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The pathogenic relevance of the prognostic markers CD38 and CD49d in chronic lymphocytic leukemia

The pathogenic relevance of the prognostic markers CD38 and CD49d in chronic lymphocytic leukemia Ann Hematol (2014) 93:361–374 DOI 10.1007/s00277-013-1967-y REVIEW ARTICLE The pathogenic relevance of the prognostic markers CD38 and CD49d in chronic lymphocytic leukemia Gabriele Brachtl & Josefina Piñón Hofbauer & Richard Greil & Tanja Nicole Hartmann Received: 26 July 2013 /Accepted: 13 November 2013 /Published online: 28 November 2013 Springer-Verlag Berlin Heidelberg 2013 Abstract The interactions of chronic lymphocytic leukemia clinical course a patient will experience. Several more recently cells with the microenvironment in secondary lymphoid tis- suggested prognostic markers, apparently involved in the sues and the bone marrow are known to promote CLL cell cellular processes underlying CLL pathogenesis, may aid to survival and proliferation. CD38 and CD49d are both inde- classify patients according to clinical risk. These markers pendent prognostic risk parameters in CLL with important include chromosomal aberrations such as deletion of 17p13, roles in shaping these interactions. Both are reported to influ- 11q22-23, or 13q1, and trisomy 12 [4] that directly influence ence CLL cell trafficking between blood and lymphoid organs cell fate or transformation, as well as molecular markers for as well as their survival and proliferation within the lymphoid CLL cell interactions with the tumor microenvironment [5]. organs, thereby impacting the pathophysiology of the disease. Among the molecular prognostic factors, the mutational status The expression of CD38 and CD49d is associated in the of immunoglobulin variable region (IGHV) genes [6, 7], the majority of cases, and they exist as part of macromolecular expression of CD38 on the surface of CLL cells [6], and the complexes. Here, we review the current evidence for the intracellular expression of zeta-associated protein 70 [8, 9]are individual and associated contributions of these molecules to the best-established ones. A more recently discovered marker CLL pathophysiology. is CD49d, the alpha4 subunit of the VLA-4 integrin (alpha4beta1). High CD49d expression predicts reduced over- all survival and time to first treatment in CLL patients [10, 11]. Keywords Chronic lymphocytic leukemia . . . The pathogenic significance of CLL cell interactions with Microenvironment CD38 CD49d VLA-4 the lymphoid microenvironment has become increasingly ac- knowledged in recent years. CLL cell proliferation is sup- posed to take place in lymph nodes and, to a lesser extent in The heterogeneity of CLL in regard to the tumor bone marrow, with up to 2 % of the entire clone being newly microenvironment generated per day [12], and is most likely supported by activated T lymphocytes that express CD40 ligand [13–15]. Chronic lymphocytic leukemia (CLL), a B cell non- Signals from T lymphocytes and from other accessory cells in Hodgkin’s lymphoma with a leukemic appearance, is a re- this environment, such as stromal or nurse-like cells, also markably heterogeneous disease that can follow a wide variety provide pro-survival support to the malignant cells [16–19]. of courses. Patients with an indolent course survive for many Not only does the microenvironment influence CLL cells, but years. Others, however, show a rapidly fatal disease despite CLL cells alter their microenvironment to their advantage by aggressive therapy and die within 2–3 years of diagnosis priming T cells towards an immune suppressive phenotype (reviewed in [1]). Mere staging by Rai [2] or Binet [3]is not [20] or inducing stromal cells to provide pro-survival signals sufficient to predict at an early stage of the disease which [21–24], which contributes to chemoresistance and treatment : : : failure. Minimal residual disease after therapy is attributed to G. Brachtl J. Piñón Hofbauer R. Greil T. N. Hartmann (*) Laboratory for Immunological and Molecular Cancer Research, supportive microenvironmental signals and prognostically as- Third Medical Department with Hematology, Oncology, sociated with shortened progression-free and overall survival Hemostaseology, Infectiology and Rheumatology, Paracelsus rates of CLL patients [25–27]. Eradicating residual CLL cells Medical University, Müllner Haupstraße 48, 5020 Salzburg, Austria within their protective niches in secondary lymphoid tissues e-mail: t.hartmann@salk.at 362 Ann Hematol (2014) 93:361–374 and bone marrow is thus considered a major therapeutic goal receptor vary greatly and depend on its association with other for achieving permanent remission. surface molecules. CD38 is laterally associated with the main As dissected in the following chapters, the prognostic signaling complexes of lymphocytes that are organized in markers CD49d and CD38 have been reported to be involved lipid rafts. In T cells, CD38 is capable of interacting with the in various cellular functions relevant to CLL pathogenesis: T cell receptor (TCR)/CD3 complex; in B cells, with the BCR/ CLL cell homing to lymphoid organs, survival, and prolifer- CD19 complex; and in NK cells, with the CD16/CD81 com- ation. However, CD49d and CD38 expression is associated in plex [31]. CD31 (PECAM-1), expressed by, e.g., endothelial about 80 % of CLL patients, and these molecules are reported cells has been described as a CD38 ligand [48]. CD38+ to physically interact within multi-protein complexes. Be- lymphocytes show a weak, selectin-like adhesion to endothe- cause of this, it is difficult to gauge the individual contribution lial cells [49], which appears to be mediated by CD38–CD31 of each molecule to key pathogenic functions in CLL. In light interactions [48]. of the high heterogeneity of reports dealing with either mole- In B cells, ligation of CD38 by agonistic antibodies triggers cule and the fact that CD49d is still a newcomer among the different in vitro responses depending on the differentiation prognostic factors, we review the current evidence for the stage of the cells. In immature B cell precursors, CD38 liga- individual and associated contributions of these molecules to tion inhibited DNA synthesis and induced apoptosis, thereby CLL pathophysiology. blocking B cell hematopoiesis [50]. In tonsillar germinal center B cells, CD38 mediated pro-survival signaling [51]. In mature circulating B cells, CD38 ligation induced prolifer- CD38 and VLA-4 in general ation by promoting the expression of CD25, MHC-II, and certain cytokines [31, 52]. CD38 In summary, CD38 is a widely expressed enzyme and receptor, involved in various cellular functions, making it CD38 is a highly conserved 45 kDa transmembrane type II difficult to pinpoint one particular function that would most glycoprotein with a short cytoplasmatic tail, a single-spanning critically impact CLL pathobiology. transmembrane domain, and a large extracellular domain (257 aa) [28, 29]. CD38 can be localized on the plasma membrane, CD49d in the cytoplasm, and in the inner nuclear membrane of cells [30, 31]. It is expressed in numerous cells types of the hema- CD49d belongs to the family of integrin alpha subunits. topoietic system, such as lymphocytes, myeloid cells, natural Integrins are heterodimers of non-covalently linked alpha killer (NK) cells, platelets, and erythrocytes, as well as in solid and beta subunits. The human CD49d (alpha4 integrin) sub- tissues, including various cell types of the brain, the eye, in unit can associate with either CD29 (beta1 integrin) or with pancreatic islet cells, smooth muscle cells, and osteoclasts and beta7 integrin [53, 54]. The combination of CD49d with beta7 osteoblasts [31]. CD38 is an important enzyme for the regu- mediates lymphocyte binding to mucosal addressin cell adhe- lation of calcium signaling and the cell’s energy transfer sion molecule-1, and its expression defines lymphocytes ca- homeostasis [29]. The products of the enzymatic reactions pable of trafficking through the intestines and the intestinal catalyzed by CD38 are all involved in the release of different lymphoid tissues [55, 56]. In contrast, very late antigen-4 intracellular calcium stores, mostly independent of the tradi- (VLA-4) is formed by the combination CD49d/CD29 [53] tional inositol triphosphate (IP3) pathway [32–37]. As such, and is expressed on leukocytes, including B and T cells, and CD38 has been shown to play a critical role in diverse immune on CD34+ hematopoietic stem/progenitor cells (HSPCs). functions: in T cell activation [34], neutrophil chemotaxis VLA-4 is the major CD49d-containing combination found [38], dendritic cell migration [39], and monocyte chemokine in resting CLL cells [57]. VLA-4 has two major ligands: production [40]. Furthermore, CD38-mediated calcium con- VCAM-1 [58], expressed on endothelial cells and bone mar- trol has also been implicated in various other functions of row stromal cells [59], and the extracellular matrix molecule different cell types: in the insulin secretion of pancreatic beta fibronectin [60]. cells [41, 42], in the oxytocin production of neurons [43–45], The use of mouse models has proved fundamental in in bone resorption of osteoclasts [46], as well as the respon- revealing the essential role of VLA-4 in fetal and adult hema- siveness of airway smooth muscle cells [47]. topoiesis (reviewed in [56, 61]). Germline deletion of either In addition to these enzymatic functions, CD38 is a well- VLA-4 subunit, CD49d or CD29, resulted in embryonic le- known lymphocyte differentiation antigen with proposed re- thality in mice. Chimeric mouse models generated with ceptor and adhesion molecule functions. This versatility of CD29 integrin-expressing or CD29 integrin-deficient embry- CD38 and its characteristic to function as dimers, multimers, onic stem (ES) cells indicated that loss of CD29 does not or as part of multi-protein complexes makes it difficult to fully impact haematopoietic stem cell formation or their differenti- comprehend its biology. The proposed functions of CD38 as a ation into different lineages but severely compromises their Ann Hematol (2014) 93:361–374 363 ability to colonize the fetal liver [62, 63]. Adult murine hema- as the best cut-off, others proposed 20 % [78, 79]or even 7 % topoiesis, however, did not seem to rely on CD29 [64]. Similar [80, 81]. Further studies are still necessary to define a common chimeric models generated with CD49d-positive