Serena Matis

B lymphocytes in humans express ZAP‐70 when activated in vivo

ZAP‐70 is a protein tyrosine kinase initially found in T and NK cells. Recently, this important signaling element was detected in leukemic B cells from a subgroup of patients with B cell chronic lymphocytic leukemia (B‐CLL). In this study, ZAP‐70 was detected in normal B cells from human tonsils, but not from peripheral blood. The cDNA sequence of B cell ZAP‐70 was the same as that in T cells. Germinal center B cells and plasma cells had a substantial proportion of ZAP‐70+ cells, while memory and follicular mantle B cells, which contain low numbers of activated B cells, expressed relatively little ZAP‐70. A cell fraction of IgD+, CD38+ B cells, which are comprised of many in vivo activated B cells, exhibited the highest levels of ZAP‐70. Density gradient fractionation of tonsil B cells confirmed that ZAP‐70 was not expressed by resting B cells, but was expressed by buoyant, in vivo activated B cells. In these B cells, the expression of ZAP‐70 correlated with that of CD38 and not with that of CD5, a hallmark of B‐CLL cells. B‐CLL cells are activated cells and their ZAP‐70 expression reflects a normal property of activated B cells populations rather than a neoplastic aberration.

Molecular and Transcriptional Characterization of 17p Loss in B-Cell Chronic Lymphocytic Leukemia

Distinct genetic abnormalities, such as TP53 deletion at 17p13.1, have been identified as having adverse prognostic relevance in B‐cell chronic lymphocytic leukemia (B‐CLL), and conventional cytogenetic studies have shown that TP53 deletion in B‐CLL is mainly associated with the loss of 17p due to complex chromosomal rearrangements. We used an integrative genomic approach to investigate the significance of 17p loss in 18 B‐CLLs in Binet stage A, carrying a TP53 monoallelic deletion detected by means of fluorescence in situ hybridization (FISH). Genome‐wide DNA analysis using single nucleotide polymorphism (SNP) arrays of 12 of 18 samples showed 17p loss in 11 cases, with breakpoints scattered along the 17p11.2 region. FISH analysis confirmed these findings and revealed 17p loss in a small fraction of leukemic cells in the remaining TP53‐deleted case, and it also indicated 17p loss in the six cases not investigated by means of SNP arrays. Mutations in exons 2–11 of the remaining TP53 allele were found in 9 of 12 deleted samples. Gene‐expression profiling of 60 B‐CLLs, including seven patients with 17p loss, identified 40 differentially expressed genes in 17p‐ versus 17p normal samples, 35 of which were downregulated in 17p‐tumors. The majority (30 of 35) of these transcripts, including putative tumor suppressor genes, mapped to 17p, thus indicating a remarkable gene‐dosage effect. Our data provide evidence that 17p loss may play an additional pathogenetic role in B‐CLL and suggest that the concomitant loss of multiple tumor suppressor genes could be responsible for the highly adverse prognostic relevance associated with TP53 loss.

Definition of progression risk based on combinations of cellular and molecular markers in patients with Binet stage A chronic lymphocytic leukaemia

IGHV mutational status and ZAP‐70 or CD38 expression correlate with clinical course in B‐cell chronic lymphocytic leukaemia (CLL). The three markers may be discordant in the single case and there is no consensus on their combined use in clinical practise. This multicenter study investigated this issue. Two‐hundred and sixty‐two Binet stage A patients were studied for the three markers. Sixty patients were profiled with HG‐U133A gene expression chips. Disease progression was determined by time from diagnosis to treatment (TTT). The probability of being treatment‐free at 3 years was significantly shorter in patients with unmutated IGHV genes (IGHVunmut 66% vs. 93%, chi square of log‐rank = 30, P < 0·0001), ZAP‐70 positive (ZAP‐70pos 73% vs. 96%, chi square of log‐rank = 8·2, P = 0·004) or CD38‐positive cells (CD38pos 68% vs. 91%, chi square of log‐rank = 21, P < 0·0001). Cox multivariate regression analysis showed that the three markers had an independent predictive value for TTT of similar power. A prognostic system based on presence of none (low‐risk), one (intermediate‐risk) or two or three (high‐risk) markers was generated. Based on such criteria, 56%, 23% and 21% of cases were clustered in low (HR = 1), intermediate [HR = 2·8, 95% confidence interval (CI) 2·4–5·8] and high‐risk group (HR = 8·0, 95% CI 3·9–16·2). Specific transcriptional patterns were significantly associated with risk groups.

