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In vitro studies have demonstrated that surface expression of CD49d on chronic lymphocytic leukaemia (CLL) B cells facilitates leukaemic cell–stromal interactions by binding to fibronectin. This interaction reduces both spontaneous and drug-induced apoptosis. The present study measured CD49d expression by flow cytometry in a cohort of untreated CLL patients previously accrued to a prospective observational study and evaluated the relationship with overall survival (OS). Among the 158 CLL patients tested, the percentage of leukaemic B cells expressing CD49d ranged from 0 to 100%. When all risk factors were treated as continuous variables, CD49d expression showed moderate correlation with expression of ZAP-70 (r = 0·54; P < 0·0001) and CD38 (r = 0·58; P < 0·0001) but not %IGHV mutation. As a continuous variable, CD49d expression strongly correlated with OS (P < 0·0001). Recursive partitioning analysis suggested the 45% threshold of CD49d expression best predicted OS. Multivariate analysis, controlling for disease stage, ZAP-70, IGHV status and fluorescent in situ hybridization defects identified CD49d as an independent predictor of OS and was a better predictor of clinical outcome than ZAP-70, IGHV, or cytogenetics. This observational cohort study suggests that CLL B-cell expression of CD49d is an easily measurable and independent predictor of OS and CD49d expression in CLL. Importantly, anti-CD49d antibodies are already approved for treatment of other human diseases. Clinical testing of anti-CD49d therapy in CLL appears warranted.
Chronic lymphocytic leukaemia (CLL) is a common lymphoid malignancy with a variable clinical course. While the majority of patients with CLL are asymptomatic and have early stage disease at the time of diagnosis, some experience an aggressive disease course that leads to premature death while others live for decades and never require therapy. A number of recently identified prognostic parameters, including ZAP-70, IGHV mutation status, CD38 and cytogenetic abnormalities, can predict which early stage patients will experience early disease progression (Shanafelt et al, 2004).
The ideal prognostic parameter would not only identify patients at risk for worse clinical outcome, but also be relevant to disease biology and provide a therapeutic target. Her2-Neu overexpression in breast cancer patients is one example of a prognostic parameter associated with aggressive disease behaviour that can be directly targeted to improve clinical outcomes (Slamon et al, 2001) (Romond et al, 2005). Although the novel prognostic parameters identified for CLL are linked to leukaemic cell biology, the reason these characteristics are associated with worse clinical outcome is poorly understood and none are currently a direct target of available anti-neoplastic therapies.
Although CLL B cells relentlessly accumulate and are resistant to cell death in vivo, these cells rapidly become apoptotic on in vitro culture, suggesting that survival signals in the in vivo environment prevent apoptosis (Kay et al, 2007) (Ghia et al, 2002) (Caligaris-Cappio, 2003). Potentially supportive interactions between CLL B cells and various nurturing environments are complex and involve cytokine-mediated effects, cell–cell interactions, and cell–extracellular matrix interactions. As the bone marrow is invariably infiltrated by CLL B cells and is typically the site of relapse in patients who achieve treatment-induced complete remission, it is believed this tissue site provides a critical nurturing environment for CLL B cells in vivo (Caligaris-Cappio, 2003).
A limited number of studies have begun to explore specific interactions between the CLL B-cell and its environment. A variety of chemokines, such as stromal derived factor-1 (SDF1), are thought to attract leukaemic B cells to the marrow micro-environment by binding to receptors on the CLL B-cell surface (e.g. CXCR4) (Burger et al, 1999, 2000, 2001; Till et al, 2002). Once in the marrow, physical contact between stromal elements and leukaemic cells have been shown to promote CLL B-cell survival (Panayiotidis et al, 1996; Lagneaux et al, 1998; Burger et al, 1999, 2000). This protective effect is mediated in part through integrins on CLL B cells [e.g. alpha4 beta1 integrin (also known as very late antigen-4, VLA-4, CD49d) (Lagneaux et al, 1999)] and ligands [e.g. vascular cell adhesion molecule 1 (VCAM1) and fibronectin] expressed on marrow stromal cells (de la Fuente et al, 1999; Plate et al, 2000; Burger et al, 2001; Pedersen et al, 2002).
