Activation of the PI3K/AKT pathway by microRNA-22 results in CLL B-cell proliferation.

Chronic lymphocytic leukemia (CLL) is characterized by accumulation of clonal B cells arrested in G0/G1 stages that coexist, in different proportions, with proliferative B cells. Understanding the crosstalk between the proliferative subsets and their milieu could provide clues on CLL biology. We previously identified one of these subpopulations in the peripheral blood from unmutated patients that appears to be a hallmark of a progressive disease. Aiming to characterize the molecular mechanism underlying this proliferative behavior, we performed gene expression analysis comparing the global mRNA and microRNA expression of this leukemic subpopulation, and compared it with their quiescent counterparts. Our results suggest that proliferation of this fraction depend on microRNA-22 overexpression that induces phosphatase and tensin homolog downregulation and phosphoinositide 3-kinase (PI3K)/AKT pathway activation. Transfection experiments demonstrated that miR-22 overexpression in CLL B cells switches on PI3K/AKT, leading to downregulation of p27(-Kip1) and overexpression of Survivin and Ki-67 proteins. We also demonstrated that this pathway could be triggered by microenvironment signals like CD40 ligand/interleukin-4 and, more importantly, that this regulatory loop is also present in lymph nodes from progressive unmutated patients. Altogether, these results underline the key role of PI3K/AKT pathway in the generation of the CLL proliferative pool and provide additional rationale for the usage of PI3K inhibitors.

"Role of the B-cell receptor and the microenvironment in chronic lymphocytic leukemia''.

Despite significant progress in treatment, chronic lymphocytic leukemia (CLL) remains an incurable disease. Advances have been made to understand the molecular pathogenesis underlying CLL progression and treatment resistance. We here review the available evidences concerning the role of the B-cell receptor (BCR) and the tumor microenvironment interactions in CLL pathogenesis. Antigen likely has a key role in the selection of the tumoral clone, the mutational status of immunoglobulin genes is a strong prognostic predictor and BCR signaling has been postulated to have a role for CLL trafficking and interaction with the stromal microenvironment. There is also important evidence, favoring a role for the microenvironment in CLL pathogenesis. Most, if not all, proliferative events occur in the lymph nodes and bone marrow, where leukemic cells receive through microenvironment interactions survival signals aiming to avoid apoptosis and acquire favorable tumoral growing conditions. In addition, the tumoral microenvironment appears to be the site where the acquisition of additional genetic lesions in the clone occur, which should greatly influence clinical outcome. The advent of new tyrosine kinase inhibitors which seem to be able to modulate microenvironment interactions and circumvent the p53 deletion have generated significant promise by raising the possibility that they could provide significant progress in disease treatment.

Chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia is the commonest form of leukaemia in Europe and North America, and mainly, though not exclusively, affects older individuals. It has a very variable course, with survival ranging from months to decades. Major progress has been made in identification of molecular and cellular markers that could predict disease progression in patients with chronic lymphocytic leukaemia. In particular, the mutational profile of immunoglobulin genes and some cytogenetic abnormalities are important predictors of prognosis. However, these advances have raised new questions about the biology, prognosis, and management of chronic lymphocytic leukaemia, some of which are addressed here. In particular, we discuss how better understanding of the function of the B-cell receptor, the nature of genetic lesions, and the balance between proliferation and apoptosis have affected our ability to assess prognosis and to manage chronic lymphocytic leukaemia. Available treatments generally induce remission, although nearly all patients relapse, and chronic lymphocytic leukaemia remains an incurable disease. Advances in molecular biology have enhanced our understanding of the pathophysiology of the disease and, together with development of new therapeutic agents, have made management of chronic lymphocytic leukaemia more rational and more effective than previously. Unfortunately, we know of no way that chronic lymphocytic leukaemia can be prevented. Early detection is practised widely, but seemingly makes no difference to the patient's eventual outcome.

Small RNAs analysis in CLL reveals a deregulation of miRNA expression and novel miRNA candidates of putative relevance in CLL pathogenesis.

