Predicting survival in chronic lymphocytic leukemia

There is increasing interest in the use of prognostic markers that may predict survival and guide management in patients diagnosed with the early stages of chronic lymphocytic leukemia (CLL). Currently, the most important traditional prognostic factors include clinical staging, lymphocyte doubling time and β2-microglobulin/thymidine kinase; and the most important novel markers include karyotypic aberrations (typically assessed by FISH probes or CpG oligonucleotide karyotyping) and IgVH mutation status. Although each of these factors have individually shown significant correlations with survival, there is increasing appreciation that the most complete information may be obtained by using a combination of several factors in prognostic normograms or models. In this article, we review the current state-of-the-art with regards to CLL prognostic factors and discuss how they can be applied in the clinic.     

CXCL12-induced VLA-4 activation is impaired in trisomy 12 chronic lymphocytic leukemia cells: a role for CCL21

Homing to distinct lymphoid organs enables chronic lymphocytic leukemia (CLL) cells to receive pro-survival and proliferative signals. Cytogenetic aberrations can significantly affect CLL cell compartmentalization. Trisomy 12 (tri12) defines a CLL subgroup with specific clinical features and increased levels of the negative prognostic marker CD49d, the α4-subunit of the integrin VLA-4, which is a key regulator of CLL cell homing to bone marrow (BM). Chemokine-induced inside-out VLA-4 activation, particularly via the CXCL12-CXCR4 axis, increases the arrest of various cell types on VCAM-1 presenting endothelium. Here, we demonstrate that high CD49d expression in tri12 CLL is accompanied by decreased CXCR4 expression. Dissecting functional consequences of these alterations, we observed that tri12 CLL cell homing to murine BM is not affected by CXCR4-CXCL12 blockage using AMD3100 or olaptesed pegol/NOX-A12. In line, CCL21-CCR7 rather than CXCL12-CXCR4 interactions triggered VLA-4-mediated arrests of tri12 CLL cells to VCAM-1 under blood flow conditions. Concordantly, in real-time kinetic analyses we found CCL21 but not CXCL12 being capable to induce inside-out VLA-4 conformational changes in this CLL subgroup. Our results provide novel insights into the peculiar clinico-biological behaviour of tri12 CLL and emphasize its specific chemokine and integrin utilization during pathophysiologically and therapeutically relevant interactions with the microenvironment.     

ID helix-loop-helix proteins as determinants of cell survival in B-cell chronic lymphocytic leukemia cells in vitro

Background

Members of the inhibitor of DNA-binding (ID) family of helix-loop-helix proteins have been causally implicated in the pathogenesis of several types of B-cell lineage malignancy, either on the basis of mutation or by altered expression. B-cell chronic lymphocytic leukemia encompasses a heterogeneous group of disorders and is the commonest leukaemia type in the Western world. In this study, we have investigated the pathobiological functions of the ID2 and ID3 proteins in this disease with an emphasis on their role in regulating leukemic cell death/survival.

Methods

Bioinformatics analysis of microarray gene expression data was used to investigate expression of ID2/ID3 in leukemic versus normal B cells, their association with clinical course of disease and molecular sub-type and to reconstruct a gene regulatory network using the ‘maximum information coefficient’ (MIC) for target gene inference. In vitro cultured primary leukemia cells, either in isolation or co-cultured with accessory vascular endothelial cells, were used to investigate ID2/ID3 protein expression by western blotting and to assess the cytotoxic response of different drugs (fludarabine, chlorambucil, ethacrynic acid) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. ID2/ID3 protein levels in primary leukemia cells and in MEC1 cells were manipulated by transduction with siRNA reagents.

Results

Datamining showed that the expression profiles of ID2 and ID3 are associated with distinct pathobiological features of disease and implicated both genes in regulating cell death/survival by targeting multiple non-overlapping sets of apoptosis effecter genes. Consistent with microarray data, the overall pattern of ID2/ID3 protein expression in relation to cell death/survival responses of primary leukemia cells was suggestive of a pro-survival function for both ID proteins. This was confirmed by siRNA knock-down experiments in MEC1 cells and in primary leukemia cells, but with variability in the dependence of leukemic cells from different patients on ID protein expression for cell survival. Vascular endothelial cells rescued leukemia cells from spontaneous and cytotoxic drug-induced cell death at least in part, via an ID protein-coupled redox-dependent mechanism.