Integrative Genomics Analyses Reveal Molecularly Distinct Subgroups of B-Cell Chronic Lymphocytic Leukemia Patients with 13q14 Deletion

Purpose: Chromosome 13q14 deletion occurs in a substantial number of chronic lymphocytic leukemia (CLL) patients and it is believed to play a pathogenetic role. The exact mechanisms involved in this lesion have not yet been fully elucidated because of its heterogeneity and the imprecise knowledge of the implicated genes. This study was addressed to further contribute to the molecular definition of this lesion in CLL. Experimental Design: We applied single-nucleotide polymorphism (SNP)-array technology and gene expression profiling data to investigate the 13q14 deletion occurring in a panel of 100 untreated, early-stage (Binet A) patients representative of the major genetics, molecular, and biological features of the disease. Results: Concordantly with FISH analysis, SNP arrays identified 44 patients with del(13)(q14) including 11 cases with a biallelic deletion. The shorter monoallelic deletion was 635-kb long. The loss of the miR-15a/16-1 cluster occurred in all del(13)(q14) cases except in 2 patients with a monoallelic deletion, who retained both copies. MiR-15a/16 expression was significantly downregulated only in patients with the biallelic loss of the miRNA cluster compared to 13q normal cases. Finally, the natural grouping of SNP profiles by nonnegative matrix factorization algorithm showed that patients could be classified into 2 separate clusters, mainly characterized by short/biallelic versus wide/monoallelic 13q14 deletions. Supervised analyses of expression data showed that specific transcriptional profiles are correlated with these 2 genomic subgroups. Conclusions: Overall, our data highlight the presence of 2 distinct molecular types of 13q14 deletions, which may be of clinical relevance in CLL. Clin Cancer Res; 16(23); 5641–53. ©2010 AACR.

Biological and clinical relevance of quantitative global methylation of repetitive DNA sequences in chronic lymphocytic leukemia

Global DNA hypomethylation affecting repeat sequences has been reported in different cancer types. Herein, we investigated the methylation levels of repetitive DNA elements in chronic lymphocytic leukemia (CLL), their correlation with the major cytogenetic and molecular features, and clinical relevance in predicting therapy-free survival (TFS). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu, long interspersed nuclear elements-1 (LINE-1) and satellite-α (SAT-α) sequences in 77 untreated early-stage (Binet A) CLL patients. Peripheral B-cells from 7 healthy donors were used as controls. Methylation levels (median %5mC) were lower in B-CLLs compared with controls (21.4 vs. 25.9; 66.8 vs. 85.7; 84.0, vs. 88.2 for Alu, LINE-1 and SAT-α, respectively) (p < 0.001). Among CLL patients, a significant association was observed with 17p13.1 deletion (16.8 vs. 22.4; 51.2 vs. 68.5; 52.6 vs. 85.0, for Alu, LINE-1 and SAT-α) but not with other major genetic lesions, IgVH mutation status, CD38 or ZAP-70 expression. Follow-up analyses showed that lower SAT-α methylation levels appeared to be an independent prognostic marker significantly associated with shorter TFS. Our study extended previous limited evidences in methylation of repetitive sequences in CLL suggesting an important biological and clinical relevance in the disease.

