Molecular characterization of neoplastic and normal "sister" lymphoblastoid B-cell lines from chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) B-cells resemble self-renewing CD5 + B-cells carrying auto/xeno-antigen-reactive B-cell receptors (BCRs) and multiple innate pattern-recognition receptors, such as Toll-like receptors and scavenger receptors. Integration of signals from BCRs with multiple surface membrane receptors determines whether the cells will be proliferating, anergic or apoptotic. To better understand the role of antigen in leukemogenesis, CLL cell lines producing monoclonal antibodies (mAbs) will facilitate structural analysis of antigens and supply DNA for genetic studies. We present here a comprehensive genotypic and phenotypic characterization of available CLL and normal B-cell-derived lymphoblastoid cell lines (LCLs) from the same individuals (n = 17). Authenticity and verification studies of CLL-patient origin were done by IGHV sequencing, fluorescence in situ hybridization (FISH) and DNA/short tandem repeat (STR) fingerprinting. Innate B-cell features, i.e. natural Ab production and CD5 receptors, were present in most CLL cell lines, but in none of the normal LCLs. This panel of immortalized CLL-derived cell lines is a valuable reference representing a renewable source of authentic Abs and DNA.

CLLU1 expression has prognostic value in chronic lymphocytic leukemia after first-line therapy in younger patients and in those with mutated IGHV genes.

CLLU1, located at chromosome 12q22, encodes a transcript specific to chronic lymphocytic leukemia and has potential prognostic value. We assessed the value of CLLU1 expression in the LRF CLL4 randomized trial. Samples from 515 patients with chronic lymphocytic leukemia were collected immediately before the start of treatment. After RNA extraction and cDNA synthesis, CLLU1 expression was assessed by quantitative polymerase chain reaction. In total, 247 and 268 samples were identified as having low and high CLLU1 expression, respectively. The median follow-up was 88 months. High CLLU1 expression was significantly correlated with unmutated IGHV genes, ZAP-70 and CD38 positivity, and absence of 13q deletion (all r>0.2, P<0.0001). At 6 years, patients with high CLLU1 expression had significantly worse progression-free survival (9% versus 17%; P=0.03) and overall survival (42% versus 57%; P=0.0003) than patients with low CLLU1 expression. Among patients with mutated IGHV genes, overall survival at 6 years was 50% in those with high CLLU1 expression and 76% in those with low CLLU1 expression (P=0.005). However, CLLU1 expression was not an independent predictor of overall survival in a multivariate model including TP53 aberrations, beta-2 microglobulin level, age and IGHV mutation status. Nor did it predict response to treatment. CLLU1 expression analysis helps to refine the prognosis of patients with chronic lymphocytic leukemia who have mutated IGHV genes.

Array-based genomic screening at diagnosis and during follow-up in chronic lymphocytic leukemia.

BACKGROUND: High-resolution genomic microarrays enable simultaneous detection of copy-number aberrations such as the known recurrent aberrations in chronic lymphocytic leukemia [del(11q), del(13q), del(17p) and trisomy 12], and copy-number neutral loss of heterozygosity. Moreover, comparison of genomic profiles from sequential patients' samples allows detection of clonal evolution. DESIGN AND METHODS: We screened samples from 369 patients with newly diagnosed chronic lymphocytic leukemia from a population-based cohort using 250K single nucleotide polymorphism-arrays. Clonal evolution was evaluated in 59 follow-up samples obtained after 5-9 years. RESULTS: At diagnosis, copy-number aberrations were identified in 90% of patients; 70% carried known recurrent alterations, including del(13q) (55%), trisomy 12 (10.5%), del(11q) (10%), and del(17p) (4%). Additional recurrent aberrations were detected on chromosomes 2 (1.9%), 4 (1.4%), 8 (1.6%) and 14 (1.6%). Thirteen patients (3.5%) displayed recurrent copy-number neutral loss of heterozygosity on 13q, of whom 11 had concurrent homozygous del(13q). Genomic complexity and large 13q deletions correlated with inferior outcome, while the former was linked to poor-prognostic aberrations. In the follow-up study, clonal evolution developed in 8/24 (33%) patients with unmutated IGHV, and in 4/25 (16%) IGHV-mutated and treated patients. In contrast, untreated patients with mutated IGHV (n=10) did not acquire additional aberrations. The most common secondary event, del(13q), was detected in 6/12 (50%) of all patients with acquired alterations. Interestingly, aberrations on, for example, chromosome 6q, 8p, 9p and 10q developed exclusively in patients with unmutated IGHV. CONCLUSIONS: Whole-genome screening revealed a high frequency of genomic aberrations in newly diagnosed chronic lymphocytic leukemia. Clonal evolution was associated with other markers of aggressive disease and commonly included the known recurrent aberrations.