Conclusions

Our study provides evidence for a pro-survival function of the ID2/ID3 proteins in chronic lymphocytic leukemia cells and also highlights these proteins as potential determinants of the pathobiology of this disorder.

Electronic supplementary material

The online version of this article (doi:10.1186/s12943-014-0286-9) contains supplementary material, which is available to authorized users.     

Monocyte-derived dendritic cells in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) results from the accumulation of small mature, slowly dividing, monoclonal B lymphocytes. The clinical course of this disease is heterogeneous, with some patients progressing rapidly with early death whilst others exhibit a more stable, possibly, non-progressing disease lasting many years. Despite progress in therapy, relapse invariably occurs and the disease remains incurable. The clinical management of CLL is therefore challenging and considerable effort has been directed towards novel therapeutic strategies aimed at reducing the disease relapse rate. Recent insights into the role of dendritic cells as the pivotal antigen-presenting cells that initiate immune responses may provide the basis for generating more effective antitumor immune responses. Consequently, dendritic cells constitute an attractive approach in the context of CLL. However, understanding the relation between dendritic cells and the cellular immune response is crucial to elucidation of how to manipulate immune responses. After summarizing general properties of dendritic cells, this review focus on the approaches exploiting monocyte-derived dendritic cells in CLL, which should help design of novel treatment strategies in this disease.     

JAK2 tyrosine kinase mediates integrin activation induced by CXCL12 in B-cell chronic lymphocytic leukemia

Chemokines participate to B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis by promoting cell adhesion and survival in bone marrow stromal niches and mediating cell dissemination to secondary lymphoid organs. In this study we investigated the role of JAK protein tyrosine kinases (PTK) in adhesion triggering by the CXC chemokine CXCL12 in normal versus CLL B-lymphocytes. We demonstrate that CXCL12 activates JAK2 in normal as well as CLL B-lymphocytes, with kinetics consistent with rapid adhesion triggering. By using complementary methodologies of signal transduction interference, we found that JAK2 mediates CXCL12-triggered activation of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. We also show that JAK2 mediates the activation of the small GTP-binding protein RhoA, in turn controlling LFA-1 affinity triggering by CXCL12. Importantly, comparative analysis of 41 B-CLL patients did not evidence JAK2 functional variability between subjects, thus suggesting that JAK2, differently from other signaling events involved in adhesion regulation in B-CLL, is a signaling molecule downstream to CXCR4 characterized by a conserved regulatory role. Our results reveal JAK2 as critical component of chemokine signaling in CLL B-lymphocytes and indicate JAK inhibition as a potentially useful new pharmacological approach to B-CLL treatment.     

Bcl-2 and apoptosis in chronic lymphocytic leukemia

Opinion statement The Bcl-2 family of pro- and antiapoptotic proteins are key regulators of the apoptosis cascade and the mitochondrial-mediated pathway of caspase activation. Of this family, Bcl-2 was the first identified and remains the best characterized. Aberrant expression of Bcl-2 is common in chronic lymphocytic leukemia (CLL) and is associated with poor response to chemotherapy and decreased overall survival. Bcl-2 is an attractive target for novel therapeutic agents. Antisense oligonucleotides directed against Bcl-2 are effective in vitro and are being evaluated in clinical trials in CLL. Small molecule Bcl-2 inhibitors are in preclinical development and should be ready for clinical evaluation in the next few years. Strategies that induce apoptosis and bypass Bcl-2 may also be therapeutically useful in CLL. Thus, over the next decade, one can envision incorporating measurements of apoptotic proteins such as Bcl-2 into the risk assessment and treatment algorithms for individual patients. In addition, we anticipate that in the next decade, rationally designed therapies targeting specific molecular defects in the malignant CLL lymphocytes will be introduced into the clinic.     

