[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptor (BCR) belonging to subset #1 (IGHV1-5-7/IGKV1-39) display a poor outcome. To characterize their genetic and genomic features and BCR function, we selected 20 subset #1 CLL from a series of 579 cases. Subset #1 CLL, all showing unmutated IGHV, were associated with the presence of del(11q) (50%) in comparison with unmutated CLL, unmutated stereotyped CLL other than subset #1 and with cases utilizing the same IGHV genes but a heterogeneous VH CDR3 (non-subset #1 CLL). There were no distinctive features regarding CD38, ZAP-70 and TP53 disruption. NOTCH1, SF3B1 and BIRC3 were mutated in 15%, 0% and 5% of cases, respectively, while BIRC3 was deleted in 22% of cases. Microarray unsupervised analysis on 80 unmutated/mutated/stereotyped/non-stereotyped CLL showed a tight clustering of subset #1 cases. Their genomic signature exhibited several differentially expressed transcripts involved in BCR signal transduction, apoptosis regulation, cell proliferation and oxidative processes, regardless of del(11q). Accordingly, BCR ligation with anti-IgM revealed a significant higher proliferation of subset #1 vs. unmutated non-subset #1 CLL, both at baseline and after 24-48 hours stimulation. Subset #1 CLL represent a paradigmatic example of the direct link between BCR structure, function and patients prognosis.
American Journal of Hematology 03/2014; 89(1). DOI:10.1002/ajh.23591 · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gene profile and functional changes upon IgD cross-linking were evaluated in chronic lymphocytic leukemia (CLL). Microarrays highlighted responsiveness to IgD in all cases, independently of clinico-biological characteristics. Stimulated samples exhibited the down-regulation of transcripts of B-cell receptor signaling and cell-adhesion at 24h and the up-modulation of differentiation and apoptosis genes at 48h. A significant increase in apoptosis upon ligation was also documented. Furthermore, comparison between IgD and IgM stimulation displayed a differential transcriptional/functional response. In conclusion, CLL respond to IgD displaying expression changes and cell-death enhancement, indicating the apoptosis induction via-IgD as an alternative approach for CLL management.
Leukemia research 01/2013; 37(4). DOI:10.1016/j.leukres.2012.12.019 · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The genetic characterization of chronic lymphocytic leukemia cells correlates with the behavior, progression and response to treatment of the disease.
Our aim was to investigate the role of ATM gene alterations, their biological consequences and their value in predicting disease progression. The ATM gene was analyzed by denaturing high performance liquid chromatography and multiplex ligation probe amplification in a series of patients at diagnosis. The results were correlated with immunoglobulin gene mutations, cytogenetic abnormalities, ZAP-70 and CD38 expression, TP53 mutations, gene expression profile and treatment-free interval.
Mutational screening of the ATM gene identified point mutations in 8/57 cases (14%). Multiplex ligation probe amplification analysis identified six patients with 11q deletion: all of them had at least 20% of deleted cells, analyzed by fluorescent in situ hybridization. Overall, ATM point mutations and deletions were detected in 14/57 (24.6%) cases at presentation, representing the most common unfavorable genetic anomalies in chronic lymphocytic leukemia, also in stage A patients. Patients with deleted or mutated ATM had a significantly shorter treatment-free interval compared to patients without ATM alterations. ATM-mutated cases had a peculiar gene expression profile characterized by the deregulation of genes involved in apoptosis and DNA repair. Finally, definition of the structure of the ATM-mutated protein led to a hypothesis that functional abnormalities are responsible for the unfavorable clinical course of patients carrying these point mutations.
ATM alterations are present at diagnosis in about 25% of individuals with chronic lymphocytic leukemia; these alterations are associated with a peculiar gene expression pattern and a shorter treatment-free interval.
[Show abstract][Hide abstract] ABSTRACT: Mantle cell lymphoma (MCL) is an aggressive neoplasm with a short survival. Cases with leukaemic MCL and splenomegaly without adenopathies (non-nodal MCL) may have a more indolent course. To gain insights into the biological features underlying this presentation, we investigated the gene expression profile (GEP) and the IGHV mutational status in a cohort of leukaemic MCL cases. Comparison of MCL with other lymphoproliferative disorders (i.e. splenic marginal zone lymphoma, follicular lymphoma, chronic lymphocytic leukaemia) revealed a MCL signature enriched for the following gene categories: mitochondrion, oxidoreductase activity, response to stress, to DNA damage and TP53-pathway. Furthermore, GEP analysis revealed that non-nodal MCL cases were characterized by the down-modulation of the following gene categories: cell projection, actin cytoskeleton organization, cell adhesion (ITGAE, CELSR1, PCDH9) and tumour invasion/progression (PGF, ST14, ETS1, OCIAD1, EZR). Many down-modulated genes were related to the TP53-pathway and to DNA damage response. IGHV status proved unmutated in all nodal and mutated in all non-nodal MCL. Non-nodal leukaemic MCLs display a peculiar clinical presentation, with distinctive biological features, such as mutated IGHV and a transcriptional profile lacking tumour invasion properties, that might contribute to the absence of nodal involvement and to the less aggressive clinical course.