or CD49d- cut-off level (reviewed in [82, 83]). negative ES cells demonstrated that this integrin subunit is In 2008, two studies concluded that high expression of critical for proper lineage differentiation and maturation of the CD49d is a robust adverse prognostic marker in CLL [10, hematopoietic system [65, 66]. Later, more refined inducible 11]. When analyzed retrospectively, CLL patients with ≥30 % knockout models revealed that CD49d is essential for bone CD49d-positive tumor cells revealed significantly shorter marrow homing and retention of progenitor cells [67, 68]. treatment-free and overall survival than patients with <30 % Moreover, absence of CD49d in HSPCs hindered both their CD49d positivity [10]. A prospective analysis indicated that self-renewal capacity and their ability to reconstitute hemato- an alternative cut-off level of 45 % CD49d expression might poiesis [69]. be superior to the 30 % level [11]. Following these first Homing is a rapid process that describes the active migra- reports, the prognostic relevance of increased CD49d expres- tion of cells from the blood, through the vascular endothelium, sion was rapidly and unequivocally confirmed by several into lymphoid organs. It differs from engraftment, for which groups, using the 30 % cut-off level [84–88](Table 1). Com- cell proliferation within the lymphoid tissue environment is parative analyses of CD49d mRNA and protein levels dem- essential [70]. Using adoptive transfers of human cells into onstrated its transcriptional–translational consistency [57, 84] immune-deficient mice, VLA-4 was identified as a key mol- which allows its determination by flow cytometry as well as ecule for both the bone marrow homing and the engraftment PCR-based assays for risk categorization. As with CD38, high of normal and leukemic human HSPCs [71, 72]. VLA-4 exists CD49d expression acts as an independent prognostic marker in multiple conformational states, including high-affinity- but is highly associated with other risk parameters such as activated but also low-affinity-extended states [73]. These IGHV, ZAP70, CD38, and the presence of chromosomal specific conformations allow VLA-4, in contrast to other aberrations [10, 11]. integrins, to support not only firm adhesion but also rolling Remarkably, the finding of differential CD49d expression of lymphocytes on VCAM-1 displaying endothelial cells. in CLL is an older discovery than anticipated. In 1996, it had Chemokine-induced inside-out activation of VLA-4, e.g., by already been demonstrated that CD49d expression in CLL is the bone marrow chemokine CXCL12, induces an upregula- variable, with higher expression of CLL samples of advanced tion of its adhesive properties to VCAM-1. This mediates the (Rai III, IV) than early stages [89]. Zucchetto and colleagues arrest of HSPCs on the BM vessels, which is a prerequisite for were the first in 2006 that reported the strong association of their BM homing [72]. Functional VLA-4 expression is also CD38 and CD49d expression on CLL cells using both param- indispensable for retention of normal HSPCs as well as leu- eters as categorical variables [90]. Comparing overall survival kemic blasts in bone marrow [74, 75]. Consequently, targeted rates, a combined CD38 low/CD49d low phenotype was disruption of VLA-4 function by anti-CD49d antibodies or attributed to the best prognosis. Out of the 115 investigated small-molecular-weight VLA-4 antagonists is known to result samples, 27 cases (23 %), however, displayed a discordant in rapid release of HSPCs into the peripheral circulation and to CD38 low/CD49d high or CD38 high/CD49d low phenotype. act synergistically or additive with conventional mobilization Patients with this discordant phenotype showed better overall regimes (for review, see [61]). survival rates compared with the combined CD38 high/ Thus, in contrast to CD38, the biological functions of CD49d high phenotype. Recently, we found a comparable CD49d and the CD49d/CD29 integrin combination VLA-4 21.5 % rate of discordant cases when analyzing 144 samples are well defined, with a principal involvement in bone marrow [86]. In our analysis, both a CD38 high or CD49d high homing and retention of hematopoietic cells, processes impor- phenotype were sufficient to predict shortened time to first tant to CLL pathophysiology. treatment, even when the presence of the second marker was low. This implies that a relevant proportion of our patients would be misclassified with regards to risk if we were to base CD38 and CD49d as prognostic markers in CLL our stratificationsolelyonCD38expression. Ourdatathus support the previous suggestion of a scoring system based on The prognostic role of CD38 in CLL was first proposed on the several antigens, including CD38 and CD49d, as an additional basis of an immunophenotypic study of CLL cases with tool for accurate risk categorization in CLL [91]. known IGHV sequences [6]. CD38 predicted shorter overall survival rates when expressed on 30 % or more CLL cells [6]. Since this first report in 1999, CD38 expression has been well CD38 and CD49d in CLL cell migration and homing established as an independent prognostic factor in CLL by numerous reports, however, with various cut-off levels. While In light of the current evidence that suggests that CLL local- Hamblin et al. [76] and Del Poeta et al. [77]concur with 30 % ization within supportive lymphoid niches is critical to disease 364 Ann Hematol (2014) 93:361–374 Table 1 High CD49d expression in CLL predicting shortened treatment free survival and overall survival Authors Single-/multi- Number of Cut-off p value TFS/median TFS p value OS/median OS Notes center study patients Majid et al. 2011 [84] Multi 652 30 % <0.0001 <0.0001 CD49d ≥30 %, 3.8 years CD49d ≥30 %, 9.7 years CD49d <30 %, 15.3 years CD49d <30 %, not reached Rossi et al. 2010 [86] Single 128 30 % 0.002 – Work in correlation with telomere length CD49d ≥30 %, 2.6 years CD49d <30 %, 9.2 years Nuckel et al. 2009 [83] Single 101 45 % 0.015 0.018 CD49d ≥45 %, 3.6 years Not reached CD49d <45 %, 9.7 years −5 Rossi et al. 2008 [87] Single 140 30 % 8.3×10 – In early stage CLL CD49d ≥30 %, 4.2 years CD49d <30 %, not reached (5-year TFS, 38.5 % vs −5 79.0 %, p =8.3×10 ) Gattei et al. 2008 [9] Single 232 (TFS) 30 % <0.001 <0.001 303 (OS) CD49d ≥30 %, 4.2 years n.a. CD49d <30 %, 9.0 years Shanafelt et al. 2008 [10] Single 158 45 % <0.0001 <0.0001 CD49d ≥45 %, 4.0 years CD49d ≥45 %, 20.0 years CD49d <45 %, 18.0 years CD49d <45 %, not reached −5 Zucchetto et al. 2006 [90] – 122 30 % – 1.66×10 Proposed scoring system of 6 surface antigens −6 Zucchetto et al. 2006 [89] – 115 30 % – 7.1×10 Work in correlation with CD38 TFS treatment-free survival, OS overall survival, n.a. not available progression, it seems logical that at least a part of the periph- between high CD49d expression and lymphadenopathy was eral blood CLL pool constantly recirculates into bone marrow demonstrated [95]. In these patients, high CD49d levels ap- and secondary lymphoid organs (Scheme 1). Nevertheless, pear to overcome some of the migratory defects of the CLL compared with healthy B lymphocytes, CLL cells dis- cells [96]. While entry of normal B lymphocytes into lymph play an impaired in vitro transendothelial migratory nodes is dependent on LFA-1, CLL cells with reduced LFA-1 (TEM) capacity over human umbilical vein endothelial levels (compared with normal B lymphocytes) were able to cells [92], a widely accepted endothelial model. These cross human umbilical vein endothelial cells in vitro by a data are in line with the early in vivo observation that Cr- mechanism that required an interplay between VLA-4 and labeled CLL cells of a CLL patient left the circulation at a LFA-1 [57, 96]. CD49d expression was decisive for in vivo dramatically diminished degree compared with healthy lym- homing of human CLL cells into the bone marrow of immune- deficient mice [57, 86]. Moreover, analyzing human bone phocytes [93]. This transmigratory defect was firstly attributed to low L- marrow CLL specimens, we also observed an association of selection expression [94], which is a major mediator of lym- CD49d expression and high leukemic BM infiltration [86]. phocyte tethering on endothelial cells. Subsequently, it be- The association of CD49d and MMP9 (see in more detail in came clear that the transmigratory capacity of CLL cells chapters below) may further contribute to invasiveness of varied between patients, with CLL cells from patients with CLL cells in the dense BM environment. advanced disease and bearing lymph node enlargement CD38 expression was also described to define CLL cells displaying increased TEM rates [95]. CCR7 and VLA-4 were with an increased migratory potential as tested by transwell identified as key factors in this process and a correlation chemotaxis assays [97]. In addition, lentiviral transfection of Ann Hematol (2014) 93:361–374 365 Scheme 1 Hypothetical model of CD49d (VLA-4) and CD38 implica- individual anti-apoptotic contributions of the molecules. (6) CLL prolif- tions in the distinct steps of the CLL life cycle. CD49d is a key molecule eration is strongly associated with CD38 expression, with a presumable for homing of CLL cells with a major mechanistical role in (1) rolling and but yet-to-be-described role of CD49d. Mobilization of CLL cells from (2) arrest of CLL cells on the bone marrow and lymph node endothelia. the lymphoid organs likely requires downregulation of CD49d expression CD38 may contribute to rolling of lymphocytes on the endothelium cells or function. CD49d stands for the functional CD49d/CD29 (VLA-4). and to (3) transendothelial migration. A macromolecular complex MMC: macromolecular complex including CD49d/CD29/CD38/MMP9/ (MMC), involving both CD38 and CD49d, is relevant for (4) invasion CD44v within the lymphoid tissue and (5) survival of CLL cells, with additional CLL cells with CD38 resulted in their enhanced motility to Thus, the evidence indisputably demonstrates that CLL CXCL12 [98]. By using an inhibitory anti-CD38 antibody cells require a functional VLA-4 to enter the bone marrow. (clone: SUN-4B7), the authors uncovered a CD38 contribu- Additionally, VLA-4 potentially compensates for CLL- tion to BM homing of CLL cells. In light of the expression of associated LFA-1 defects during lymph node entry. While CD31, reported to be a ligand of CD38, in the endothelium, a the role for CD38 in homing is still ambiguous, CD38- role of CD38 in CLL cell extravasation appears logical. How- associated proliferation (discussed in more detail below) ever, when comparing the integrin expression on CD38 high may play a role in the successful engraftment of the tumor or low CLL cells, increased VLA-4 (CD49d subunit) and cells within these lymphoid tissues. LFA-1 (CD18 subunit) expression was found in the CD38 high subtype [99], making it difficult to ascribe enhanced migration functions solely to CD38. In short-term homing CD38 and CD49d in CLL cell survival assays wherein CLL cells from discordant CD38+/VLA-4− and CD38-/VLA-4+ cases were transplanted into NOD/SCID Both, CD38 and CD49d have been described to contribute to mice, only cells expressing VLA-4 were capable of entering CLL survival, in a direct or indirect manner. A direct contri- the BM [86]. Importantly, the enhanced engraftment of CD38 bution would involve the induction of anti-apoptotic signaling positive samples in NOD/SCID mice and the higher prolifer- cascades upon ligand binding, whereas an indirect contribu- ation rates in the spleens of these xenogeneic animals have tion would be to ensure that the tumor cells are in the right been attributed to CD38-dependent proliferation rather than place at the right time in order to avail of the favorable signals homing of the CLL cells [100]. emanating from the microenvironment. 