Rapid and efficient nonviral gene delivery of CD154 to primary chronic lymphocytic leukemia cells

Interactions between CD40 and CD40 ligand (CD154) are essential in the regulation of both humoral and cellular immune responses. Forced expression of human CD154 in B chronic lymphocytic leukemia (B-CLL) cells can upregulate costimulatory and adhesion molecules and restore antigen-presenting capacity. Unfortunately, B-CLL cells are resistant to direct gene manipulation with most currently available gene transfer systems. In this report, we describe the use of a nonviral, clinical-grade, electroporation-based gene delivery system and a standard plasmid carrying CD154 cDNA, which achieved efficient (64+/-15%) and rapid (within 3 h) transfection of primary B-CLL cells. Consistent results were obtained from multiple human donors. Transfection of CD154 was functional in that it led to upregulated expression of CD80, CD86, ICAM-I and MHC class II (HLA-DR) on the B-CLL cells and induction of allogeneic immune responses in MLR assays. Furthermore, sustained transgene expression was demonstrated in long-term cryopreserved transfected cells. This simple and rapid gene delivery technology has been validated under the current Good Manufacturing Practice conditions, and multiple doses of CD154-expressing cells were prepared for CLL patients from one DNA transfection. Vaccination strategies using autologous tumor cells manipulated ex vivo for patients with B-CLL and perhaps with other hematopoietic malignancies could be practically implemented using this rapid and efficient nonviral gene delivery system.     

Immunologic characterization of chronic lymphocytic leukemia cells.

Immunologic characterization of the neoplastic cells in the circulation of patients with CLL suggests these cells show significant differences in membrane characteristics from normal B lymphocytes. Although the leukemic cells bear a homogenous membrane-associated immunoglobulin, they also react with an anti-human T cell serum. In all patients studied, 60-90% of the cells, were stained by this antiserum. This suggests that the leukemic cells share antigenic determinants with T lymphocytes. CLL cells, unlike normal B cells, showed a marked increase in mouse-complement receptors. No increase in receptors for guinea pig complement was observed in the leukemic cells. The population of SIg-bearing lymphocytes was significantly greater than that of complement-receptor bearing lymphocytes. The total number of E-rosetting cells was increased in all CLL patients. Mitogenic responses of the leukemic cells were depressed and delayed. These results suggest that neoplastic lymphocytes cannot be classified as T- or B-derived on the basis of criteria used to define normal lymphocytes.     

Diagnostic features and survival in typical and prolymphocytoid variants of chronic lymphocytic leukemia

Diagnostic features were evaluated with regards to survival in 203 cases of typical and prolymphocytoid chronic lymphocytic leukemia. Excluding 27 (13 per cent) patients with second malignancies, survival analyses indicated that the prognostic factors with greatest significance were age at presentation (less than 65 years and 65 years or more: p = 0.0004), proportions of prolymphocytoid cells (less than 10 per cent and 10 per cent or more: p less than 0.0001 age corrected) and surface immunoglobulin (SIg) density (weak and more than weak: p = 0.001 age corrected). In contrast, sex, absolute numbers of prolymphocytoid cells, SIg light chain type and FMC7 expression were not prognostically significant. These observations suggest that the recognition and delineation of prolymphocytoid CLL variants by morphological and immunophenotypic criteria, although important in diagnostic terms, may have less relevance with respect to prognosis and patient management.     

Altered membrane-associated functions in chronic lymphocytic leukemia cells.

Peripheral blood lymphocytes consisting mainly of neoplastic B cells from patients with chronic lymphocytic leukemia (CLL cells) showed a markedly reduced response to the human B-cell mitogens anti-beta2 microglobulin, Sepharose-bound protein A and Sepharose-bound anti-human immunoglobulin (anti F(ab')2) in all of nine patients studied. On the other hand, CLL cells from three out of eight patients tested responded well to the calcium ionophore A23187. Sepharose-bound protein A and anti-beta2 microglobulin also failed to induce increased uptake of 86Rubidium (potassium analogue) in CLL cells as compared to B-cell-enriched preparations of normal peripheral blood lymphocytes. The capacity of CLL cells to cap various surface markers including beta2 microglobulin was reduced. On the other hand, surface concentrations of beta2 microglobulin were not reduced as measured by fluorescein-labelled anti-beta2-microglobulin in single-cell cytofluorometry. It is concluded that various membrane-associated events elicited by ligand-receptor interactions are altered or blocked in CLL cells.     