LPL is the strongest prognostic factor in a comparative analysis of RNA-based markers in early chronic lymphocytic leukemia.

BACKGROUND: The expression levels of LPL, ZAP70, TCL1A, CLLU1 and MCL1 have recently been proposed as prognostic factors in chronic lymphocytic leukemia. However, few studies have systematically compared these different RNA-based markers. DESIGN AND METHODS: Using real-time quantitative PCR, we measured the mRNA expression levels of these genes in unsorted samples from 252 newly diagnosed chronic lymphocytic leukemia patients and correlated our data with established prognostic markers (for example Binet stage, CD38, IGHV gene mutational status and genomic aberrations) and clinical outcome. RESULTS: High expression levels of all RNA-based markers, except MCL1, predicted shorter overall survival and time to treatment, with LPL being the most significant. In multivariate analysis including the RNA-based markers, LPL expression was the only independent prognostic marker for overall survival and time to treatment. When studying LPL expression and the established markers, LPL expression retained its independent prognostic strength for overall survival. All of the RNA-based markers, albeit with varying ability, added prognostic information to established markers, with LPL expression giving the most significant results. Notably, high LPL expression predicted a worse outcome in good-prognosis subgroups, such as patients with mutated IGHV genes, Binet stage A, CD38 negativity or favorable cytogenetics. In particular, the combination of LPL expression and CD38 could further stratify Binet stage A patients. CONCLUSIONS: LPL expression is the strongest RNA-based prognostic marker in chronic lymphocytic leukemia that could potentially be applied to predict outcome in the clinical setting, particularly in the large group of patients with favorable prognosis.

Analysis of CLLU1 expression levels before and after therapy in patients with chronic lymphocytic leukemia.

OBJECTIVE: Chronic lymphocytic leukemia (CLL) is incurable, but therapy leading to eradication of minimal residual disease (MRD) in CLL is associated with improved clinical outcomes. CLL upregulated gene 1 (CLLU1) is solely upregulated in CLL patient samples. We hypothesized that CLLU1 could be used to monitor for residual disease in CLL patient samples after therapy. METHODS: We examined whether the CLLU1 real-time quantitative PCR (RQ-PCR) could detect small numbers of CLL cells in mixtures of normal peripheral blood mononuclear (PBMC) cells. We then performed a retrospective analysis on time-matched cryo-preserved specimens from patients who achieved MRD-negative remissions that underwent serial marrow biopsies for evaluation of residual disease by 4-color flow cytometry. RNA from PBMC samples collected at the time of the marrow assessments was analyzed for CLLU1. Nine patients underwent a total of 46 paired blood and marrow evaluations (median 5 assessments per patient). RESULTS: CLLU1 RQ-PCR on PBMCs of healthy donors reconstituted with varying amounts of CLL cells demonstrated leukemia cells could be reliably detected with high sensitivities depending on the CLLU1 expression level. Analysis of time-matched samples assessed for CLLU1 levels in the blood by RQ-PCR and residual disease of the marrow determined by 4-color flow cytometry revealed a correlation coefficient of 0.96 (P < 0.0001). CONCLUSION: The CLLU1 RQ-PCR is a sensitive and specific assay for detecting residual CLL cells after therapy. Assessment of blood CLLU1 levels can be used as a reliable marker of tumor burden and has the potential to complement currently used techniques for MRD monitoring in patients with CLL.

Screening for copy-number alterations and loss of heterozygosity in chronic lymphocytic leukemia--a comparative study of four differently designed, high resolution microarray platforms.

Screening for gene copy-number alterations (CNAs) has improved by applying genome-wide microarrays, where SNP arrays also allow analysis of loss of heterozygozity (LOH). We here analyzed 10 chronic lymphocytic leukemia (CLL) samples using four different high-resolution platforms: BAC arrays (32K), oligonucleotide arrays (185K, Agilent), and two SNP arrays (250K, Affymetrix and 317K, Illumina). Cross-platform comparison revealed 29 concordantly detected CNAs, including known recurrent alterations, which confirmed that all platforms are powerful tools when screening for large aberrations. However, detection of 32 additional regions present in 2-3 platforms illustrated a discrepancy in detection of small CNAs, which often involved reported copy-number variations. LOH analysis using dChip revealed concordance of mainly large regions, but showed numerous, small nonoverlapping regions and LOH escaping detection. Evaluation of baseline variation and copy-number ratio response showed the best performance for the Agilent platform and confirmed the robustness of BAC arrays. Accordingly, these platforms demonstrated a higher degree of platform-specific CNAs. The SNP arrays displayed higher technical variation, although this was compensated by high density of elements. Affymetrix detected a higher degree of CNAs compared to Illumina, while the latter showed a lower noise level and higher detection rate in the LOH analysis. Large-scale studies of genomic aberrations are now feasible, but new tools for LOH analysis are requested.