366 Ann Hematol (2014) 93:361–374 Ligation of CD38 in IL-2-treated CLL cells led to an As we found VLA-4/CD38 low-risk samples to be less increase in long-term survival [101]. Whether engagement sensitive towards spontaneous apoptosis ex vivo [86], they of CD38 by its supposed ligand CD31 induces direct pro- appear less dependent on continuous external stimulation. survival signals in CLL is still controversially discussed. Hypothetically, they are in a more quiescent mitotic state, Co-culture of CD38 expressing CLL cells with CD31- based on their lower basal Ki-67 expression. Our data are transfected murine cell lines or CD31+ nurse like cells en- consistent with the report by Coscia and colleagues [111] hanced survival of CLL cells and induced their proliferation who observed that high-risk CLL cells with unmutated IGHV [102, 103]. The CD31-specificity of these results could be genes were extremely vulnerable when removed from the demonstrated by addition of an antagonistic anti-CD31 mAb microenvironmental signals that induce supportive NF–kB that abrogated the effects [102]. Other authors, however, have signaling in these cells. Furthermore, NF–kB signaling via reported that they found no increase in cell survival or prolif- its transcriptional target TAp63 has been shown to result in eration of either CD38 high or low expressing CLL cells co- increased VLA-4 expression on high-risk samples [112], thus cultured with endothelial cells or CD31-transfected fibroblasts potentially creating a positive feedback loop leading to the in the presence or absence of either anti-CD31 or anti-CD38 accumulation of survival signals in the tumor cells. Of note, blocking mAbs [104]. Discordant results were also obtained other intercellular interactions leading to the activation of CLL when studying the modulation of apoptosis regulators upon cells, such as their interaction with vascular endothelium or incubation of CLL cells from CD38 high-risk patients with CD40L-transfected fibroblasts, also result in the upregulation CD31-transfected fibroblasts [104, 105]. Whether these find- of VLA-4 expression on the tumor cells [103, 113]. Whether ings reflect the in vivo situation remains to be confirmed. these interactions result in the concomitant increase of the Similarly, a direct ligand-triggered survival function of ligands VCAM-1 and fibronectin has not been investigated VLA-4was suggested inCLL,as tumor cells couldbe rescued yet. However, the implication of these findings is that VLA-4- from spontaneous or drug-induced in vitro apoptosis by direct mediated adhesion may help to strengthen the intercellular cell–cell contact with stromal cells [19, 106]. These studies contacts, thereby allowing stable and long-term bi-directional suggested that beta1 integrins contribute to this protective signaling between the tumor cells and the microenvironment. adhesion [106]. Furthermore, another series of studies reported VLA-4 may also support survival of CLL cells as part of a that culturing CLL cells on fibronectin- or VCAM-1-coated larger protein complex including MMP9 and CD44v [114, plates increased their in vitro viability and chemoresistance, 115]. In this setting, VLA-4 supports the adhesion of CLL which was accompanied by an increased Bcl-2/Bax ratio and cells to proMMP9, which results in pro-survival signals to- elevated Bcl-xL levels [107, 108]. The authors also suggested wards the CLL cells via the hemopexin domain of MMP9 that a general chemoresistant phenotype is related to high [115]. Intriguingly, proMMP9 hereby acts as a non-canonical VLA-4 expression of CLL cells. However, Majid and col- ligand for VLA-4 inducing a Lyn-Stat3-Mcl-1 pro-survival leagues [85] did not find a correlation between CD49d expres- signaling cascade distinct from VCAM-1 induced survival sion and in vitro resistance to fludarabine in liquid cultures signals. albeit they still observed a protective adhesion of CD49d high cells to fibronectin-coated plates. Similarly, we confirmed higher adhesion rates of VLA-4+ CLL cells to protective CD38 and CD49d in CLL cell proliferation stromal cells than of VLA-4− CLL cells [86]. Nevertheless, VLA-4 low CLL cells were still protected from spontaneous An enormous amount of effort has been put into defining the apoptosis to a similar extent by the presence of stroma, sug- link between CD38 and CLL cell proliferation and determin- gesting that this CLL subgroup uses alternative viability sig- ing whether CD38 plays an active or passive role in this nals. In line with these observations, cell adhesion-mediated process. The most recent findings suggest that CD38 expres- drug resistance, induced by culturing CLL cells on a follicular sion marks an activated and recently born CLL cell subset dendritic cell line, was shown to be independent of VCAM-1 [116–118]. The proliferation marker Ki-67 that characterizes [109] but dependent on other signals, e.g., CD44. One possible cell cycle entry and the mitosis regulating anaphase- explanation for these divergent findings is that different groups promoting complex/cyclosome (APC/C) are significantly in- investigated the protective effects of VLA-4-mediated cell creased in CLL cells of CD38 high-risk patients compared adhesion over different lengths of time. Of note, Zucchetto with low-risk samples [86, 118][99]. Moreover, CD38+ CLL et al. [110] found that protective VLA-4/VCAM-1 interactions subclones within individual patient samples express increased between CLL cells and VCAM-1-transfected fibroblasts first expression of the early activation marker CD69, the B cell become apparent after 7 days of co-culture and continuously activation marker CD27, and of Ki-67 [118–120]. Elegant increased with the most dramatic effects being observed after in vivo labeling studies verified that the CD38+ peripheral 21 days, a much longer time period than studied in most blood CLL cell pool comprises more newly proliferated CLL previous reports. cells than the CD38- pool [116]. Consistently, Ki-67 positive Ann Hematol (2014) 93:361–374 367 cells were frequently positive for CD38 in CLL proliferation complexity: Buggins et al. reported [123] a multimer- centers in lymph nodes [15]. In vitro, CLL cell proliferation complex involving CD38, CD49d, MMP9, and CD44 and can be induced by activation of T lymphocytes or CD40 observed a co-immunoprecipitation of CD38 with CD49d in ligand stimulation, and this is accompanied by an increase in the majority, but of CD38 with MMP9 in only about half of CD38 expression on the CLL cell [15, 121]. These observa- the investigated samples. Redondo-Munoz et al. reported tions, however, are correlative rather than proof of a direct the association of CD49d and MMP9 particularly with functional role for CD38 in this cellular function. A single CD44 variant forms instead of pan CD44 [124]. All the study implicated an active role for CD38 in CLL proliferation: reported complex structures may represent novel CLL CD38 ligation on CLL cells by an agonistic antibody in the high-risk-specific therapeutic targets as they do not ap- presence of IL-2 provoked intracellular calcium signals and pear to form in normal B cells or in low-risk cases. Notably, proliferation of the tumor cells [101]. What is clear, however, besides direct interactions, CD38 and CD49d may also indi- is that CD38 expression can be regulated by the microenvi- rectly influence each other as parts of a consecutive chain of ronment and can serve as a marker for an activated or recently events [125]. activated CLL phenotype [15, 99, 101, 122]. It is likely that Given this complexity, it is difficult to separate association- CD38 expression within the CLL clone is transient and CD38- intrinsic from molecule-specific functions, particularly when positive tumor cells eventually become CD38-negative, as interpreting correlative analyses. In addition, the use of proposed by Calissano et al. [116]. This is supported by the blocking antibodies in functional studies bears the risk of co- fact that CD38+ and CD38− subclones do not show any capping, crosslinking, or steric hindrance of the partner mol- difference in telomere length [118, 119] and that there is no ecule, which could be overcome by the use of small molecule clonal evolution of the CD38+ subclone [119]. This would inhibitors, which are increasingly being developed. Further- also imply that a CD38− subclone could become CD38+ more, a genetic modulation of CD38 and VLA-4 expression given the proper stimuli. Whether the upregulation of CD38 could help to correctly define their individual contributions. is a prerequisite to proliferation remains to be determined and The successful lentiviral introduction of CD38 in CD38- is hindered by the lack of small molecule specific inhibitors of negative CLL cells has only recently been achieved [126] CD38, as well as difficulties in achieving stable and long-term and contributes to a better understanding of the molecule. knockdowns in CLL cells. Furthermore, introduction of CD38 into cells of the CLL- In contrast to CD38, there is much less data linking VLA-4 derived prolymphocytic leukemia cell line MEC1 