Clonal heterogeneity in chronic lymphocytic leukemia cells: superior response to surface IgM cross-linking in CD38, ZAP-70-positive cells

The reasons why immunoglobulin gene mutation status and expression of ZAP-70 and CD38 influence disease progression in chronic lymphocytic leukemia are still undefined. This study shows that CD38+, ZAP-70+ cells have a greater capacity for signalling through the B-cell receptor and suggests a function for B-cell receptor signaling in promoting cell expansion. Background Patients with chronic lymphocytic leukemia whose cells express CD38 and ZAP-70 and utilize unmutated Ig VH region genes have a very poor prognosis. We studied whether cells expressing CD38 and ZAP-70 are more susceptible to stimulation through B-cell receptors than are cells that do not express CD38 and ZAP-70. Design and Methods CD38-positive and CD38-negative leukemic cells were separated from single cases and compared for their response to B-cell receptor cross-linking and ZAP-70 expression. Cohort studies were also carried out by measuring the apoptotic response to surface immunoglobulin M (IgM) cross-linking in 82 patients with chronic lymphocytic leukemia and the protein tyrosine phosphorylation induced by surface IgM in 21 patients. Results CD38-positive cells, isolated from cases of chronic lymphocytic leukemia classified as CD38-positive or CD38-negative, expressed more ZAP-70 than the corresponding CD38-negative cells, exhibited more robust protein tyrosine phosphorylation and had a greater tendency to apoptosis upon B-cell receptor cross-linking. In the cohort studies, surface IgM-induced protein tyrosine phosphorylation correlated significantly with CD38 and ZAP-70 expression and with the absence of Ig VH gene mutations. Apoptosis induced by surface IgM cross-linking correlated significantly only with the proportion of CD38-positive cells. Difficulties in finding more definitive correlations were probably related to imprecision in the in vitro test system and in the definition of cases as positive or negative. Conclusions Collectively, these data indicate that CD38-positive, ZAP-70-positive cells have a greater capacity for signaling through the B-cell receptor and suggest a function for B-cell receptor signaling in promoting chronic lymphocytic leukemia cell expansion, especially within the CD38-positive fraction of the leukemic clone.

Clinical Monoclonal B lymphocytosis versus Rai 0 Chronic Lymphocytic Leukemia: a Comparison of Cellular, Cytogenetic, Molecular, and Clinical Features

Purpose: To investigate the incidence and clinical relevance of classic and new prognostic markers, IGHV gene mutational status, and chromosomal abnormalities in clinical monoclonal B lymphocytosis (cMBL) compared with Rai stage 0 chronic lymphocytic leukemia (Rai0-CLL). Experimental Design: A group of 136 patients with cMBL and a group of 216 Rai0-CLL cases were investigated prospectively. Results: IGHV-mutated cases were significantly more frequent among cMBLs (P = 0.005), whereas the distribution of CD38 and ZAP-70 positive cases, of patients with NOTCH1 and SF3B1 mutations or exhibiting the major CLL cytogenetic abnormalities, was similar in the two groups. Moreover, no significant differences were found either in IGHV/IGHD/IGHJ gene usage or in the overall prevalence of stereotyped IGHV gene sequences. Cells from cMBL and Rai0-CLL exhibited similar gene and microRNA (miRNA) signatures; in addition, when grouped according to the IGHV mutational status, IGHV-unmutated cases showed different transcriptional signatures compared with IGHV-mutated patients, irrespective of the cMBL or Rai0-CLL classification. cMBL diagnosis per se was predictive of longer progression-free survival. Conclusions: Our study based on a prospective series of patients indicates that no major differences exist between the circulating cells from cMBL and Rai0-CLL, at least based on a comparison of the markers used in the study. This possibly suggests that the two conditions mainly differ in the initial size of the monoclonal cell population, which may influence the subsequent timing of clonal expansion and clinical manifestations. Clin Cancer Res; 19(21); 5890–900. ©2013 AACR.