MicroRNAs (miRNAs) are a novel class of small noncoding RNA molecules that regulate gene expression by inducing degradation or translational inhibition of target mRNAs. There are more than 500 miRNA genes reported in the human genome, constituting one of the largest classes of regulatory genes. Increasing experimental evidence supports the idea of aberrant miRNA expression in cancer pathogenesis. We analyzed the pattern of miRNA expression in chronic lymphocytic leukemia (CLL) cells and our results showed a global reduction in miRNA expression levels in CLL cells associated to a consistent underexpression of miR-181a, let-7a and miR-30d. We observed overexpression of miR-155 and a set of five miRNAs that are differentially expressed between patients with different clinical outcomes. Five novel miRNA candidates cloned from leukemic cells are reported. Surprisingly, predicted mRNA targets for these novel miRNA revealed a high proportion of targets located in a small region of chromosome 1, which is frequently altered in human cancer. Additionally, several targets were shared by at least two of miRNA candidates. Predicted targets included several genes recently described as tumor suppressors. These data could afford new avenues for exploring innovative pathways in CLL biology and therapy.

What do somatic hypermutation and class switch recombination teach us about chronic lymphocytic leukaemia pathogenesis?

B-CLL cells express CD5 and IgM/IgD and thus have a mantle zone-like phenotype of naive cells, which, in normal conditions express unmutated Ig genes. However, recent studies have shown that 50%-70% of CLL harbour somatic mutations of VH genes, as if they had matured in a lymphoid follicle. Interestingly, the presence or absence of somatic hypermutation (SHM) process is associated with the use of particular VH genes. Particular alleles of the VH1-69 gene and the VH4-39 gene are preferentially expressed in an unmutated form, while VH4-34 or the majority of VH3 family genes frequently contain somatic mutations. The fact that some genes like VH1-69 and VH3-07 recombine this VH segment to particular JH segments and the restricted use of CDR3 sequences by CLLs expressing the VH4-39 gene suggest that the observed differences in BCR structure in B-CLL could result from selection by distinct antigenic epitopes. It is currently unclear whether this putative antigen-driven process could occur prior to leukaemic transformation and/or that the precursors were transformed into leukaemic cells at distinct maturational stages. The mutational profile of Ig genes has been shown to be associated with disease prognosis. These results could favour the idea that CLL could correspond to two different diseases that look alike in morphologic and phenotypic terms. In CLL with mutated Ig genes, the proliferating B cell may have transited through germinal centres, the physiologic site of hypermutation, whereas in CLL with unmutated Ig genes the malignant B cell may derive from a pre-germinal centre naïve B cell. Despite these clinical and molecular differences, recent studies on gene expression profiling of B-CLL cells showed that CLL is characterized by a common gene expression signature that is irrespective of Ig mutational status and differs from other lymphoid cancers and normal lymphoid subpopulations, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Activation induced cytidine deaminase (AID) plays a key role in SHM and class switch recombination (CSR). However, the mechanisms accounting for AID action and control of its expression remain unclear. In a recent work we have shown that in contrast to normal circulating B-cells, AID transcripts are expressed constitutively in CLL patients undergoing active CSR, but interestingly this expression occurs predominately in unmutated CLL B-cells. These data favour the view that AID protein may act differentially on CSR and SHM pathways, but the role-played by AID in both processes remains to be elucidated. Recent work indicates that AID is expressed in a small fraction of tumoral cells, which could suggest that this small fraction of cells may correspond to B-CLL cells that would have recently experienced an AID-inducing stimulus occurring in a specific microenvironment.

hTERT expression and prognosis in B-chronic lymphocytic leukemia.

BACKGROUND: In B-chronic lymphocytic leukemia (B-CLL), there is a need for molecular markers to predict the evolution of this heterogeneous disease in individual patients. The level of expression of the human telomerase reverse transcriptase (hTERT) gene has been associated with disease aggressiveness in human cancers. The purpose of the present study was to examine the prognostic significance of hTERT expression in B-CLL. PATIENTS AND METHODS: We used real-time reverse transcription-PCR to quantitate the amount of hTERT transcripts in mononuclear blood cells from 90 B-CLL patients. In addition, samples were analyzed for somatic mutations in the immunoglobulin V (IgV) genes. RESULTS: The expression of hTERT gene was detected in 59% of patients. The level of expression increased with advancing B-CLL stage (P=0.0064). Patients expressing hTERT showed significantly shorter survival than hTERT-negative patients (P=0.000034), irrespective of the disease stage. On average, the level hTERT mRNA expression was seven-fold higher in the poor-prognosis B-CLL group with unmutated IgV than in the Ig-mutated group (P<10(-7)). The level of hTERT expression discriminated the Ig-unmutated from Ig-mutated B-CLL in 89% of cases. CONCLUSION: Our data indicate that hTERT expression in B-CLL may serve as a molecular prognostic marker.