increased to CLL proliferation. We recently demonstrated that, similar to their adhesion to VLA-4 ligands, indicating functional CD38- CD38, VLA-4 expression of bone marrow-derived CLL cells VLA-4 interaction [110]. Conversely, lentiviral transfer of is higher than that of peripheral blood CLL cells and that the short hairpin RNA (shRNA) could be used for stable and proliferating CLL cell fraction was enriched in the VLA-4+ specific reduction of CD38 expression in high-risk CLL. subclone [86]. Notably, VLA-4 high-risk CLL cells also However, genetic manipulations of the cell cycle-arrested displayed increased in vitro proliferation rates upon co- primary peripheral blood CLL cells are still a challenge. culture with CD40L-transfected fibroblasts [121]. How To achieve an efficient knockdown, it would likely be VLA-4 impacts CLL cell proliferation remains to be necessary to combine shRNA approaches with long-term elucidated. culture and cell cycle induction of CLL cells. To this end, co-culture techniques that mimic the proliferative and sup- portive microenvironment in CLL are continuously being Challenges in separating individual and shared improved [121]. contributions of VLA-4 and CD38 in CLL An alternative approach to separate the functions of CD38 pathophysiology and VLA-4, feasible in the absence of efficient knockdown techniques, is the analysis of discordant cases. Notably, the A possible molecular basis for the high correlation of CD49d correlation of risk factors in CLL samples is not absolute with and CD38 expression in CLL could be their physical associ- a considerable rate of discordancy. Functional analyses using ation in multi-protein-complexes. Recent reports suggest a these discordant samples are useful to define the dispensabil- variety of possible protein combinations [98, 110, 114, 115, ity or compensation of a specific molecule in a cellular func- 123, 124]. Two recent reports demonstrate a colocalization tion. Taking this road, we have been able to demonstrate that and physical association of CD38 and CD49d/CD29 by a CD38 is not required for BM homing of CLL cells while combination of immunofluorescence and immunoprecipita- VLA-4 is indispensable. However, in light of the potential tion approaches [110, 123]. Whether this interaction occurs enzymatic function of CD38, it is still conceivable that in via the CD49d or the CD29d subunit of the VLA-4 integrin CD38/VLA-4 double-positive cases CD38 can exert a sup- could not unequivocally be clarified [110, 123]. Interpatient portive function in energy-dependent VLA-4 activation. The variability, which is usually high in CLL, adds further basis of this crosstalk remains to be elucidated. 368 Ann Hematol (2014) 93:361–374 Therapeutic implications and lymph nodes. Recently, the recombinant anti-VLA-4 an- tibody natalizumab demonstrated the potential to overcome Collectively, the data demonstrate that CLL cells with high- stromal cell-induced resistance of B cell lymphoma cells risk features are in fact those that are most exquisitely depen- against cytotoxic drugs and rituximab in vitro [127]. dent on microenvironmental stimuli for their survival and Natalizumab is already approved as an anti-inflammatory proliferation. Notably, the pathophysiological and prognostic drug, and a number of small molecule inhibitors for VLA-4 importance of this crosstalk bears therapeutical consequences. have been developed [61], primarily for use in multiple scle- Finding a therapeutic means of interfering with the bi- rosis or asthma. However, most of the clinical trials using directional communication between CLL cells and the sup- these small-molecular-weight antagonists for VLA-4 have portive microenvironment would go a long way to finding a been terminated due to low efficacy or side effects of the definite cure for this disease. Besides single targeting of CD38 substances. A new generation of currently developed VLA-4 or VLA-4, disrupting the macromolecular complexes housing inhibitors might overcome the previous problems and widen these proteins, or inhibiting downstream signaling, are all the therapeutic spectrum of VLA-4 antagonism towards tumor conceivable strategies. Additionally, immunomodulatory therapy. In fact, VLA-4 antagonizing nanoparticles recently drugs such as lenalidomide, whose molecular mechanism of demonstrated adhesion-inhibitory and cytotoxic effects that action is still unclear, may indirectly impact CD38 and VLA-4 resulted in reduced tumor growth in a multiple myeloma expression and function, and this should be further mouse model [128]. investigated. Moreover, targeting of VLA-4 downstream signals might The ubiquitous expression of CD38 in many different cell provide an alternative approach. VLA-4 antagonism is known types and tissues obviously raises concerns regarding the safe- to mobilize stem and progenitor cells from bone marrow [61], ty of widespread inhibition of CD38 function. Currently, there and its ligand VCAM-1 is highly expressed in both CLL BM are three different anti-CD38 antibodies under evaluation for and lymph nodes (unpublished observation). It is therefore safety in clinical trials (Table 2). Two monoclonal antibodies expected that VLA-4 targeting will not only impede CLL cell are being tested in multiple myeloma (daratumumab, identifi- recirculation to these lymphoid niches, but additionally mobi- er: NCT00574288 and MOR03087, identifier: NCT01421186; lize tumor cells from lymphoid organs, similar to the effects http://clinicaltrials.gov) and a third in selected CD38+ seen with novel small molecule inhibitors. Notably, ibrutinib, hematological malignancies including CLL (SAR650984, the clinically active BTK inhibitor PCI-32765, was recently identifier: NCT01084252; http://clinicaltrials.gov). These shown to impair VLA-4-mediated adhesion of CLL cells antibodies are supposed to bind to CD38+ tumor cells and [129]. This is consistent with the clinical observation of a trigger antibody-dependent cellular cytotoxicity rather than transient lymphocytosis of ibrutinib-treated patients due to mobilization of CLL cells from lymphoid organs into the inhibit the biological CD38 functions. Treatment strategies using VLA-4 inhibitors supposedly peripheral blood [130]. Other small molecule antagonists used interfere with the recirculation of CLL cells into bone marrow in clinical trials for treatment of CLL, e.g., the phos- Table 2 Anti-CD38 strategies currently investigated in clinical trials for treatment of haematologic malignancies Substance Single/combination Study phase Company Disease entities Identifier Status Daratumumab Human anti-CD38 Single I/II Genmab MM NCT00574288 Recruiting (HuMax®- CD38) Daratumumab Human anti-CD38 Lenalidomide+ I/II Genmab MM NCT01615029 Recruiting dexamethasone MOR03087 Human anti-CD38 Single I/II MorphoSys MM NCT01421186 Recruiting AG Lenalidomid/dexamethasone Bortezomib/dexamethasone SAR650984 Humanized IgG1 Single I Sanofi B-NHL, AML, NCT01084252 Recruiting anti-CD38 B-ALL, CLL; MM SAR650984 Humanized IgG1 Lenalidomide+ I Sanofi MM NCT01749969 Recruiting anti-CD38 dexamethasone MM multiple myeloma, AML acute myeloid leukemia, ALL acute lymphoblastic leukemia All trials: refractory/relapsed patients Ann Hematol (2014) 93:361–374 369 7. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK (1999) phatidylinositol 3-kinase inhibitor CAL-101 [131], might also Unmutated Ig V(H) genes are associated with a more aggressive affect VLA-4-mediated cellular functions in CLL. form of chronic lymphocytic leukemia. Blood 94(6):1848–1854 Conclusively, it is evident that CD38 and VLA-4 are more 8. Chen L, Widhopf G, Huynh L, Rassenti L, Rai KR, Weiss A, Kipps than just markers of an aggressive CLL cell type and that they TJ (2002) Expression of ZAP-70 is associated with increased B-cell receptor signaling in chronic lymphocytic leukemia. Blood 100(13): play functional roles in the pathobiology of the disease. As 4609–4614. doi:10.1182/blood-2002-06-1683 such, they represent therapeutic targets that may be exploited 9. Wiestner A, Rosenwald A, Barry TS, Wright G, Davis RE, in addition to, or in combination with, the currently developed Henrickson SE, Zhao H, Ibbotson RE, Orchard JA, Davis Z, novel approaches of interfering with CLL cell–tumor host Stetler-Stevenson M, Raffeld M, Arthur DC, Marti GE, Wilson WH, Hamblin TJ, Oscier DG, Staudt LM (2003) ZAP-70 expres- interactions. Targeting these molecules should also be tested sion identifies a chronic lymphocytic leukemia subtype with for its potential in avoiding the frequent relapses and devel- unmutated immunoglobulin genes, inferior clinical outcome, and opment of chemoresistance in CLL. distinct gene expression profile. Blood 101(12):4944–4951. doi:10. 1182/blood-2002-10-3306 10. Gattei V, Bulian P, Del Principe MI, Zucchetto A, Maurillo L, Buccisano F, Bomben R, Dal-Bo M, Luciano F, Rossi FM, Degan Acknowledgments Work of the authors is supported by the Austrian M, Amadori S, Del Poeta G (2008) Relevance of CD49d protein Science Fund FWF (project W1213 and SFB program P021 to R.G., expression as overall survival and progressive disease prognostica- project P25015-B13 to T.N.H.), the Austrian National Bank (projects tor in chronic lymphocytic leukemia. Blood 111(2):865–873. doi: 13420 to T.N.H., 14311 to R.G., the Paracelsus Medical University 10.1182/blood-2007-05-092486 Salzburg (projects E-10/11/058-HAR and E-12/15/074-HAH to T.N.H), 11. Shanafelt TD, Geyer SM, Bone ND, Tschumper RC, Witzig TE, the “Klinische Malignom und Zytokinforschung Salzburg-Innsbruck Nowakowski GS, Zent CS, Call TG, Laplant B, Dewald GW, GmbH”, and the province of Salzburg. Jelinek DF, Kay NE (2008) CD49d expression is an independent predictor of overall survival in patients with chronic lymphocytic Authors G.B., J.P.H., R.G., and T.N.H wrote the paper and approve of leukaemia: a prognostic parameter with therapeutic potential. Br J the submitted and final version. Haematol 140(5):537–546. doi:10.1111/j.1365-2141.2007.06965.x 12. Messmer BT, Messmer D, Allen SL, Kolitz JE, Kudalkar P, Cesar Conflict of interest The authors declare no competing financial conflict D, Murphy EJ, Koduru P, Ferrarini M, Zupo S, Cutrona G, Damle of interest. RN, Wasil T, Rai KR, Hellerstein MK, Chiorazzi N (2005) In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells. J Clin Invest 115(3):755–764. doi: 10.1172/JCI23409 13. 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The pathogenic relevance of the prognostic markers CD38 and CD49d in chronic lymphocytic leukemia