British Journal of Haematology 12/2011; 156(5):601-11. DOI:10.1111/j.1365-2141.2011.08962.x · 4.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) is a heterogeneous disease characterized by defects in the DNA damage response and apoptosis. Among the factors involved in these pathways, we focused on the enzyme poly(ADP-ribose) polymerase 1 (PARP1) and on its substrate Che-1 by evaluating their basal expression and functional changes upon irradiation (IR). Microarray experiments were performed on 98 untreated CLL cases. Next, freshly isolated primary cells from 21 untreated patients were analyzed for in vitro response to irradiation through Western blot, PARP activity assay, Annexin-V analysis, and PARP1 basal expression by quantitative polymerase chain reaction. Microarray analysis showed that PARP1 and CHE1 were constitutively expressed in CLL and had a high degree of correlation with each other and with TP53. PARP1 and TP53 downmodulation was associated with worse clinical outcomes, especially in TP53-mutated cases. Next, CLL samples from 21 untreated patients were classified as responders and nonresponders based on IR-induced PARP1 cleavage. Notably, while responder samples were characterized by Che-1 and p53 induction at 8 hours and reduction at 24 hours post-IR, nonresponders included both samples with p53 dysfunctions and cases with a normal IR-induced Che-1 and/or p53 response. Finally, we observed that PARP1 was downregulated in nonresponder vs responder samples and that its basal expression was positively correlated with PARP1 cleavage after IR. In conclusion, we showed that reduced expression of PARP1 is associated with an impairment of CLL responsiveness to cell death.
[Show abstract][Hide abstract] ABSTRACT: Given that TP53 alterations predict prognosis and response to therapy in chronic lymphocytic leukemia (CLL), screening for TP53 mutations has an increasing role in patient management. TP53 direct sequencing is a time-consuming method, while the AmpliChip p53 Research Test is a novel non time-consuming microarray-based resequencing assay and queries Exons 2-11. We evaluated the impact of TP53 mutations on clinical outcome by analyzing 98 untreated CLL using the AmpliChip p53 Research Test and direct sequencing and performed microarrays analysis on TP53 mutated and/or deleted cases. The AmpliChip p53 Research Test detected 17 mutations in 14 patients (17.3%); a significant association between TP53 mutations and del(17p) was recorded. From a clinical standpoint, a higher percentage of mutation was found in CLL with unfavorable outcome (17.2% vs. 7.1% in progressive vs. stable cases). Detection of TP53 mutations by the AmpliChip p53 Research Test was associated with a significantly worse survival (P = 0.0002). Comparison of the array and direct sequencing tests showed that the p53 Research Test detected more mutations, although it failed to identify two microdeletions. Finally, microarrays analysis showed a more distinctive signature associated with del(17p) than with TP53 mutations, likely due to a concomitant gene dosage effect. The AmpliChip p53 Research Test is a straightforward method that bears prognostic value. This study confirms a high percentage of TP53 mutations in CLL with unfavorable outcome and a significant association between TP53 aberrations and del(17p). Finally, specific gene expression profiles are recognized for TP53 alterations.
Genes Chromosomes and Cancer 04/2011; 50(4):263-74. DOI:10.1002/gcc.20852 · 4.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation-induced cytidine deaminase (AICDA) initiates somatic hypermutation and class-switch recombination of immunoglobulin (Ig) genes and induces mutations also in non-Ig genes. AICDA aberrant expression was detected in B-lineage acute lymphoblastic leukaemia (B-ALL), particularly BCR/ABL1+ B-ALL; patients expressing AICDA carried more copy number alterations than 'AICDA-negative' cases. To determine the role of AICDA, AICDA expression and gene expression profiling were studied in adult BCR/ABL1+ B-ALL. Patients displaying the full-length isoform AICDA are characterized by up-regulation of DNA repair/replication and cell cycle genes, suggesting their involvement in the genetic instability of BCR/ABL1+ B-ALL.
British Journal of Haematology 03/2011; 152(6):727-32. DOI:10.1111/j.1365-2141.2010.08449.x · 4.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A comprehensive panel of clinical-biological parameters was prospectively evaluated at presentation in 112 patients with chronic lymphocytic leukemia (<65 years), to predict the risk of progression in early stage disease. Eighty-one percent were in Binet stage A, 19% in stages B/C. Treatment-free survival was evaluated as the time from diagnosis to first treatment, death or last follow up. In univariate analysis, advanced stage, hemoglobin, platelets, white blood cell, leukemic lymphocyte count, raised beta 2-microglobulin and LDH, unmutated immunoglobulin variable region genes, CD38, del(17p), del(11q) and +12, were significantly associated with a short treatment-free survival; the T/leukemic lymphocyte ratio was associated with a better outcome. Multivariate analysis of treatment-free survival in stage A patients selected a high white blood cell count and unmutated immunoglobulin variable region genes as unfavorable prognostic factors and a high T/leukemic lymphocyte ratio as a favorable one. At diagnosis, these parameters independently predict the risk of progression in stage A chronic lymphocytic leukemia patients.