Post-transcriptional regulation of inducible nitric oxide synthase in chronic lymphocytic leukemia B cells in pro- and antiapoptotic culture conditions.

Functional inducible NOS (iNOS) may be involved in the prolonged lifespan of chronic lymphocytic leukemia cells (B-CLL), although the exact mechanisms implicated remain elusive as yet. In this work, we have examined iNOS expression in normal B lymphocytes and B-CLL cells in pro- and antiapoptotic conditions. Our results demonstrate: (1) The existence of a new splice variant characterized by a complete deletion of exon 14 (iNOS 13-16(14del)), which was preferentially detected in normal B lymphocytes and may represent an isoform that could play a role in the regulation of enzyme activity. (2) The existence of another alternatively spliced iNOS mRNA transcript involving a partial deletion of the flavodoxin region (iNOS 13-16(neg)) was correlated to a decreased B-CLL cell viability. The 9-beta-D-arabinofuranosyl-2-fluoradenine or fludarabine (F-ara) treatment induced iNOS 13-16(neg) transcript variants, whereas IL-4 enhanced both the transcription of variants, including these exons (iNOS 13-16(pos)), and the expression of a 122 kDa iNOS protein. These results suggest that in B-CLL, a regulation process involving nitric oxide (.- NO) levels could occur by a post-transcriptional mechanism mediated by soluble factors. Our results also provide an insight into a new complementary proapoptotic action of F-ara in B-CLL by the induction of particular iNOS splice variants, leading to the activation of a caspase-3-dependent apoptotic pathway.

Predictive value of serum thymidine kinase level for Ig-V mutational status in B-CLL.

In B-CLL IgV(H) genes mutational status is a major prognostic factor. Since sequencing of IgV(H) genes is not available in most laboratories, an easily performed surrogate assay is desirable. To identify the best surrogate assay, and to better discriminate prognostic subgroups we analyzed clinical and biological data from 58 typical CLL cases. A higher serum thymidine kinase level (>15 U/l) proved to be a strong predictor of mutational status, and the only independent one among the studied parameters. To further identify prognostic subgroups, cluster analysis was employed on 38 cases on which all data were available, which segregated two groups including 25 and 13 patients, respectively. These two clusters differed by their proliferative potential and appeared to discriminate patients with very different clinical course and outcome. s-TK was strikingly different among these two clusters, suggesting that s-TK level could be used routinely to identify patients at risk of progression.

Do CLL B cells correspond to naive or memory B-lymphocytes? Evidence for an active Ig switch unrelated to phenotype expression and Ig mutational pattern in B-CLL cells.

Recent work suggests that chronic lymphocytic leukemia (B-CLL) expressing unmutated immunoglobulin V genes could correspond to the proliferation of naive B cells whereas those expressing mutated genes, may correspond to the proliferation of post-germinal center B cells. Current data from gene profiling expression have failed to demonstrate a clear-cut distinction between these two forms of B-CLL disease. In the present study, we have investigated the complete V(H) nucleotide sequence and the presence of RNA transcripts from different C(H) domains in 25 B-CLL patients. Our results demonstrate that: (1) expression of IgD is not related to the mutational frequency and activation of the isotype switch pathway; (2) isotype switch, leading to simultaneous expression at the transcriptional and protein level of IgM, IgD, IgG and IgA, occurs in a small percentage of patients, and (3) different mechanisms such as VDJ duplication and trans-splicing or RNA splicing of long nuclear transcript, could be involved in isotype switch. Our results highlight the difficulty in assigning a normal counterpart to B-CLL cells and raise the possibility that a different B cell development pathway, independent from classical germinal centers, might exist in B-CLL.