Annals of Hematology , Volume 93 (3) – Nov 28, 2013

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Abstract

Ann Hematol (2014) 93:361–374 DOI 10.1007/s00277-013-1967-y REVIEW ARTICLE The pathogenic relevance of the prognostic markers CD38 and CD49d in chronic lymphocytic leukemia Gabriele Brachtl & Josefina Piñón Hofbauer & Richard Greil & Tanja Nicole Hartmann Received: 26 July 2013 /Accepted: 13 November 2013 /Published online: 28 November 2013 Springer-Verlag Berlin Heidelberg 2013 Abstract The interactions of chronic lymphocytic leukemia clinical course a patient will experience. Several more recently cells with the microenvironment in secondary lymphoid tis- suggested prognostic markers, apparently involved in the sues and the bone marrow are known to promote CLL cell cellular processes underlying CLL pathogenesis, may aid to survival and proliferation. CD38 and CD49d are both inde- classify patients according to clinical risk. These markers pendent prognostic risk parameters in CLL with important include chromosomal aberrations such as deletion of 17p13, roles in shaping these interactions. Both are reported to influ- 11q22-23, or 13q1, and trisomy 12 [4] that directly influence ence CLL cell trafficking between blood and lymphoid organs cell fate or transformation, as well as molecular markers for as well as their survival and proliferation within the lymphoid CLL cell interactions with the tumor microenvironment [5]. organs, thereby impacting the pathophysiology of the disease. Among the molecular prognostic factors, the mutational status The expression of CD38 and CD49d is associated in the of immunoglobulin variable region (IGHV) genes [6, 7], the majority of cases, and they exist as part of macromolecular expression of CD38 on the surface of CLL cells [6], and the complexes. Here, we review the current evidence for the intracellular expression of zeta-associated protein 70 [8, 9]are individual and associated contributions of these molecules to the best-established ones. A more recently discovered marker CLL pathophysiology. is CD49d, the alpha4 subunit of the VLA-4 integrin (alpha4beta1). High CD49d expression predicts reduced over- all survival and time to first treatment in CLL patients [10, 11]. Keywords Chronic lymphocytic leukemia . . . The pathogenic significance of CLL cell interactions with Microenvironment CD38 CD49d VLA-4 the lymphoid microenvironment has become increasingly ac- knowledged in recent years. CLL cell proliferation is sup- posed to take place in lymph nodes and, to a lesser extent in The heterogeneity of CLL in regard to the tumor bone marrow, with up to 2 % of the entire clone being newly microenvironment generated per day [12], and is most likely supported by activated T lymphocytes that express CD40 ligand [13–15]. Chronic lymphocytic leukemia (CLL), a B cell non- Signals from T lymphocytes and from other accessory cells in Hodgkin’s lymphoma with a leukemic appearance, is a re- this environment, such as stromal or nurse-like cells, also markably heterogeneous disease that can follow a wide variety provide pro-survival support to the malignant cells [16–19]. of courses. Patients with an indolent course survive for many Not only does the microenvironment influence CLL cells, but years. Others, however, show a rapidly fatal disease despite CLL cells alter their microenvironment to their advantage by aggressive therapy and die within 2–3 years of diagnosis priming T cells towards an immune suppressive phenotype (reviewed in [1]). Mere staging by Rai [2] or Binet [3]is not [20] or inducing stromal cells to provide pro-survival signals sufficient to predict at an early stage of the disease which [21–24], which contributes to chemoresistance and treatment : : : failure. Minimal residual disease after therapy is attributed to G. Brachtl J. Piñón Hofbauer R. Greil T. N. Hartmann (*) Laboratory for Immunological and Molecular Cancer Research, supportive microenvironmental signals and prognostically as- Third Medical Department with Hematology, Oncology, sociated with shortened progression-free and overall survival Hemostaseology, Infectiology and Rheumatology, Paracelsus rates of CLL patients [25–27]. Eradicating residual CLL cells Medical University, Müllner Haupstraße 48, 5020 Salzburg, Austria within their protective niches in secondary lymphoid tissues e-mail: t.hartmann@salk.at 362 Ann Hematol (2014) 93:361–374 and bone marrow is thus considered a major therapeutic goal receptor vary greatly and depend on its association with other for achieving permanent remission. surface molecules. CD38 is laterally associated with the main As dissected in the following chapters, the prognostic signaling complexes of lymphocytes that are organized in markers CD49d and CD38 have been reported to be involved lipid rafts. In T cells, CD38 is capable of interacting with the in various cellular functions relevant to CLL pathogenesis: T cell receptor (TCR)/CD3 complex; in B cells, with the BCR/ CLL cell homing to lymphoid organs, survival, and prolifer- CD19 complex; and in NK cells, with the CD16/CD81 com- ation. However, CD49d and CD38 expression is associated in plex [31]. CD31 (PECAM-1), expressed by, e.g., endothelial about 80 % of CLL patients, and these molecules are reported cells has been described as a CD38 ligand [48]. CD38+ to physically interact within multi-protein complexes. Be- lymphocytes show a weak, selectin-like adhesion to endothe- cause of this, it is difficult to gauge the individual contribution lial cells [49], which appears to be mediated by CD38–CD31 of each molecule to key pathogenic functions in CLL. In light interactions [48]. of the high heterogeneity of reports dealing with either mole- In B cells, ligation of CD38 by agonistic antibodies triggers cule and the fact that CD49d is still a newcomer among the different in vitro responses depending on the differentiation prognostic factors, we review the current evidence for the stage of the cells. In immature B cell precursors, CD38 liga- individual and associated contributions of these molecules to tion inhibited DNA synthesis and induced apoptosis, thereby CLL pathophysiology. blocking B cell hematopoiesis [50]. In tonsillar germinal center B cells, CD38 mediated pro-survival signaling [51]. In mature circulating B cells, CD38 ligation induced prolifer- CD38 and VLA-4 in general ation by promoting the expression of CD25, MHC-II, and certain cytokines [31, 52]. CD38 In summary, CD38 is a widely expressed enzyme and receptor, involved in various cellular functions, making it CD38 is a highly conserved 45 kDa transmembrane type II difficult to pinpoint one particular function that would most glycoprotein with a short cytoplasmatic tail, a single-spanning critically impact CLL pathobiology. transmembrane domain, and a large extracellular domain (257 aa) [28, 29]. CD38 can be localized on the plasma membrane, CD49d in the cytoplasm, and in the inner nuclear membrane of cells [30, 31]. It is expressed in numerous cells types of the hema- CD49d belongs to the family of integrin alpha subunits. topoietic system, such as lymphocytes, myeloid cells, natural Integrins are heterodimers of non-covalently linked alpha killer (NK) cells, platelets, and erythrocytes, as well as in solid and beta subunits. The human CD49d (alpha4 integrin) sub- tissues, including various cell types of the brain, the eye, in unit can associate with either CD29 (beta1 integrin) or with pancreatic islet cells, smooth muscle cells, and osteoclasts and beta7 integrin [53, 54]. The combination of CD49d with beta7 osteoblasts [31]. CD38 is an important enzyme for the regu- mediates lymphocyte binding to mucosal addressin cell adhe- lation of calcium signaling and the cell’s energy transfer sion molecule-1, and its expression defines lymphocytes ca- homeostasis [29]. The products of the enzymatic reactions pable of trafficking through the intestines and the intestinal catalyzed by CD38 are all involved in the release of different lymphoid tissues [55, 56]. In contrast, very late antigen-4 intracellular calcium stores, mostly independent of the tradi- (VLA-4) is formed by the combination CD49d/CD29 [53] tional inositol triphosphate (IP3) pathway [32–37]. As such, and is expressed on leukocytes, including B and T cells, and CD38 has been shown to play a critical role in diverse immune on CD34+ hematopoietic stem/progenitor cells (HSPCs). functions: in T cell activation [34], neutrophil chemotaxis VLA-4 is the major CD49d-containing combination found [38], dendritic cell migration [39], and monocyte chemokine in resting CLL cells [57]. VLA-4 has two major ligands: production [40]. Furthermore, CD38-mediated calcium con- VCAM-1 [58], expressed on endothelial cells and bone mar- trol has also been implicated in various other functions of row stromal cells [59], and the extracellular matrix molecule different cell types: in the insulin secretion of pancreatic beta fibronectin [60]. cells [41, 42], in the oxytocin production of neurons [43–45], The use of mouse models has proved fundamental in in bone resorption of osteoclasts [46], as well as the respon- revealing the essential role of VLA-4 in fetal and adult hema- siveness of airway smooth muscle cells [47]. topoiesis (reviewed in [56, 61]). Germline deletion of either In addition to these enzymatic functions, CD38 is a well- VLA-4 subunit, CD49d or CD29, resulted in embryonic le- known lymphocyte differentiation antigen with proposed re- thality in mice. Chimeric mouse models generated with ceptor and adhesion molecule functions. This versatility of CD29 integrin-expressing or CD29 integrin-deficient embry- CD38 and its characteristic to function as dimers, multimers, onic stem (ES) cells indicated that loss of CD29 does not or as part of multi-protein complexes makes it difficult to fully impact haematopoietic stem cell formation or their differenti- comprehend its biology. The proposed functions of CD38 as a ation into different lineages but severely compromises their Ann Hematol (2014) 93:361–374 363 ability to colonize the fetal liver [62, 63]. Adult murine hema- as the best cut-off, others proposed 20 % [78, 79]or even 7 % topoiesis, however, did not seem to rely on CD29 [64]. Similar [80, 81]. Further studies are still necessary to define a common chimeric models generated with CD49d-positive or CD49d- cut-off level (reviewed in [82, 83]). negative ES cells