CD49d (integrin alpha 4) plays a critical role in leucocyte trafficking, activation, and survival, and also facilitates interactions between leucocytes and stromal cells found in the marrow or germinal center of lymphoid follicles via VCAM-1 and fibronectin (Rose et al, 2002). Notably, in addition to these adhesion functions, CD49d can also serve as a signalling receptor that influences B-cell survival via upregulation of Bcl-2 family members (Koopman et al, 1994; Hayashida et al, 2000). Other studies suggest that CD49d expression on CLL B cells is lower than normal B cells (Baldini et al, 1992; Eksioglu-Demiralp et al, 1996; Lucio et al, 1998), differs from other low grade B-cell malignancies (Baldini et al, 1992; Pinto et al, 1993; Eksioglu-Demiralp et al, 1996; Csanaky et al, 1997; Lucio et al, 1998), demonstrates intra-patient variation (Baldini et al, 1992; Eksioglu-Demiralp et al, 1996; Vincent et al, 1996; Sembries et al, 1999; Zucchetto et al, 2006a), and is associated with disease stage and the presence of lymphadenopathy (Eksioglu-Demiralp et al, 1996; Behr et al, 1998; Lucio et al, 1998; Till et al, 2002).
While several investigators have demonstrated that signalling via CD49d in CLL B cells reduces both spontaneous and drug-induced apoptosis in vitro (de la Fuente et al, 1999, 2002) little is known about the prognostic importance of CD49d in patients with CLL. We and others recently reported that CD49d gene (ITGA4) expression in CLL B cells correlates with CD38 expression (Durig et al, 2003) (Pittner et al, 2005) and have confirmed the correlation between CD49d and CD38 in CLL B cells at the protein level (Pittner et al, 2005). Preliminary studies suggest that CD49d expression in CLL B cells may relate to overall survival (OS) among CLL patients (Sembries et al, 1999) (Zucchetto et al, 2006a) independent of CD38 status (Zucchetto et al, 2006a) or 11q- (Sembries et al, 1999). Others studies have used CD49d as one of six factors used to assign a prognostic ‘surface antigen profiling’ score (Zucchetto et al, 2005)(Zucchetto et al, 2006b). However, there is no published data on the correlation of CD49d expression with a wider range of established prognostic parameters and its value as an independent prognostic factor. We report that the level of expression of CD49d protein on CLL B cells is prognostically important in a cohort of patients with untreated CLL previously enroled on a prospective observational study.
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In this observational cohort study, there was wide inter-patient variation in CLL B-cell CD49d protein expression (range 0–100% CD19+ cells). Increased CD49d expression was an independent predictor of OS on multivariate analysis including the other novel biological or molecular prognostic parameters. This is notable since multiple prior studies have suggested that ZAP-70 (Rassenti et al, 2004) (Crespo et al, 2003) (Orchard et al, 2004) (Rassenti et al, 2006), IGHV (Rassenti et al, 2004) (Krober et al, 2002) (Oscier et al, 2002) (Hamblin et al, 2002) and FISH (Shanafelt et al, 2006b) (Krober et al, 2002) (Oscier et al, 2002) (Dohner et al, 2000) are the most powerful molecular prognostic parameters identified to date.
Although a number of studies have looked at the correlation between CD49d expression in CLL B cells and surrogate markers of clinical outcome (i.e. disease stage), only a few have directly evaluated the relationship with OS (Sembries et al, 1999) (Zucchetto et al, 2006a). These small studies did not include comprehensive assessment of the other relevant prognostic parameters and could not evaluate the independent prognostic value of CD49d relative to disease stage, ZAP-70 status, FISH analysis, and IGHV mutation status or provide insight on how to optimally combine CD49d with these factors to predict clinical outcome. In the present study, CD49d was the single strongest prognostic factor in both univariate and MV analysis including these factors and a two factor prognostic model using CD49d and chromosome analysis by FISH could not be significantly improved upon by adding any additional factor(s).
