma development.     

IGHV3‐21 gene usage is associated with high TCL1 expression in chronic lymphocytic leukemia

T‐cell leukemia/lymphoma protein 1 (TCL1) was recently shown to display an expression pattern in chronic lymphocytic leukemia (CLL) corresponding to molecular subtypes, where poor‐risk patients demonstrated higher expression levels. Here, we examined the mRNA expression pattern of TCL1 in 144 patients with CLL, including 67 immunoglobulin heavy‐chain variable (IGHV) mutated, 58 IGHV unmutated and 19 patients with IGHV3‐21 usage. A higher TCL1 expression level was detected in patients with CLL with unmutated vs. mutated IGHV genes (P < 0.001), whereas no difference was demonstrated within the IGHV3‐21 cohort (i.e., mutated vs. unmutated and stereotyped vs. non‐stereotyped complementarity determining region 3). The IGHV3‐21 subgroup displayed high TCL1 mRNA expression, differing significantly from other IGHV mutated cases (P < 0.001), although 11/19 had mutated IGHV genes. Furthermore, high TCL1 expression levels were associated with significantly shorter overall survival (P < 0.001). Altogether, we show that TCL1 mRNA expression may predict clinical outcome in CLL and that the IGHV3‐21 subset, regardless of mutational status, displays high TCL1 expression.     

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.