demonstrated that this integrin subunit is In 2008, two studies concluded that high expression of critical for proper lineage differentiation and maturation of the CD49d is a robust adverse prognostic marker in CLL [10, hematopoietic system [65, 66]. Later, more refined inducible 11]. When analyzed retrospectively, CLL patients with ≥30 % knockout models revealed that CD49d is essential for bone CD49d-positive tumor cells revealed significantly shorter marrow homing and retention of progenitor cells [67, 68]. treatment-free and overall survival than patients with <30 % Moreover, absence of CD49d in HSPCs hindered both their CD49d positivity [10]. A prospective analysis indicated that self-renewal capacity and their ability to reconstitute hemato- an alternative cut-off level of 45 % CD49d expression might poiesis [69]. be superior to the 30 % level [11]. Following these first Homing is a rapid process that describes the active migra- reports, the prognostic relevance of increased CD49d expres- tion of cells from the blood, through the vascular endothelium, sion was rapidly and unequivocally confirmed by several into lymphoid organs. It differs from engraftment, for which groups, using the 30 % cut-off level [84–88](Table 1). Com- cell proliferation within the lymphoid tissue environment is parative analyses of CD49d mRNA and protein levels dem- essential [70]. Using adoptive transfers of human cells into onstrated its transcriptional–translational consistency [57, 84] immune-deficient mice, VLA-4 was identified as a key mol- which allows its determination by flow cytometry as well as ecule for both the bone marrow homing and the engraftment PCR-based assays for risk categorization. As with CD38, high of normal and leukemic human HSPCs [71, 72]. VLA-4 exists CD49d expression acts as an independent prognostic marker in multiple conformational states, including high-affinity- but is highly associated with other risk parameters such as activated but also low-affinity-extended states [73]. These IGHV, ZAP70, CD38, and the presence of chromosomal specific conformations allow VLA-4, in contrast to other aberrations [10, 11]. integrins, to support not only firm adhesion but also rolling Remarkably, the finding of differential CD49d expression of lymphocytes on VCAM-1 displaying endothelial cells. in CLL is an older discovery than anticipated. In 1996, it had Chemokine-induced inside-out activation of VLA-4, e.g., by already been demonstrated that CD49d expression in CLL is the bone marrow chemokine CXCL12, induces an upregula- variable, with higher expression of CLL samples of advanced tion of its adhesive properties to VCAM-1. This mediates the (Rai III, IV) than early stages [89]. Zucchetto and colleagues arrest of HSPCs on the BM vessels, which is a prerequisite for were the first in 2006 that reported the strong association of their BM homing [72]. Functional VLA-4 expression is also CD38 and CD49d expression on CLL cells using both param- indispensable for retention of normal HSPCs as well as leu- eters as categorical variables [90]. Comparing overall survival kemic blasts in bone marrow [74, 75]. Consequently, targeted rates, a combined CD38 low/CD49d low phenotype was disruption of VLA-4 function by anti-CD49d antibodies or attributed to the best prognosis. Out of the 115 investigated small-molecular-weight VLA-4 antagonists is known to result samples, 27 cases (23 %), however, displayed a discordant in rapid release of HSPCs into the peripheral circulation and to CD38 low/CD49d high or CD38 high/CD49d low phenotype. act synergistically or additive with conventional mobilization Patients with this discordant phenotype showed better overall regimes (for review, see [61]). survival rates compared with the combined CD38 high/ Thus, in contrast to CD38, the biological functions of CD49d high phenotype. Recently, we found a comparable CD49d and the CD49d/CD29 integrin combination VLA-4 21.5 % rate of discordant cases when analyzing 144 samples are well defined, with a principal involvement in bone marrow [86]. In our analysis, both a CD38 high or CD49d high homing and retention of hematopoietic cells, processes impor- phenotype were sufficient to predict shortened time to first tant to CLL pathophysiology. treatment, even when the presence of the second marker was low. This implies that a relevant proportion of our patients would be misclassified with regards to risk if we were to base CD38 and CD49d as prognostic markers in CLL our stratificationsolelyonCD38expression. Ourdatathus support the previous suggestion of a scoring system based on The prognostic role of CD38 in CLL was first proposed on the several antigens, including CD38 and CD49d, as an additional basis of an immunophenotypic study of CLL cases with tool for accurate risk categorization in CLL [91]. known IGHV sequences [6]. CD38 predicted shorter overall survival rates when expressed on 30 % or more CLL cells [6]. Since this first report in 1999, CD38 expression has been well CD38 and CD49d in CLL cell migration and homing established as an independent prognostic factor in CLL by numerous reports, however, with various cut-off levels. While In light of the current evidence that suggests that CLL local- Hamblin et al. [76] and Del Poeta et al. [77]concur with 30 % ization within supportive lymphoid niches is critical to disease 364 Ann Hematol (2014) 93:361–374 Table 1 High CD49d expression in CLL predicting shortened treatment free survival and overall survival Authors Single-/multi- Number of Cut-off p value TFS/median TFS p value OS/median OS Notes center study patients Majid et al. 2011 [84] Multi 652 30 % <0.0001 <0.0001 CD49d ≥30 %, 3.8 years CD49d ≥30 %, 9.7 years CD49d <30 %, 15.3 years CD49d <30 %, not reached Rossi et al. 2010 [86] Single 128 30 % 0.002 – Work in correlation with telomere length CD49d ≥30 %, 2.6 years CD49d <30 %, 9.2 years Nuckel et al. 2009 [83] Single 101 45 % 0.015 0.018 CD49d ≥45 %, 3.6 years Not reached CD49d <45 %, 9.7 years −5 Rossi et al. 2008 [87] Single 140 30 % 8.3×10 – In early stage CLL CD49d ≥30 %, 4.2 years CD49d <30 %, not reached (5-year TFS, 38.5 % vs −5 79.0 %, p =8.3×10 ) Gattei et al. 2008 [9] Single 232 (TFS) 30 % <0.001 <0.001 303 (OS) CD49d ≥30 %, 4.2 years n.a. CD49d <30 %, 9.0 years Shanafelt et al. 2008 [10] Single 158 45 % <0.0001 <0.0001 CD49d ≥45 %, 4.0 years CD49d ≥45 %, 20.0 years CD49d <45 %, 18.0 years CD49d <45 %, not reached −5 Zucchetto et al. 2006 [90] – 122 30 % – 1.66×10 Proposed scoring system of 6 surface antigens −6 Zucchetto et al. 2006 [89] – 115 30 % – 7.1×10 Work in correlation with CD38 TFS treatment-free survival, OS overall survival, n.a. not available progression, it seems logical that at least a part of the periph- between high CD49d expression and lymphadenopathy was eral blood CLL pool constantly recirculates into bone marrow demonstrated [95]. In these patients, high CD49d levels ap- and secondary lymphoid organs (Scheme 1). Nevertheless, pear to overcome some of the migratory defects of the CLL compared with healthy B lymphocytes, CLL cells dis- cells [96]. While entry of normal B lymphocytes into lymph play an impaired in vitro transendothelial migratory nodes is dependent on LFA-1, CLL cells with reduced LFA-1 (TEM) capacity over human umbilical vein endothelial levels (compared with normal B lymphocytes) were able to cells [92], a widely accepted endothelial model. These cross human umbilical vein endothelial cells in vitro by a data are in line with the early in vivo observation that Cr- mechanism that required an interplay between VLA-4 and labeled CLL cells of a CLL patient left the circulation at a LFA-1 [57, 96]. CD49d expression was decisive for in vivo dramatically diminished degree compared with healthy lym- homing of human CLL cells into the bone marrow of immune- deficient mice [57, 86]. Moreover, analyzing human bone phocytes [93]. This transmigratory defect was firstly attributed to low L- marrow CLL specimens, we also observed an association of selection expression [94], which is a major mediator of lym- CD49d expression and high leukemic BM infiltration [86]. phocyte tethering on endothelial cells. Subsequently, it be- The association of CD49d and MMP9 (see in more detail in came clear that the transmigratory capacity of CLL cells chapters below) may further contribute to invasiveness of varied between patients, with CLL cells from patients with CLL cells in the dense BM environment. advanced disease and bearing lymph node enlargement CD38 expression was also described to define CLL cells displaying increased TEM rates [95]. CCR7 and VLA-4 were with an increased migratory potential as tested by transwell identified as key factors in this process and a correlation chemotaxis assays [97]. In addition, lentiviral transfection of Ann Hematol (2014) 93:361–374 365 Scheme 1 Hypothetical model of CD49d (VLA-4) and CD38 implica- individual anti-apoptotic contributions of the molecules. (6) CLL prolif- tions in the distinct steps of the CLL life cycle. CD49d is a key molecule eration is strongly associated with CD38 expression, with a presumable for homing of CLL cells with a major mechanistical role in (1) rolling and but yet-to-be-described role of CD49d. Mobilization of CLL cells from (2) arrest of CLL cells on the bone marrow and lymph node endothelia. the lymphoid organs likely requires downregulation of CD49d expression CD38 may contribute to rolling of lymphocytes on the endothelium cells or function. CD49d stands for the functional CD49d/CD29 (VLA-4). and to (3) transendothelial migration. A macromolecular complex MMC: macromolecular complex including CD49d/CD29/CD38/MMP9/ (MMC), involving both CD38 and CD49d, is relevant for (4) invasion CD44v within the lymphoid tissue and (5) survival of CLL cells, with additional CLL cells with CD38 resulted in their enhanced motility to Thus, the evidence indisputably demonstrates that CLL CXCL12 [98]. By using an inhibitory anti-CD38 antibody cells require a functional VLA-4 to enter the bone marrow. (clone: SUN-4B7), the authors uncovered a CD38 contribu- Additionally, VLA-4 potentially compensates for CLL- tion to BM homing of CLL cells. In light of the expression of associated LFA-1 defects during lymph node entry. While CD31, reported to be a ligand of CD38, in the endothelium, a the role for CD38 in homing is still ambiguous, CD38- role of CD38 in CLL cell extravasation appears logical. How- associated proliferation (discussed in more detail below) ever, when comparing the integrin expression on CD38 high may play a role in the successful engraftment of the tumor or low CLL cells, increased VLA-4 (CD49d subunit) and cells within these lymphoid tissues. LFA-1 (CD18 subunit) expression was found in the CD38 high subtype [99], making it difficult to ascribe enhanced migration functions solely to CD38. In short-term homing CD38 and CD49d in CLL cell survival assays wherein CLL cells from discordant CD38+/VLA-4− and CD38-/VLA-4+ cases were transplanted into NOD/SCID Both, CD38 and CD49d have been described to contribute to mice, only cells expressing VLA-4 were capable of entering CLL survival, in a direct or indirect manner. A direct contri- the BM [86]. Importantly, the enhanced engraftment of CD38 bution would involve the induction of anti-apoptotic signaling positive samples in NOD/SCID mice and the higher prolifer- cascades upon ligand binding, whereas an indirect contribu- ation rates in the spleens of these xenogeneic animals have tion would be to ensure that the tumor cells are in the right been attributed to CD38-dependent proliferation rather than place at the right time in order to avail of the favorable signals homing of the CLL cells [100]. emanating from the microenvironment. 