Riccardo Bomben

Biological and clinical risk factors of chronic lymphocytic leukaemia transformation to Richter syndrome

Predictors of chronic lymphocytic leukaemia (CLL) transformation to Richter syndrome (RS) are not established and were investigated in 185 consecutive CLL cases. Actuarial incidence of RS (n = 17; all diffuse large B‐cell lymphomas) at 10 years was 16·2% (95% confidence interval: 8·0–24·4%). At CLL diagnosis, prognosticators of RS by univariate analysis were IGHV homology ≥98% (P = 0·006), IGHV4‐39 usage (P < 0·001), del13q14 absence (P = 0·004), expression of CD38 (P < 0·001) and ZAP70 (P = 0·004), size (P < 0·001) and number (P < 0·001) of lymph nodes, advanced Binet stage (P = 0·002), and lactate dehydrogenase (P < 0·001). Multivariate analysis, performed separately for biological and clinical variables, identified CD38 expression [Hazard ratio (HR) = 4·26; P = 0·018], IGHV4‐39 usage (HR = 4·29; P = 0·018), and lymph node size ≥3 cm (HR = 9·07; P < 0·001) as independent RS prognosticators. A multivariate model simultaneously analysing biological and clinical variables identified lymph node size ≥3 cm (HR = 6·51; P = 0·001) and del13q14 absence (HR = 4·08; P = 0·031) as independent RS prognosticators. Risk factors of CLL transformation differed from risk factors of CLL progression. These results suggest that CD38 and del13q14 may identify biological subsets of CLL with different RS predisposition. Predominant nodal disease, CD38 expression, IGHV4‐39 usage, and absence of del13q14 may help in predicting RS at CLL diagnosis. Close monitoring and a careful biopsy policy are needed in patients carrying transformation risk factors.

CD38/CD31, the CCL3 and CCL4 Chemokines, and CD49d/Vascular Cell Adhesion Molecule-1 Are Interchained by Sequential Events Sustaining Chronic Lymphocytic Leukemia Cell Survival

CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Despite evidence that both molecules are involved in interactions occurring between CLL and normal cells in the context of CLL-involved tissues, a functional link is still missing. Using gene expression profiles comparing CD38(+)CD49d(+) versus CD38(-)CD49d(-) CLL cells, we showed overexpression of the CCL3 and CCL4 chemokines in cells from the former group. These chemokines were also up-regulated by CD38 signals in CLL; moreover, CCL3 was expressed by CLL cells from bone marrow biopsies (BMB) of CD38(+)CD49d(+) but not CD38(-)CD49d(-) cases. High levels of CCR1 and, to a lesser extent, CCR5, the receptors for CCL3 and CCL4, were found in CLL-derived monocyte-macrophages. Consistently, CCL3 increased monocyte migration, and CD68(+) macrophage infiltration was particularly high in BMB from CD38(+)CD49d(+) CLL. Conditioned media from CCL3-stimulated macrophages induced endothelial cells to express vascular cell adhesion molecule-1 (VCAM-1), the CD49d ligand, likely through tumor necrosis factor alpha overproduction. These effects were apparent in BMB from CD38(+)CD49d(+) CLL, where lymphoid infiltrates were characterized by a prominent meshwork of VCAM-1(+) stromal/endothelial cells. Lastly, CD49d engagement by VCAM-1 transfectants increased viability of CD38(+)CD49d(+) CLL cells. Altogether, CD38 and CD49d can be thought of as parts of a consecutive chain of events ultimately leading to improved survival of CLL cells.

Molecular and clinical features of chronic lymphocytic leukaemia with stereotyped B cell receptors: Results from an Italian multicentre study

A fraction of chronic lymphocytic leukaemia (CLL) cases carry highly homologous B‐cell receptors (BCR), i.e. characterized by non‐random combinations of immunoglobulin heavy‐chain variable (IGHV) genes and heavy‐chain complementarity determining region‐3 (HCDR3), often associated with a restricted selection of IGVK/L light chains. Such ‘stereotyped’ BCR occur more frequently in CLL with unmutated (UM) than mutated (M) IGHV genes. We analysed 1426 IG rearrangements (from 1398 CLL cases) by a clustering driven by HCDR3 similarities. Molecular findings were correlated to time‐to‐treatment (TTT) and presence of known prognosticators. Sixty‐nine clusters (319 IG‐rearrangements, 22·4%) with stereotyped BCR were identified. Among 30 confirmed clusters (≥3 IG‐rearrangements/cluster), we found 14 novel clusters, of which 11 had M IG rearrangements (M clusters) and predominantly (8/11) used IGHV3 subgroup genes. Recurrent cluster‐biased amino acid changes were found throughout IGHV sequences of these ‘M clusters’. Regarding clinical outcome: (i) UM CLL from the IGHV1‐2/1‐3/1‐18/1‐46/7‐4‐1/IGKV1‐39 cluster had poorer prognosis than UM/M cases, or UM cases using the same IGHV genes but not in clusters; (ii) M CLL from the IGHV3‐21/IGLV3‐21 cluster had TTT similar to UM CLL, and shorter than M CLL expressing IGHV3‐21 but not in cluster. Altogether, our analysis identified additional molecular and clinical features for CLL expressing stereotyped BCR.