CLLU1 expression analysis adds prognostic information to risk prediction in chronic lymphocytic leukemia.

We recently identified a disease-specific gene CLLU1 in chronic lymphocytic leukemia (CLL) and also demonstrated that high CLLU1 expression levels predict poor clinical outcome. To validate this finding, we measured CLLU1 mRNA expression levels by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in 175 patients with CLL. Analyses of IgV(H) mutational status, ZAP-70 expression, CD38 expression, and chromosomal aberrations were also performed. High levels of CLLU1 expression were associated with shorter overall survival (P < .001), with a 7% increase in risk of early death by each doubling of the CLLU1 expression level. Stratification for age at diagnosis demonstrated a strong prognostic significance of CLLU1 expression in patients younger than 70 years (P < .001), but not in patients aged 70 or older (P = .61). The prognostic significance of IgV(H) mutational status and ZAP-70 expression had a similar age-dependent variation. Multivariate analysis in the younger age group showed that CLLU1 expression analysis added further prognostic information within all prognostic subgroups, with the exception of patients with unmutated IgV(H) CLL. Only CLLU1 expression and IgV(H) mutational status had independent predictive power. Thus, analysis of CLLU1 expression is highly applicable in risk prediction in CLL for patients of an age eligible for risk stratification.

Evaluation of ZAP-70 expression by flow cytometry in chronic lymphocytic leukemia: A multicentric international harmonization process.

The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous with some patients requiring early therapy whereas others will not be treated for years. The evaluation of an individual CLL patient's prognosis remains a problematic issue. The presence or absence of somatic mutations in the IgVH genes is currently the gold-standard prognostic factor, but this technique is labor intensive and costly. Genomic studies uncovered that 70 kDa zeta-associated protein (ZAP-70) expression was associated with unmutated IgVH genes and ZAP-70 protein expression was proposed as a surrogate for somatic mutational status. Among the available techniques for ZAP-70 detection, flow cytometry is most preferable as it allows the simultaneous quantification of ZAP-70 protein expression levels in CLL cells and residual normal lymphocyte subsets. However, several factors introduce variability in the results reported from different laboratories; these factors include the anti-ZAP-70 antibody clone and conjugate, the staining procedure, the gating strategy, and the method of reporting the results. The need for standardization of the approach led to the organization of an international working group focused on harmonizing all aspects of the technique. During this workshop, a technical consensus was reached on the methods for cell permeabilization and immunophenotyping procedures. An assay was then designed that allowed comparison of two clones of anti-ZAP-70 antibody and the identification of the expression of this molecule in B, T, and NK cells identified in a four multicolor analysis. This procedure was applied to three stabilized blood samples, provided by the UK NEQAS group to all participating members of this study, in order to minimize variability caused by sample storage and shipment. Analysis was performed in 20 laboratories providing interpretable data from 14 centers. Various gating strategies were used and the ZAP-70 levels were expressed as percentage positive (POS) relative to isotype control or normal B-cells or normal T-cells; in addition the levels were reported as a ratio of expression in CLL cells relative to T-cells. The reported level of ZAP-70 expression varied greatly depending on the antibody and the method used to express the results. The CLL/T-cell ZAP-70 expression ratio showed a much lower interlaboratory variation than other reporting strategies and is recommended for multicenter studies. Stabilization results in decreased expression of CD19 making gating more difficult and therefore stabilized samples are not optimal for multicentric analysis of ZAP-70 expression. We assessed the variation of ZAP-70 expression levels in fresh cells according to storage time, which demonstrated that ZAP-70 is labile but sufficiently stable to allow comparison using fresh samples distributed between labs in Europe. These studies have demonstrated progress toward a consensus reporting procedure, and further work is underway to harmonize the preparation and analysis procedures.

CLLU1 expression levels predict time to initiation of therapy and overall survival in chronic lymphocytic leukemia.