Relationship between progression-free survival and overall survival in chronic lymphocytic leukemia: a literature-based analysis

Background

The endpoints of progression-free survival (pfs) and time-to-progression (ttp) are frequently used to evaluate the clinical benefit of anticancer drugs. However, the surrogacy of those endpoints for overall survival (os) is not validated in all cancer settings. In the present study, we used a trial-based approach to assess the relationship between median pfs or ttp and median os in chronic lymphocytic leukemia (cll).

Methods

The pico (population, interventions, comparators, outcomes) method was used to conduct a systematic review of the literature. The population consisted of patients with cll; the interventions and comparators were standard therapies for cll; and the outcomes were median pfs, ttp, and os. Two independent reviewers screened titles, abstracts, and full papers for eligibility and then extracted data from selected studies. Correlation coefficients were calculated to assess the relationship between median pfs or ttp and median os. Subgroup correlation analyses were also conducted according to the characteristics of the selected studies (such as line of treatment and type of treatment under investigation).

Results

Of the 1263 potentially relevant articles identified during the literature search, twenty-three were included. On average, median pfs or ttp was 16.0 months (standard deviation: 12.4 months) and median os was 43.5 months (standard deviation: 31.2 months). Results of the correlation analysis indicated that median pfs or ttp is highly correlated with median os (Spearman correlation coefficient: 0.813; p ≤ 0.001). A significant correlation between median pfs or ttp and median os was observed in second- and subsequent-line therapies, but not in the first-line setting.

Conclusions

Our study demonstrates a strong correlation between median pfs or ttp and median os in previously treated cll, which reinforce the hypothesis that pfs and ttp could be adequate surrogate endpoints for os in this cancer setting.     

Coexistence of chronic myelogenous leukemia and chronic lymphocytic leukemia

A 55-year-old man is reported who initially developed chronic lymphocytic leukemia. Seven years later, after chemotherapy with chlorambucil, chronic myelogenous leukemia was diagnosed in addition to the chronic lymphocytic leukemia. Four previously reported cases with the same sequence of events are reviewed as well as cases of chronic myelogenous leukemia following chemotherapy alone.     

Colony-forming assay for circulating chronic lymphocytic leukemia cells

In these studies, we report adaptation of a colony-forming assay to chronic lymphocytic leukemia (CLL) peripheral blood cells. T-lymphocyte-depleted CLL peripheral blood cells were cultured with irradiated, normal T cells and media conditioned by normal, mitogen-stimulated T cells in methylcellulose. Colonies containing small and transformed lymphocytes appeared after 5–7 days incubation. The plating efficiency of CLL colonies was 0.15 ± 0.08% ($\text{x}\text{±}\text{S.D.}$), similar to that of other colony-forming systems. The majority of CLL colony-forming cells were in S phase (50 ± 4%, $\text{x}\text{±}\text{S.E.}$) as determined by thymidine suicide and the fraction of colony-forming cells in S phase was inversely related to the WBC. Cells harvested from CLL colonies lacked surface markers for T lymphocytes and stained positively for monoclonal surface and/or cytoplasmic immunoglobulin light chains. A 1-h incubation was used to study the in vitro response of CLL colony-forming cells to adriamycin and melphalan. Preliminary studies suggest differences in patterns of in vitro sensitivity to melphalan between patients previously treated with alkylating agents and those who had not received treatment. This system can be used to study regulation of CLL cell proliferation, and may have utility in predicting response to chemotherapeutic agents.     