Association between molecular lesions and specific B-cell receptor subsets in chronic lymphocytic leukemia.

Genetic lesions and B-cell receptor (BCR) signaling are both oncogenic drivers in chronic lymphocytic leukemia (CLL). However, scant data are available on preferential associations between specific genetic alterations and stereotyped BCR subsets. By analyzing 1419 cases, 2 CLL subsets (2 and 8) harboring stereotyped BCR are enriched in specific molecular alterations influencing disease course. SF3B1 mutations are the genetic hallmark of IGHV3-21-CLL belonging to subset 2 (52%) but are evenly represented in nonstereotyped IGHV3-21-CLL. Trisomy 12 (87%) and NOTCH1 mutations (62%) characterize IGHV4-39-CLL belonging to subset 8 but occur with the expected frequency in IGHV4-39-CLL with heterogeneous BCR. Clinically, co-occurrence of SF3B1 mutations and subset 2 BCR configuration prompts disease progression in IGHV3-21-CLL, whereas cooperation between NOTCH1 mutations, +12, and subset 8 BCR configuration invariably primes CLL transformation into Richter syndrome. These findings provide a proof of concept that specific stereotyped BCR may promote or select molecular lesions influencing outcome.

Analysis of IgVH gene mutations in B cell chronic lymphocytic leukaemia according to antigen-driven selection identifies subgroups with different prognosis and usage of the canonical somatic hypermutation machinery

Cases of B‐cell chronic lymphocytic leukaemia (B‐CLL) with mutated (M) IgVH genes have a better prognosis than unmutated (UM) cases. We analysed the IgVH mutational status of B‐CLL according to the features of a canonical somatic hypermutation (SHM) process, correlating this data with survival. In a series of 141 B‐CLLs, 124 cases were examined for IgVH gene per cent mutations and skewing of replacement/silent mutations in the framework/complementarity‐determining regions as evidence of antigen‐driven selection; this identified three B‐CLL subsets: significantly mutated (sM), with evidence of antigen‐driven selection, not significantly mutated (nsM) and UM, without such evidence and IgVH gene per cent mutations above or below the 2% cut‐off. sM B‐CLL patients had longer survival within the good prognosis subgroup that had more than 2% mutations of IgVH genes. sM, nsM and UM B‐CLL were also characterized for the biased usage of IgVH families, intraclonal IgVH gene diversification, preference of mutations to target‐specific nucleotides or hotspots, and for the expression of enzymes involved in SHM (translesion DNA polymerase ζ and η and activation‐induced cytidine deaminase). These findings indicate the activation of a canonical SHM process in nsM and sM B‐CLLs and underscore the role of the antigen in defining the specific clinical and biological features of B‐CLL.

Genome‐wide DNA analysis identifies recurrent imbalances predicting outcome in chronic lymphocytic leukaemia with 17p deletion

Deletion of 17p (TP53) identifies a rare subset of chronic lymphocytic leukaemia (17p‐ CLL) with aggressive behaviour. Genome‐wide DNA‐profiling was performed to investigate 18 patients with 17p‐ CLL. All cases had multiple copy‐number (CN) changes. Among the several recurrent CN changes identified, 8q24.13‐q24.1‐gain (MYC), 8p‐loss (TNFRSF10A/B, also known as TRAIL1/2) and 2p16.1‐p14‐gain (REL/BCL11A) appeared frequently represented. 8p‐loss and 2p16.1‐p14‐gain also appeared clinically relevant and predicted significant shorter time from diagnosis to treatment (8p‐loss) and overall survival (8p‐loss and 2p16.1‐p14‐gain, P < 0·05). These observations document a highly unstable genome in 17p‐ CLL and suggest that additional genes outside the TP53 locus may be important for tumour behaviour.