366 Ann Hematol (2014) 93:361–374 Ligation of CD38 in IL-2-treated CLL cells led to an As we found VLA-4/CD38 low-risk samples to be less increase in long-term survival [101]. Whether engagement sensitive towards spontaneous apoptosis ex vivo [86], they of CD38 by its supposed ligand CD31 induces direct pro- appear less dependent on continuous external stimulation. survival signals in CLL is still controversially discussed. Hypothetically, they are in a more quiescent mitotic state, Co-culture of CD38 expressing CLL cells with CD31- based on their lower basal Ki-67 expression. Our data are transfected murine cell lines or CD31+ nurse like cells en- consistent with the report by Coscia and colleagues [111] hanced survival of CLL cells and induced their proliferation who observed that high-risk CLL cells with unmutated IGHV [102, 103]. The CD31-specificity of these results could be genes were extremely vulnerable when removed from the demonstrated by addition of an antagonistic anti-CD31 mAb microenvironmental signals that induce supportive NF–kB that abrogated the effects [102]. Other authors, however, have signaling in these cells. Furthermore, NF–kB signaling via reported that they found no increase in cell survival or prolif- its transcriptional target TAp63 has been shown to result in eration of either CD38 high or low expressing CLL cells co- increased VLA-4 expression on high-risk samples [112], thus cultured with endothelial cells or CD31-transfected fibroblasts potentially creating a positive feedback loop leading to the in the presence or absence of either anti-CD31 or anti-CD38 accumulation of survival signals in the tumor cells. Of note, blocking mAbs [104]. Discordant results were also obtained other intercellular interactions leading to the activation of CLL when studying the modulation of apoptosis regulators upon cells, such as their interaction with vascular endothelium or incubation of CLL cells from CD38 high-risk patients with CD40L-transfected fibroblasts, also result in the upregulation CD31-transfected fibroblasts [104, 105]. Whether these find- of VLA-4 expression on the tumor cells [103, 113]. Whether ings reflect the in vivo situation remains to be confirmed. these interactions result in the concomitant increase of the Similarly, a direct ligand-triggered survival function of ligands VCAM-1 and fibronectin has not been investigated VLA-4was suggested inCLL,as tumor cells couldbe rescued yet. However, the implication of these findings is that VLA-4- from spontaneous or drug-induced in vitro apoptosis by direct mediated adhesion may help to strengthen the intercellular cell–cell contact with stromal cells [19, 106]. These studies contacts, thereby allowing stable and long-term bi-directional suggested that beta1 integrins contribute to this protective signaling between the tumor cells and the microenvironment. adhesion [106]. Furthermore, another series of studies reported VLA-4 may also support survival of CLL cells as part of a that culturing CLL cells on fibronectin- or VCAM-1-coated larger protein complex including MMP9 and CD44v [114, plates increased their in vitro viability and chemoresistance, 115]. In this setting, VLA-4 supports the adhesion of CLL which was accompanied by an increased Bcl-2/Bax ratio and cells to proMMP9, which results in pro-survival signals to- elevated Bcl-xL levels [107, 108]. The authors also suggested wards the CLL cells via the hemopexin domain of MMP9 that a general chemoresistant phenotype is related to high [115]. Intriguingly, proMMP9 hereby acts as a non-canonical VLA-4 expression of CLL cells. However, Majid and col- ligand for VLA-4 inducing a Lyn-Stat3-Mcl-1 pro-survival leagues [85] did not find a correlation between CD49d expres- signaling cascade distinct from VCAM-1 induced survival sion and in vitro resistance to fludarabine in liquid cultures signals. albeit they still observed a protective adhesion of CD49d high cells to fibronectin-coated plates. Similarly, we confirmed higher adhesion rates of VLA-4+ CLL cells to protective CD38 and CD49d in CLL cell proliferation stromal cells than of VLA-4− CLL cells [86]. Nevertheless, VLA-4 low CLL cells were still protected from spontaneous An enormous amount of effort has been put into defining the apoptosis to a similar extent by the presence of stroma, sug- link between CD38 and CLL cell proliferation and determin- gesting that this CLL subgroup uses alternative viability sig- ing whether CD38 plays an active or passive role in this nals. In line with these observations, cell adhesion-mediated process. The most recent findings suggest that CD38 expres- drug resistance, induced by culturing CLL cells on a follicular sion marks an activated and recently born CLL cell subset dendritic cell line, was shown to be independent of VCAM-1 [116–118]. The proliferation marker Ki-67 that characterizes [109] but dependent on other signals, e.g., CD44. One possible cell cycle entry and the mitosis regulating anaphase- explanation for these divergent findings is that different groups promoting complex/cyclosome (APC/C) are significantly in- investigated the protective effects of VLA-4-mediated cell creased in CLL cells of CD38 high-risk patients compared adhesion over different lengths of time. Of note, Zucchetto with low-risk samples [86, 118][99]. Moreover, CD38+ CLL et al. [110] found that protective VLA-4/VCAM-1 interactions subclones within individual patient samples express increased between CLL cells and VCAM-1-transfected fibroblasts first expression of the early activation marker CD69, the B cell become apparent after 7 days of co-culture and continuously activation marker CD27, and of Ki-67 [118–120]. Elegant increased with the most dramatic effects being observed after in vivo labeling studies verified that the CD38+ peripheral 21 days, a much longer time period than studied in most blood CLL cell pool comprises more newly proliferated CLL previous reports. cells than the CD38- pool [116]. Consistently, Ki-67 positive Ann Hematol (2014) 93:361–374 367 cells were frequently positive for CD38 in CLL proliferation complexity: Buggins et al. reported [123] a multimer- centers in lymph nodes [15]. In vitro, CLL cell proliferation complex involving CD38, CD49d, MMP9, and CD44 and can be induced by activation of T lymphocytes or CD40 observed a co-immunoprecipitation of CD38 with CD49d in ligand stimulation, and this is accompanied by an increase in the majority, but of CD38 with MMP9 in only about half of CD38 expression on the CLL cell [15, 121]. These observa- the investigated samples. Redondo-Munoz et al. reported tions, however, are correlative rather than proof of a direct the association of CD49d and MMP9 particularly with functional role for CD38 in this cellular function. A single CD44 variant forms instead of pan CD44 [124]. All the study implicated an active role for CD38 in CLL proliferation: reported complex structures may represent novel CLL CD38 ligation on CLL cells by an agonistic antibody in the high-risk-specific therapeutic targets as they do not ap- presence of IL-2 provoked intracellular calcium signals and pear to form in normal B cells or in low-risk cases. Notably, proliferation of the tumor cells [101]. What is clear, however, besides direct interactions, CD38 and CD49d may also indi- is that CD38 expression can be regulated by the microenvi- rectly influence each other as parts of a consecutive chain of ronment and can serve as a marker for an activated or recently events [125]. activated CLL phenotype [15, 99, 101, 122]. It is likely that Given this complexity, it is difficult to separate association- CD38 expression within the CLL clone is transient and CD38- intrinsic from molecule-specific functions, particularly when positive tumor cells eventually become CD38-negative, as interpreting correlative analyses. In addition, the use of proposed by Calissano et al. [116]. This is supported by the blocking antibodies in functional studies bears the risk of co- fact that CD38+ and CD38− subclones do not show any capping, crosslinking, or steric hindrance of the partner mol- difference in telomere length [118, 119] and that there is no ecule, which could be overcome by the use of small molecule clonal evolution of the CD38+ subclone [119]. This would inhibitors, which are increasingly being developed. Further- also imply that a CD38− subclone could become CD38+ more, a genetic modulation of CD38 and VLA-4 expression given the proper stimuli. Whether the upregulation of CD38 could help to correctly define their individual contributions. is a prerequisite to proliferation remains to be determined and The successful lentiviral introduction of CD38 in CD38- is hindered by the lack of small molecule specific inhibitors of negative CLL cells has only recently been achieved [126] CD38, as well as difficulties in achieving stable and long-term and contributes to a better understanding of the molecule. knockdowns in CLL cells. Furthermore, introduction of CD38 into cells of the CLL- In contrast to CD38, there is much less data linking VLA-4 derived prolymphocytic leukemia cell line MEC1 increased to CLL proliferation. We recently demonstrated that, similar to