Exosomal NAMPT from chronic lymphocytic leukemia cells orchestrate monocyte survival and phenotype under endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress has been reported to be transmitted from tumor cells to immune cells via exosome and implicated in immune escape. However, the influence of ER stress on monocytes in chronic lymphocytic leukemia (CLL) cells is largely unknown. Here, we observed the expression of ER stress markers (GRP78, ATF6, PERK, IRE1a, and XBP1s) in CLL cells. The increasing mRNA expression of these ER stress response components was positively correlated with more aggressive disease. Exosome from ER stress inducer tunicamycin (TM)-primed CLL cells (ERS-exo) up-regulated the expression of ER stress marker on monocytes, indicating ER stress is transmissible in vitro via exosome. Treatment with ERS-exo promoted the survival of monocytes and induced phenotypic changes with a significantly larger percentage of CD14⁺CD16⁺monocytes. Finally, we identified exosome-mediated transfer of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) from ER stressed CLL cells into monocytes as a novel mechanism through which ERS-exo regulated monocytes. Exosomal eNAMPT up-regulated nicotinamide adenine dinucleotide (NAD⁺) production which subsequently activated SIRT1-C/EBPβ signaling pathway in monocytes. Our results suggest the role of ER stress in mediating immunological dysfunction in CLL.     

Differential expression of survivin in bone marrow cells from patients with acute lymphocytic leukemia and chronic lymphocytic leukemia

Survivin, a member of the inhibitor of apoptosis protein (IAP) gene family, has been detected widely in fetal tissue and in a variety of human malignancies. In the current study, we investigated the expression of IAP family proteins in bone marrow samples from acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and control cases by quantitative real-time RT-PCR method and an immunohistochemical approach. Overexpression of survivin and cIAP2 mRNA was significant in CLL bone marrow cells (P < 0.05, respectively) compared with control samples. By immunohistochemistry, survivin was detected in a few scattered myeloid cells in all cases of control bone marrow. Concerning the ALL bone marrow, more than half the cases demonstrated positive expression of survivin (8 out of 13), while the majority of CLL cases (20 out of 21) exhibited intense expression of survivin. The differential subcellular localization of survivin was distinct between ALL and CLL cases. ALL cells essentially revealed nuclear localization of survivin as well as cytoplasmic signals in some cases, while CLL cells from the majority of cases predominantly showed cytoplasmic expression. Next, RT-PCR was performed for the expression of survivin and its splicing variant, survivin-2B and survivin-deltaEx3 in ALL and CLL cells, as the distribution of these variants would be regulated by nuclear/cytoplasmic transport system. In both ALL and CLL bone marrow samples, the expression of wild-type survivin was more predominant than that of survivin-2B or survivin-deltaEx3, although the expression of survivin-deltaEx3 was prominent in samples from survivin-expressing ALL cases. Thus, the splicing of survivin mRNA may be differently regulated in ALL and CLL cells, causing distinct manners of nuclear/cytoplasmic transport of survivin protein. In conclusion, our observations indicate a differential regulatory mechanism for the expression of IAP family proteins in ALL and CLL cells, although the functions of IAP families and the mechanisms of nuclear/cytoplasmic transport of survivin should be clarified in future studies.     

Deoxynucleoside anabolic enzyme levels in acute myelocytic leukemia and chronic lymphocytic leukemia cells

The deoxynucleoside kinase reaction is often rate-limiting in the anabolism of pharmacologically active anti-cancer nucleosides. The levels of thymidine kinase (TK), deoxycytidine kinase, deoxyguanosine kinase (dGK), and thymidylate kinase were determined in leukocyte extracts from patients with chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML). The extracts from AML patients showed significantly higher TK activity than the ones from CLL patients. There were no differences in the levels of the other three kinases. In the case of dGK, the determinations were carried out with both an immunoblotting assay and selective enzyme activity measurements.     

Epigenetics in chronic lymphocytic leukemia

Enormous evidence has accumulated in the past decades that establishes the importance of epigenetic modifications in cancer and has resulted in shifting the focus from entirely genetic-based studies to integrated studies involving both genetic and epigenetic alterations. Chronic lymphocytic leukemia (CLL) is one such example where studies involving epigenetic aberrations have accelerated the search for affected genes, which was initially restricted to commonly deleted chromosomal regions. Many novel genes that are epigenetically silenced in CLL have been identified. Advances in the understanding of post-translational histone modifications and DNA methylation in normal and in CLL cells have proven to be extremely beneficial in finding powerful diagnostic markers, as well as in exploring novel therapies. At present, the field of epigenetics is at an evolving stage, but there is no doubt that further unraveling of its cause and effects in transformed cells will bring a new revolution in cancer therapeutics.