The CD49d/CD29 complex is physically and functionally associated with CD38 in B-cell chronic lymphocytic leukemia cells

CD49d and CD38 are independent negative prognostic markers in chronic lymphocytic leukemia (CLL). Their associated expression marks a disease subset with a highly aggressive clinical course. Here, we demonstrate a constitutive physical association between the CD49d/CD29 integrin complex and CD38 in primary CLL cells and B-cell lines by (i) cocapping, (ii) coimmunoprecipitation and (iii) cell adhesion experiments using CD49d-specific substrates (vascular-cell adhesion molecule-1 or CS-1/H89 fibronectin fragments). The role of CD38 in CD49d-mediated cell adhesion was studied in CD49d+CD38+ and CD49d+CD38− primary CLL cells, and confirmed using CD38 transfectants of the originally CD49d+CD38− CLL-derived cell line Mec-1. Results indicate that CD49d+CD38+ cells adhered more efficiently onto CD49d-specific substrates than CD49d+CD38− cells (P<0.001). Upon adhesion, CD49d+CD38+ cells underwent distinctive changes in cell shape and morphology, with higher levels of phosphorylated Vav-1 than CD49d+CD38− cells (P=0.0006) and a more complex distribution of F-actin to the adhesion sites. Lastly, adherent CD49d+CD38+ cells were more resistant to serum-deprivation-induced (P<0.001) and spontaneous (P=0.03) apoptosis than the CD49d+CD38− counterpart. Altogether, our results point to a direct role for CD38 in enhancing CD49d-mediated adhesion processes in CLL, thus providing an explanation for the negative clinical impact exerted by these molecules when coexpressed in neoplastic cells.

CD49d in B-cell chronic lymphocytic leukemia: correlated expression with CD38 and prognostic relevance.

CD49d in B-cell chronic lymphocytic leukemia: correlated expression with CD38 and prognostic relevance

The miR-17 ∼ 92 family regulates the response to Toll-like receptor 9 triggering of CLL cells with unmutated IGHV genes

Chronic lymphocytic leukemia (CLL) cells from clinically aggressive cases have a greater capacity to respond to external microenvironmental stimuli, including those transduced through Toll-like-receptor-9 (TLR9). Concomitant microRNA and gene expression profiling in purified CLL cells (n=17) expressing either unmutated (UM) or mutated (M) IGHV genes selected microRNAs from the miR-17∼92 family as significantly upregulated and in part responsible for modifications in the gene expression profile of UM CLL cells stimulated with the TLR9 agonist CpG. Notably, the stable and sustained upregulation of miR-17∼92 microRNAs by CpG was preceded by a transient induction of the proto-oncogene MYC. The enforced expression of miR-17, a major member from this family, reduced the expression of the tumor suppressor genes E2F5, TP53INP1, TRIM8 and ZBTB4, and protected cells from serum-free-induced apoptosis (P⩽0.05). Consistently, transfection with miR-17∼92 family antagomiRs reduced Bromo-deoxy-uridine incorporation in CpG-stimulated UM CLL cells. Finally, miR-17 expression levels, evaluated in 83 CLL samples, were significantly higher in UM (P=0.03) and ZAP-70high (P=0.02) cases. Altogether, these data reveal a role for microRNAs of the miR-17∼92 family in regulating pro-survival and growth-promoting responses of CLL cells to TLR9 triggering. Overall, targeting of this pathway may represent a novel therapeutic option for management of aggressive CLL.

A scoring system based on the expression of six surface molecules allows the identification of three prognostic risk groups in B‐cell chronic lymphocytic leukemia

We have previously identified 12 surface antigens whose differential expression represented the signature of B‐cell chronic lymphocytic leukemia (B‐CLL) subsets with different prognosis. In the present study, expression data for these antigens, as determined in 137 B‐CLL cases, all with survivals, were utilized to devise a comprehensive immunophenotypic scoring system of prognostic relevance for B‐CLL patients. In particular, univariate z score was employed to identify the markers with greater prognostic impact, while maximally selected log‐rank statistics were chosen to define the optimal cut‐off points capable to split patients into two groups with different survivals. A weighted immunophenotypic scoring system was developed by integrating results from these analyses. Six antigens were selected: three positive prognosticators (CD62L, CD54, CD49c) and three negative prognosticators (CD49d, CD38, CD79b), with cut‐off values ranging from 30% to 50% of positive cells. By weighing the expression of each marker according to its statistical power, a complete scoring system, with point values comprised between 0 (complete absence of phenotypic conditions associated with good prognosis) and 9 (all the phenotypic conditions associated with good prognosis fulfilled), allowed to split the whole set of B‐CLL patients, into three distinctive prognostic groups (P = 4.78 × 10−11) with high‐ (score 0–3), intermediate‐ (score 4–6), and low‐ (score 7–9) risk of death. The three risk groups showed different distribution of cases as for Rais stages, IgVH mutations, and ZAP‐70 expression. The proposed immunophenotypic scoring system may be an additional useful tool in routine diagnostic/prognostic procedures for B‐CLL. J. Cell. Physiol. 207: 354–363, 2006.