Giovanna Cutrona

In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells

Due to its relatively slow clinical progression, B cell chronic lymphocytic leukemia (B-CLL) is classically described as a disease of accumulation rather than proliferation. However, evidence for various forms of clonal evolution suggests that B-CLL clones may be more dynamic than previously assumed. We used a nonradioactive, stable isotopic labeling method to measure B-CLL cell kinetics in vivo. Nineteen patients drank an aliquot of deuterated water (2H2O) daily for 84 days, and 2H incorporation into the deoxyribose moiety of DNA of newly divided B-CLL cells was measured by gas chromatography/mass spectrometry, during and after the labeling period. Birth rates were calculated from the kinetic profiles. Death rates were defined as the difference between calculated birth and growth rates. These analyses demonstrated that the leukemic cells of each patient had definable and often substantial birth rates, varying from 0.1% to greater than 1.0% of the entire clone per day. Those patients with birth rates greater than 0.35% per day were much more likely to exhibit active or to develop progressive disease than those with lower birth rates Thus, B-CLL is not a static disease that results simply from accumulation of long-lived lymphocytes. Rather, it is a dynamic process composed also of cells that proliferate and die, often at appreciable levels. The extent to which this turnover occurs has not been previously appreciated. A correlation between birth rates and disease activity and progression appears to exist, which may help identify patients at risk for worsening disease in advance of clinical deterioration.

Effects in live cells of a c- myc anti-gene PNA linked to a nuclear localization signal

Peptide nucleic acids (PNA) are synthetic homologs of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. In this study, a PNA construct was employed as an anti-gene agent in intact cells in culture. The cell lines studied were derived from Burkitts lymphomas (BL) that presented a translocated and hyperexpressed c-myc oncogene. A 17-mer anti-myc PNA, complementary to a unique sequence located at the beginning of the second exon of the oncogene, and was covalently linked at its N terminus to a nuclear localization signal (NLS) (PNA-mycwt-NLS). When BL cells were exposed to PNA-mycwt-NLS, the anti-gene construct was localized predominantly in the cell nuclei and a rapid consequent downregulation of c-myc expression occurred. Under these conditions, both completion of a productive cell cycle and apoptosis were inhibited.

CD38 and chronic lymphocytic leukemia: a decade later

This review highlights a decade of investigations into the role of CD38 in CLL. CD38 is accepted as a dependable marker of unfavorable prognosis and as an indicator of activation and proliferation of cells when tested. Leukemic clones with higher numbers of CD38(+) cells are more responsive to BCR signaling and are characterized by enhanced migration. In vitro activation through CD38 drives CLL proliferation and chemotaxis via a signaling pathway that includes ZAP-70 and ERK1/2. Finally, CD38 is under a polymorphic transcriptional control after external signals. Consequently, CD38 appears to be a global molecular bridge to the environment, promoting survival/proliferation over apoptosis. Together, this evidence contributes to the current view of CLL as a chronic disease in which the hosts microenvironment promotes leukemic cell growth and also controls the sequential acquisition and accumulation of genetic alterations. This view relies on the existence of a set of surface molecules, including CD38, which support proliferation and survival of B cells on their way to and after neoplastic transformation. The second decade of studies on CD38 in CLL will tell if the molecule is an effective target for antibody-mediated therapy in this currently incurable leukemia.

Apoptotic cells overexpress vinculin and induce vinculin-specific cytotoxic T-cell cross-priming.

Here we show that apoptotic cells overexpress vinculin and are ingested by dendritic cells, which subsequently cross-prime vinculin-specific cytotoxic T lymphocytes (CTLs). Successful cross-priming requires that the apoptotic cells provide maturation signals to dendritic cells through CD40–CD40 ligand (CD40L) interactions. If apoptotic cells are CD40L−, the help of a third T cell is needed for priming, indicating a regulatory role for apoptotic cells in determining priming or tolerance. Vinculin-specific CTL priming is also related to apoptosis in vivo, given that in HIV-seropositive individuals, the frequency of specific CTLs depends on the proportion of peripheral CD40L+ apoptotic cells.

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.

CD26 expression in mature B-cell neoplasia: its possible role as a new prognostic marker in B-CLL.

CD26 (dipeptidyl peptidase IV, DPP IV) is widely expressed by T and natural killer (NK) cells, epithelial and endothelial cells of different tissues, and it is strongly upregulated in activated B‐cells; moreover it plays a regulatory role in the neoplastic transformation and progression of various types of tumours. CD26 expression was evaluated by means of flow cytometry in various peripheral B‐cell lymphoid tumours: 12 follicular and 12 mantle cell lymphomas, 20 multiple myelomas (MMs), 12 hairy cell leukaemias (HCLs), 112 chronic lymphocytic leukaemias (CLLs), 20 CD5negative B‐cell chronic lymphoproliferative diseases (CD5neg B‐CLPDs) and 12 diffuse large cell lymphomas (DLCLs). CD26 expression was absent or barely detectable in follicular and mantle cell lymphomas, high in MMs and HCLs, and variable in CLLs, in CD5neg B‐CLPDs and in DLCLs. CD26 significantly correlated with CD49d and CD38 expressions (p < 0.0001) in B‐CLLs, and there was a significant correlation between CD26 and ZAP‐70 expressions or IgVH mutational status (p < 0.0001). After a median follow‐up of 36 months, 65 B‐CLL patients were treated; taking 10% as the best CD26 cut‐off value, Kaplan–Meier curves revealed a significantly shorter time to treatment in the CD26‐positive cases (p < 0.0001). Overall, our data indicate that CD26 expression may identify subsets of B‐CLL patients with an unfavourable clinical outcome in terms of therapeutic need, thus suggesting its potential role as a marker (together with CD38 and CD49d) in a future routine cytofluorimetric panel to be validated for the prognostic stratification of B‐CLLs. Copyright