their adhesion to VLA-4 ligands, indicating functional CD38- CD38, VLA-4 expression of bone marrow-derived CLL cells VLA-4 interaction [110]. Conversely, lentiviral transfer of is higher than that of peripheral blood CLL cells and that the short hairpin RNA (shRNA) could be used for stable and proliferating CLL cell fraction was enriched in the VLA-4+ specific reduction of CD38 expression in high-risk CLL. subclone [86]. Notably, VLA-4 high-risk CLL cells also However, genetic manipulations of the cell cycle-arrested displayed increased in vitro proliferation rates upon co- primary peripheral blood CLL cells are still a challenge. culture with CD40L-transfected fibroblasts [121]. How To achieve an efficient knockdown, it would likely be VLA-4 impacts CLL cell proliferation remains to be necessary to combine shRNA approaches with long-term elucidated. culture and cell cycle induction of CLL cells. To this end, co-culture techniques that mimic the proliferative and sup- portive microenvironment in CLL are continuously being Challenges in separating individual and shared improved [121]. contributions of VLA-4 and CD38 in CLL An alternative approach to separate the functions of CD38 pathophysiology and VLA-4, feasible in the absence of efficient knockdown techniques, is the analysis of discordant cases. Notably, the A possible molecular basis for the high correlation of CD49d correlation of risk factors in CLL samples is not absolute with and CD38 expression in CLL could be their physical associ- a considerable rate of discordancy. Functional analyses using ation in multi-protein-complexes. Recent reports suggest a these discordant samples are useful to define the dispensabil- variety of possible protein combinations [98, 110, 114, 115, ity or compensation of a specific molecule in a cellular func- 123, 124]. Two recent reports demonstrate a colocalization tion. Taking this road, we have been able to demonstrate that and physical association of CD38 and CD49d/CD29 by a CD38 is not required for BM homing of CLL cells while combination of immunofluorescence and immunoprecipita- VLA-4 is indispensable. However, in light of the potential tion approaches [110, 123]. Whether this interaction occurs enzymatic function of CD38, it is still conceivable that in via the CD49d or the CD29d subunit of the VLA-4 integrin CD38/VLA-4 double-positive cases CD38 can exert a sup- could not unequivocally be clarified [110, 123]. Interpatient portive function in energy-dependent VLA-4 activation. The variability, which is usually high in CLL, adds further basis of this crosstalk remains to be elucidated. 368 Ann Hematol (2014) 93:361–374 Therapeutic implications and lymph nodes. Recently, the recombinant anti-VLA-4 an- tibody natalizumab demonstrated the potential to overcome Collectively, the data demonstrate that CLL cells with high- stromal cell-induced resistance of B cell lymphoma cells risk features are in fact those that are most exquisitely depen- against cytotoxic drugs and rituximab in vitro [127]. dent on microenvironmental stimuli for their survival and Natalizumab is already approved as an anti-inflammatory proliferation. Notably, the pathophysiological and prognostic drug, and a number of small molecule inhibitors for VLA-4 importance of this crosstalk bears therapeutical consequences. have been developed [61], primarily for use in multiple scle- Finding a therapeutic means of interfering with the bi- rosis or asthma. However, most of the clinical trials using directional communication between CLL cells and the sup- these small-molecular-weight antagonists for VLA-4 have portive microenvironment would go a long way to finding a been terminated due to low efficacy or side effects of the definite cure for this disease. Besides single targeting of CD38 substances. A new generation of currently developed VLA-4 or VLA-4, disrupting the macromolecular complexes housing inhibitors might overcome the previous problems and widen these proteins, or inhibiting downstream signaling, are all the therapeutic spectrum of VLA-4 antagonism towards tumor conceivable strategies. Additionally, immunomodulatory therapy. In fact, VLA-4 antagonizing nanoparticles recently drugs such as lenalidomide, whose molecular mechanism of demonstrated adhesion-inhibitory and cytotoxic effects that action is still unclear, may indirectly impact CD38 and VLA-4 resulted in reduced tumor growth in a multiple myeloma expression and function, and this should be further mouse model [128]. investigated. Moreover, targeting of VLA-4 downstream signals might The ubiquitous expression of CD38 in many different cell provide an alternative approach. VLA-4 antagonism is known types and tissues obviously raises concerns regarding the safe- to mobilize stem and progenitor cells from bone marrow [61], ty of widespread inhibition of CD38 function. Currently, there and its ligand VCAM-1 is highly expressed in both CLL BM are three different anti-CD38 antibodies under evaluation for and lymph nodes (unpublished observation). It is therefore safety in clinical trials (Table 2). Two monoclonal antibodies expected that VLA-4 targeting will not only impede CLL cell are being tested in multiple myeloma (daratumumab, identifi- recirculation to these lymphoid niches, but additionally mobi- er: NCT00574288 and MOR03087, identifier: NCT01421186; lize tumor cells from lymphoid organs, similar to the effects http://clinicaltrials.gov) and a third in selected CD38+ seen with novel small molecule inhibitors. Notably, ibrutinib, hematological malignancies including CLL (SAR650984, the clinically active BTK inhibitor PCI-32765, was recently identifier: NCT01084252; http://clinicaltrials.gov). These shown to impair VLA-4-mediated adhesion of CLL cells antibodies are supposed to bind to CD38+ tumor cells and [129]. This is consistent with the clinical observation of a trigger antibody-dependent cellular cytotoxicity rather than transient lymphocytosis of ibrutinib-treated patients due to mobilization of CLL cells from lymphoid organs into the inhibit the biological CD38 functions. Treatment strategies using VLA-4 inhibitors supposedly peripheral blood [130]. Other small molecule antagonists used interfere with the recirculation of CLL cells into bone marrow in clinical trials for treatment of CLL, e.g., the phos- Table 2 Anti-CD38 strategies currently investigated in clinical trials for treatment of haematologic malignancies Substance Single/combination Study phase Company Disease entities Identifier Status Daratumumab Human anti-CD38 Single I/II Genmab MM NCT00574288 Recruiting (HuMax®- CD38) Daratumumab Human anti-CD38 Lenalidomide+ I/II Genmab MM NCT01615029 Recruiting dexamethasone MOR03087 Human anti-CD38 Single I/II MorphoSys MM NCT01421186 Recruiting AG Lenalidomid/dexamethasone Bortezomib/dexamethasone SAR650984 Humanized IgG1 Single I Sanofi B-NHL, AML, NCT01084252 Recruiting anti-CD38 B-ALL, CLL; MM SAR650984 Humanized IgG1 Lenalidomide+ I Sanofi MM NCT01749969 Recruiting anti-CD38 dexamethasone MM multiple myeloma, AML acute myeloid leukemia, ALL acute lymphoblastic leukemia All trials: refractory/relapsed patients Ann Hematol (2014) 93:361–374 369 7. Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK (1999) phatidylinositol 3-kinase inhibitor CAL-101 [131], might also Unmutated Ig V(H) genes are associated with a more aggressive affect VLA-4-mediated cellular functions in CLL. form of chronic lymphocytic leukemia. Blood 94(6):1848–1854 Conclusively, it is evident that CD38 and VLA-4 are more 8. Chen L, Widhopf G, Huynh L, Rassenti L, Rai KR, Weiss A, Kipps than just markers of an aggressive CLL cell type and that they TJ (2002) Expression of ZAP-70 is associated with increased B-cell receptor signaling in chronic lymphocytic leukemia. Blood 100(13): play functional roles in the pathobiology of the disease. As 4609–4614. doi:10.1182/blood-2002-06-1683 such, they represent therapeutic targets that may be exploited 9. Wiestner A, Rosenwald A, Barry TS, Wright G, Davis RE, in addition to, or in combination with, the currently developed Henrickson SE, Zhao H, Ibbotson RE, Orchard JA, Davis Z, novel approaches of interfering with CLL cell–tumor host Stetler-Stevenson M, Raffeld M, Arthur DC, Marti GE, Wilson WH, Hamblin TJ, Oscier DG, Staudt LM (2003) ZAP-70 expres- interactions. Targeting these molecules should also be tested sion identifies a chronic lymphocytic leukemia subtype with for its potential in avoiding the frequent relapses and devel- unmutated immunoglobulin genes, inferior clinical outcome, and opment of chemoresistance in CLL. distinct gene expression profile. Blood 101(12):4944–4951. doi:10. 1182/blood-2002-10-3306 10. Gattei V, Bulian P, Del Principe MI, Zucchetto A, Maurillo L, Buccisano F, Bomben R, Dal-Bo M, Luciano F, Rossi FM, Degan Acknowledgments Work of the authors is supported by the Austrian M, Amadori S, Del Poeta G (2008) Relevance of CD49d protein Science Fund FWF (project W1213 and SFB program P021 to R.G., expression as overall survival and progressive disease prognostica- project P25015-B13 to T.N.H.), the Austrian National Bank (projects tor in chronic lymphocytic leukemia. Blood 111(2):865–873. doi: 13420 to T.N.H., 14311 to R.G., the Paracelsus Medical University 10.1182/blood-2007-05-092486 Salzburg (projects E-10/11/058-HAR and E-12/15/074-HAH to T.N.H), 11. Shanafelt TD, Geyer SM, Bone ND, Tschumper RC, Witzig TE, the “Klinische Malignom und Zytokinforschung Salzburg-Innsbruck Nowakowski GS, Zent CS, Call TG, Laplant B, Dewald GW, GmbH”, and the province of Salzburg. Jelinek DF, Kay NE (2008) CD49d expression is an independent predictor of overall survival in patients with chronic lymphocytic Authors G.B., J.P.H., R.G., and T.N.H wrote the paper and approve of leukaemia: a prognostic parameter with therapeutic potential. Br J the submitted and final version. Haematol 140(5):537–546. doi:10.1111/j.1365-2141.2007.06965.x 12. Messmer BT, Messmer D, Allen SL, Kolitz JE, Kudalkar P, Cesar Conflict of interest The authors declare no competing financial conflict D, Murphy EJ, Koduru P, Ferrarini M, Zupo S, Cutrona G, Damle of interest. RN, Wasil T, Rai KR, Hellerstein MK, Chiorazzi N (2005) In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells. J Clin Invest 115(3):755–764. doi: 10.1172/JCI23409 13. 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Annals of HematologyPubmed Central

Published: Nov 28, 2013

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