Randomized comparison of fludarabine, CAP, and ChOP in 938 previously untreated stage B and C chronic lymphocytic leukemia patients.

To comparatively assess first-line treatment with fludarabine and 2 anthracycline-containing regimens, namely CAP (cyclophosphamide, doxorubicin plus prednisone) and ChOP (cyclophosphamide, vincristine, prednisone plus doxorubicin), in advanced stages of chronic lymphocytic leukemia (CLL), previously untreated patients with stage B or C CLL were randomly allocated to receive 6 monthly courses of either ChOP, CAP, or fludarabine (FAMP), stratified based on the Binet stages. End points were overall survival, treatment response, and tolerance. From June 1, 1990 to April 15, 1998, 938 patients (651 stage B and 287 stage C) were randomized in 73 centers. Compared to ChOP and FAMP, CAP induced lower overall remission rates (58.2%; ChOP, 71.5%; FAMP; 71.1%; P <.0001 for each), including lower clinical remission rates (CAP, 15.2%; ChOP, 29.6%; FAMP, 40.1%; P =.003). By contrast, median survival time did not differ significantly according to randomization (67, 70, and 69 months in the ChOP, CAP, and FAMP groups, respectively). Incidences of infections (< 5%) and autoimmune hemolytic anemia (< 2%) during the 6 courses were similar in the randomized groups, whereas fludarabine induced, compared to ChOP and CAP, more frequent protracted thrombocytopenia (P =.003) and less frequent nausea-vomiting (P =.003) and hair loss (P <.0001). For patients with stage B and C CLL first-line fludarabine and ChOP regimens both provided similar overall survival and close response rates, and better results than CAP. However, there was an increase in clinical remission rate and a trend toward a better tolerance of fludarabine over ChOP that may influence the choice between these regimens as front-line treatments in patients with CLL.

Molecular characterization of a monoclonal murine anti-blood group A antibody.

A mouse monoclonal anti-human blood group A antigen (AC12, mu, kappa) has been generated and sequenced in order to analyze the immunoglobulin genes used to generate antibodies with anti-human blood group A specificity. Mice were immunized with human type A RBC. Anti-A producing hybridomas were detected by agglutination against human type A RBC. Total cellular RNA was extracted from hybridomas cells. PCR amplification and sequencing of anti-A heavy and light chain cDNAs were performed. The VH and VK sequences of antibody AC12 were shown to be very homologous to that used by other antibodies recognizing carbohydrates as well as glycoproteins, peptides or haptens constituting self antigens as well as nonself antigens. The VH sequence of antibody AC12 presented important homology with a previously reported monoclonal anti-blood group B antibody. The antibody AC12 also presented homology with the VH and VK sequences of a previously reported human anti-blood group A antibody which contributes additional evidence in favor of a restricted usage of V segments by antibodies directed against red blood antigens.

Functional monocyte-derived dendritic cells can be generated in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) remains an incurable disease. Although modern available treatments are able to induce disease regression, relapse almost inexorably occurs. Therefore, novel therapeutic strategies aimed at reducing the disease relapse rate are very much needed. Among these, the induction of tumour-associated antigen-specific cytotoxic T lymphocytes (CTL), through either DNA vaccines or injection of idiotype pulsed dendritic cells (DCs), has been actively investigated with encouraging preliminary results in B-cell malignancies. As the CLL B lymphocyte characteristically expresses low amounts of surface immunoglobulin (Ig) and T cells from these patients have been reported to display impaired functional activity, there are concerns related to the possibility of generating specific cytotoxic antitumoral T cells in this disease. In addition, no information is presently available regarding the functional ability of CLL-derived DCs. In the present work, freshly purified monocytes from CLL patients and normal donors were induced to differentiate in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 serum-free medium and compared for their morphological, phenotypic and functional characteristics. Our results demonstrate that: (1) functional DCs can be generated from CLL patients with similar phenotype and function to those observed from normal donors; (2) in contrast to normal control subjects, monocyte-derived DCs from CLL patients spontaneously secrete endogenous IL-10; and (3) interferon (IFN)-gamma in combination with CD40L plays a major role in priming DCs from CLL patients for IL-12 and IL-15 production. Overall, these results indicate that it is possible to derive functionally competent DCs from circulating monocytes in CLL patients.