OBJECTIVES: Chronic lymphocytic leukemia (CLL) is an incurable disease with a highly variable clinical course. IgV(H) mutational status, chromosomal aberrations, CD38 expression and ZAP-70 expression are prognostic markers in CLL, however, they are not exclusively confined to this disease. We recently identified a novel CLL-specific gene (CLL upregulated gene1, CLLU1) that is exclusively upregulated in CLL cells. Here we describe our evaluation of the prognostic significance of CLLU1 in CLL. METHODS: A cohort of 59 previously untreated CLL patients was studied. We determined the expression levels of two CLLU1 transcripts, cDNA1 and CDS, by quantitative RT-PCR. The relation between CLLU1 expression and time to therapy, overall survival and presence or absence of ZAP-70, CD38, chromosomal aberrations or IgV(H) mutations in the 59 patients was analyzed. RESULTS: Analyzed as a continuous, quantitative parameter CLLU1 levels significantly predicted time from diagnosis to initiation of therapy (P < or = 0.0003) Analyzed as a categorical parameter, by segregation of the patients into groups with cDNA1 or CDS expression above or below the median, the CLLU1 levels significantly predicted time from diagnosis to initiation of therapy (P = 0.001) and predicted overall survival with borderline significance (P < or = 0.05). Patient stratification according to clinical stage, cytogenetics, IgV(H) mutational status, ZAP-70 and CD38, demonstrated significantly increased CLLU1 expression in all investigated CLL poor risk groups. CLLU1 expression levels contributed additional prognostic information to ZAP-70-positive patients. CONCLUSIONS: CLLU1 is the first identified CLL specific gene. The CLLU1 mRNA expression level can predict time to initiation of treatment and survival in CLL patients.

Identification of a gene on chromosome 12q22 uniquely overexpressed in chronic lymphocytic leukemia.

The pathogenesis of chronic lymphocytic leukemia (CLL) is unknown but may involve aberrant activation of signaling pathways. Somatic hypermutations in rearranged immunoglobulin heavy-chain (IgVH) genes allow a division of CLL patients into 2 categories: mutated IgVH genes are associated with an indolent disease, whereas unmutated IgVH genes define an aggressive form. Using differential display to compare gene expression in CLL cells with and without IgVH hypermutations, we identified a novel gene, CLL up-regulated gene 1 (CLLU1), that was highly up-regulated in CLL cells without IgVH hypermutations. CLLU1 mapped to chromosome 12q22, within a cluster of genes that are active in germinal center B cells. However, appreciable levels of CLLU1 were detectable only in CLL cells and not in a panel of normal tissue extracts or in any other tested hematologic malignancy. High expression of CLLU1 in CLL samples occurred irrespective of trisomy 12 or large chromosomal rearrangements. CLLU1 encodes 6 mRNAs with no sequence homology to any known gene, and most transcripts appear to be noncoding. Two transcripts, however, potentially encode a peptide with remarkable structural similarity to human interleukin 4. These data, in particular the unique and restricted expression pattern, suggest that CLLU1 is the first disease-specific gene identified in CLL.

The chimeric anti-CD20 antibody rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells through a p38 mitogen activated protein-kinase-dependent mechanism.

Antibodies against CD20 can activate complement and induce antibody-dependent cellular cytotoxicity (ADCC) in B lymphocytes. In B-cell lines, such antibodies also induce apoptosis. In this study, the expression and function of CD20 on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed. Flow cytometric analysis demonstrated that B-CLL cells express CD20 with a fluorescence intensity that is significantly weaker than that of normal CD5(+) and CD5(-) B cells and that of malignant CD5(-) low-grade non-Hodgkin lymphoma cells. A small population of cells from healthy donors that have an expression pattern of CD5 and CD20 identical to that of B-CLL cells were identified, and this population was confirmed to be of T lineage, not B lineage. Culture of freshly isolated B-CLL cells in the presence of the chimeric anti-CD20 antibody rituximab and a cross-linking F(ab)(2) fragment, resulted in dose- and time-dependent induction of apoptosis. The induction of apoptosis occurred under conditions in which the influence of complement activation and ADCC was negligible. Cross-linking of rituximab induced strong and sustained phosphorylation of the 3 mitogen activated protein (MAP) kinases c-Jun NH2-terminal protein kinase, extracellular signal-regulated kinase, and p38. Introduction of the p38 inhibitor SB203580 into the system completely blocked signaling downstream of p38, as evidenced by the absence of MAPKAP K2 activity, and significantly reduced the degree of anti-CD20-induced apoptosis. These results demonstrate that cross-linking of rituximab bound to CD20 on freshly isolated B-CLL cells induces apoptosis through a signaling pathway that is dependent on p38 MAP-kinase activation.