Inhibition of Burkitt's lymphoma cells growth in SCID mice by a PNA specific for a regulatory sequence of the translocated c-myc

In Burkitts lymphoma (BL) cells due to a t(8;14) chromosomal translocation c-myc is often placed in proximity to the Eμ enhancer of the Ig locus and upregulated. We demonstrated that in BL cells a peptide nucleic acid (PNA), complementary to intronic Eμ sequences (PNAEμwt), specifically blocks the expression of the c-myc oncogene under the Eμ enhancer control and inhibits BL cell growth in culture. Here, we investigated whether PNAEμwt was also able to block tumor growth in SCID mice inoculated with human BL cell lines. After subcutaneous inoculum in mice BL cells reproducibly form tumors. Both pre-treatment of BL cells with PNAEμwt before inoculum and chronic intravenous administration of PNAEμwt to mice already inoculated with BL cells selectively caused increased latency of tumor appearance and decreased final tumor size. Tumors from PNAEμwt-treated animals showed substantial areas of cell necrosis and of c-myc downregulation. Inhibition of tumor growth was specific and was not observed with PNAEμmut carrying sequence mutations and in BL cell lines where the translocated c-myc is not under the control of the Eμ enhancer. These data confirm the potential therapeutic value of PNA targeted to regulatory non-coding regions.

Small nucleolar RNAs as new biomarkers in chronic lymphocytic leukemia

BackgroundSmall nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs are non-coding RNAs involved in the maturation of other RNA molecules. Alterations of sno/scaRNA expression may play a role in cancerogenesis. This study elucidates the patterns of sno/scaRNA expression in 211 chronic lymphocytic leukemia (CLL) patients (Binet stage A) also in comparison with those of different normal B-cell subsets.MethodsThe patterns of sno/scaRNA expression in highly purified CD19+ B-cells of 211 CLL patients and in 18 normal B-cell samples - 6 from peripheral blood, and 12 from tonsils (4 germinal center, 2 marginal zone, 3 switched memory and 3 naïve B-cells) - were analyzed on the Affymetrix GeneChip® Human Gene 1.0 ST array.ResultsCLLs display a sno/scaRNAs expression profile similar to normal memory, naïve and marginal-zone B-cells, with the exception of a few down-regulated transcripts (SNORA31, -6, -62, and -71C). Our analyses also suggest some heterogeneity in the pattern of sno/scaRNAs expression which is apparently unrelated to the major biological (ZAP-70 and CD38), molecular (IGHV mutation) and cytogenetic markers. Moreover, we found that SNORA70F was significantly down-regulated in poor prognostic subgroups and this phenomenon was associated with the down-regulation of its host gene COBLL1. Finally, we generated an independent model based on SNORA74A and SNORD116-18 expression, which appears to distinguish two different prognostic CLL groups.ConclusionsThese data extend the view of sno/scaRNAs deregulation in cancer and may contribute to discover novel biomarkers associated with the disease and potentially useful to predict the clinical outcome of early stage CLL patients.

Relevance of Stereotyped B-Cell Receptors in the Context of the Molecular, Cytogenetic and Clinical Features of Chronic Lymphocytic Leukemia

Highly homologous B-cell receptors, characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3), are expressed in a recurrent fraction of patients affected by chronic lymphocytic leukemia (CLL). We investigated the IGHV status of 1131 productive IG rearrangements from a panel of 1126 CLL patients from a multicenter Italian study group, and correlated the presence and class of HCDR3 stereotyped subsets with the major cytogenetic alterations evaluated by FISH, molecular prognostic factors, and the time to first treatment (TTFT) of patients with early stage disease (Binet A). Stereotyped HCDR3 sequences were found in 357 cases (31.7%), 231 of which (64.7%) were unmutated. In addition to the previously described subsets, 31 new putative stereotypes subsets were identified. Significant associations between different stereotyped HCDR3 sequences and molecular prognostic factors, such as CD38 and ZAP-70 expression, IGHV mutational status and genomic abnormalities were found. In particular, deletion of 17p13 was significantly represented in stereotype subset #1. Notably, subset #1 was significantly correlated with a substantially reduced TTFT compared to other CLL groups showing unmutated IGHV, ZAP-70 or CD38 positivity and unfavorable cytogenetic lesions including del(17)(p13). Moreover, subset #2 was strongly associated with deletion of 13q14, subsets #8 and #10 with trisomy 12, whereas subset #4 was characterized by the prevalent absence of the common cytogenetic abnormalities. Our data from a large and representative panel of CLL patients indicate that particular stereotyped HCDR3 sequences are associated with specific cytogenetic lesions and a distinct clinical outcome.