[Show abstract][Hide abstract] ABSTRACT: Dear Editor, In acute myeloid leukemia (AML), mutations of the FMSlike tyrosine kinase-3 (FLT3) are found in about one third of patients. The most common mutations are the internal tandem duplication (FLT3-ITD) within exon 14 coding the juxtamembrane (JM) domain and the point mutations involving exon 20 coding the second tyrosine-kinase domain (FLT3-TKD). Recently, a third class of activating point mutations in the JM (FLT3-JM-PM) has been identified in AML patients, but its biological and clinical significance remains to be clarified .
Annals of Hematology 10/2010; 90(7):845-6. DOI:10.1007/s00277-010-1092-0 · 2.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the role of protein kinases (PKs) in chronic lymphocytic leukemia (CLL), we performed gene expression profile on 505 PK genes. Comparison between CLL with acute lymphocytic leukemia (ALL) patients highlighted an homogeneous up-modulation of several PKs in CLL, 16 also overexpressed in two additional CLL cohorts. Q-PCR analysis confirmed these findings. No differences were observed in the main prognostic subclasses, indicating that PK overexpression is specific of the disease itself. Tests in vitro showed that Dasatinib partially reduced CLL cells viability, mostly in IGHV germline patients. These findings suggest that treatment with second generation tyrosine kinase (TK) inhibitors may represent an attractive therapeutic strategy for CLL patients.
Leukemia research 06/2010; 34(6):733-41. DOI:10.1016/j.leukres.2009.11.005 · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In chronic lymphocytic leukemia (CLL), spontaneous regressions are an exceptional phenomenon, whose biologic features are unknown. We describe 9 CLL patients who underwent a spontaneous clinical regression over an 11-year follow-up, despite a residual neoplastic clone detected by flow cytometry. CD38 and ZAP-70 were negative in all cases. Immunoglobulin heavy chain variable region (IgVH) genes, mutated in all 7 evaluable patients, were restricted to the VH3 family in 6, with the usage of V(H)3-30 gene in 2. The light chain variable region genes were mutated in 6 of 8 cases, with the use of V(kappa)4-1 gene in 3. Microarray analysis of CLL cells showed a distinctive genomic profile with an overrepresentation of BCR-related and ribosomal genes, regulators of signal transduction and transcription. The number of activated T lymphocytes expressing IFN-gamma, TNF-alpha, and IL-4 was similar between CLL in spontaneous regression and healthy persons. In conclusion, spontaneous clinical regressions can occur in CLL despite the persistence of the neoplastic clone, and the biologic features include negative CD38 and ZAP-70, mutated V(H)3-30 and V(kappa)4-1 genes. The peculiar gene profile suggests that BCR signaling may play an important role in this scenario as the most significant feature of the leukemic clone in regression.
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) patients exhibit a variable clinical course. To investigate the association between clinicobiologic features and responsiveness of CLL cells to anti-IgM stimulation, we evaluated gene expression changes and modifications in cell-cycle distribution, proliferation, and apoptosis of IgV(H) mutated (M) and unmutated (UM) samples upon BCR cross-linking. Unsupervised analysis highlighted a different response profile to BCR stimulation between UM and M samples. Supervised analysis identified several genes modulated exclusively in the UM cases upon BCR cross-linking. Functional gene groups, including signal transduction, transcription, cell-cycle regulation, and cytoskeleton organization, were up-regulated upon stimulation in UM cases. Cell-cycle and proliferation analyses confirmed that IgM cross-linking induced a significant progression into the G(1) phase and a moderate increase of proliferative activity exclusively in UM patients. Moreover, we observed only a small reduction in the percentage of subG(0/1) cells, without changes in apoptosis, in UM cases; contrariwise, a significant increase of apoptotic levels was observed in stimulated cells from M cases. These results document that a differential genotypic and functional response to BCR ligation between IgV(H) M and UM cases is operational in CLL, indicating that response to antigenic stimulation plays a pivotal role in disease progression.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that modulate the expression of genes at the posttranscriptional level. These small molecules have been shown to be involved in cancer, apoptosis, and cell metabolism. In the present study we provide an informative profile of the expression of miRNAs in primary chronic lymphocytic leukemia (CLL) cells using 2 independent and quantitative methods: miRNA cloning and quantitative real-time-polymerase chain reaction (qRT-PCR) of mature miRNAs. Both approaches show that miR-21 and miR-155 are dramatically overexpressed in patients with CLL, although the corresponding genomic loci are not amplified. miR-150 and miR-92 are also significantly deregulated in patients with CLL. In addition, we detected a marked miR-15a and miR-16 decrease in about 11% of cases. Finally, we identified a set of miRNAs whose expression correlates with biologic parameters of prognostic relevance, particularly with the mutational status of the IgV(H) genes. In summary, the results of this study offer for the first time a comprehensive and quantitative profile of miRNA expression in CLL and their healthy counterpart, suggesting that miRNAs could play a primary role in the disease itself.