As many prognostic parameters are now available for use in patients with early stage CLL, progress will require identification of molecular features that provide insight into disease biology and have the potential for therapeutic targeting. CD49d mediates interactions between CLL B cells and stromal cells and facilitates in vitro resistance to both spontaneous and drug induced apoptosis (de la Fuente et al, 1999, 2002). The association of CD49d with other poor prognostic parameters (IGHV unmutated, ZAP-70+, CD38+) in our study implies that leukaemic clones with these molecular characteristics may receive enhanced stromal nuturing with resultant effects on leukaemic cell viability, migration and apoptotic resistance (Panayiotidis et al, 1996; Lagneaux et al, 1998, 1999; Burger et al, 1999, 2000, 2001; Plate et al, 2000; Pedersen et al, 2002; Till et al, 2002). CD49d expression/ligation influences a number of factors that relate to the migratory and invasive properties of CLL B cells including adhesion to fibronectin and the endothelium (Eksioglu-Demiralp et al, 1996; Vincent et al, 1996), upregulation of MMP-9 through phosphatidylinositol 3 kinase/Akt signaling (Redondo-Munoz et al, 2006), podosome formation (Redondo-Munoz et al, 2006) and chemokine-dependent motility through the endothelium (Till et al, 2005). Others have demonstrated that the CD49d – fibronectin interaction reduces spontaneous apoptosis in CLL B cells by increasing the Bcl-2/Bax ratio (de la Fuente et al, 1999) and also appears to reduce fludarabine-induced apoptosis by increasing Bcl-XL levels (de la Fuente et al, 2002, 2003).
Perhaps most importantly, our observations have realistic and relatively immediate therapeutic potential since anti-CD49d anti-bodies are available for clinical use, have already been widely studied in humans for treatment of multiple sclerosis (Rudick et al, 2006) (Polman et al, 2006), and are approved for clinical use by the Food and Drug Administration (FDA) for that indication. Combining anti-CD49d antibodies with established chemotherapy or immunotherapy regimens in CLL could impair stromal nuturing of leukaemic cells and overcome apoptotic resistance. Unlike ZAP-70, IGHV mutation status, CD38 and cytogenetic abnormalities, measurement of CD49d could thus be an actionable prognostic test with opportunities for direct therapeutic targeting. Other approaches to therapeutically target CD49d are also in development including small peptide mimetics to block CD49d binding to VCAM-1 or fibronectin in the hopes of enhancing treatment efficacy (Rose et al, 2002).
Our findings are subject to a number of limitations. Although the patient group studied was a defined cohort of previously untreated patients enroled on a prospective observational trial, larger validation studies are needed before CD49d can be used as a prognostic parameter in clinical practice. Similar to other investigators (Sembries et al, 1999), we used a two-colour (CD19, CD49d) flow cytometry-based strategy analogous to that used for evaluation of CD38 and ZAP-70. Zucchetto et al (2006a) have previously reported a three-colour (CD19, CD5, CD49d) strategy to measure CD49d. The best way to measure CD49d expression in CLL B cells is unknown and will need to be determined. While 45% expression was the best threshold to classify patients at risk of shorter survival in the present cohort, the optimal cut-off point to stratify patient risk will need to be defined in future studies utilizing even larger cohorts. The stability of CD49d expression during the course of the disease is also unknown and clearly requires future evaluation.
Our study also has a number of important strengths. The individuals studied were a well-defined cohort of CLL patients participating in a prospective observational trial. Over 90% of patients had early stage disease at study entry and thus represent the patient group for whom prognostic tools are most needed. CD49d correlated with OS as a continuous variable and was an independent predictor of OS on multivariate analysis that included the other well-established prognostic parameters. Finally, the assay used for CD49d involves a two-colour flow cytometric assay for a cell surface antigen that should be available in nearly every modern clinical and research laboratory.
In conclusion, CLL B-cell membrane expression of CD49d as measured by flow cytometry is a powerful prognostic parameter in patients with CLL. The prognostic utility of CD49d expression is independent of ZAP-70, IGHV mutation status and cytogenetic abnormalities evaluated by FISH. As CD49d is functionally important to CLL B-cell biology and leukaemic cell survival, our findings suggest CD49d may have promise as a therapeutic target in CLL. Anti-CD49d antibodies are already FDA-approved for treatment of other human diseases. Clinical testing of anti-CD49d therapy in CLL appears to be warranted.