The prognostic role of CXCR3 expression by chronic lymphocytic leukemia B cells

Chemokine receptors are involved in tumor progression and several of these receptors, including CXCR3, are expressed by chronic lymphocytic leukemia (CLL) B cells. This study was aimed to examine a possible relationship between CXCR3 expression in CLL and the clinical evolution of the disease.Using flow activated cell sorting (FACS), we analyzed the level of expression of CXCR3 on blood CLL B cells from 76 consecutive patients. The results were correlated with CD38 expression, IgVH gene status and clinical outcome.CXCR3, measured as mean fluorescence intensity (MFI), was unimodally expressed by blood tumor cells at various levels (range, 3.5 to 232.3) but levels within individual patients were remarkably stable over time. Low CXCR3 expression by CLL B cells was strongly associated with Rai disease stages III and IV (p<0.0001) and a pattern of diffuse tumor infiltration of the bone marrow (p<0.0001). In the 28 cases available for genetic studies, low CXCR3 expression also showed good concordance with tumor unmutated IgVH gene status (p<0.04), and tended to correlate with high CD38 expression (p<0.06). Patients with low CXCR3 expression (MFI < or =15) had a shorter survival (p<0.0001) and, in multivariate analysis, low CXCR3 expression (MFI pound15) was an independent predictor of poor outcome (hazard ratio 24.5; p<0.01). INTERPRETATION AND CONCLUSIONSL: CXCR3 expression by CLL B cells appears to be stable within individual patients. Tests to assay this chemokine receptor are cheap and easy to perform and the results could be of prognostic value in CLL.

section_content: -cell chronic lymphocytic leukemia (CLL), the most common leukemia in adulhood, is a malignancy of B lymphocytes immunophenotypically characterized by the expression of CD19, CD5, CD23 and low-intensity monoclonal immunoglobulin (Ig) on their cell surface. 1 This disorder presents at older ages and its course is typically indolent. However, a substantial number of patients die of progressive disease and it is now clear that CLL is a heterogeneous entity, not only at the clinical level but also at molecular and cellular levels, as evidenced by differences in the number of somatic mutations present in the Ig variable heavy-chain (IgVH) gene and by a variety of cytogenetic alterations. 4] [5] However, since these classifications are based on clinical features, they fail to predict the course of the disease at the onset. Therefore, in recent years, considerable attention has been given to finding parameters that could anticipate the accelerated accumulation of and invasive behavior by CLL B cells. This has led to the analysis of many possible prognostic factors in this disease, including lactate dehydrogenase levels, β2-microglobulin, lymphocyte doubling time, diffuse pattern of bone marrow invasion, soluble CD23, CD38 surface expression, cytogenetic abnormalities, IgVH gene somatic mutations and ZAP-70 expression. 2, 6 hemokines and their receptors are essential in the initial driving of hematopoietic cells to their homing locations in physiological as well as in pathological conditions, including tumor invasion. 7, 8 CLL B cells have been shown to express CXCR3, CXCR4 and CXCR5. 9, 11 he two latter receptors are a common feature of most normal human B lymphocytes. 7, 12 In contrast, CXCR3 is only expressed by a minor fraction of normal B lymphocytes, including CD5 positive and negative subsets. 9, 11 his chemokine receptor has been shown to play a critical role in the mobilization of activated T lymphocytes, NK cells, dendritic cells and other leukocytes towards an inflammatory focus, where their interferon-γinducible ligands CXCL9, CXCL10 and CXCL11 (Mig, IP-10 and ITAC, respectively) are abundantly produced by several cell types. 7, 13 The unexpected and distinctive expression of CXCR3 by CLL B cells led us to explore its possible role in the disease.
section_name: B

section_name: Design and Methods

section_content: Seventy-six consecutive patients diagnosed with Bcell chronic lymphocytic leukemia (CLL) were included in the study. Twenty-seven of these patients were recruited at diagnosis. In all the cases, the monoclonal B cell expansion was CD19 +, CD23 + and CD5 +. Clinical data corresponding to these cases are summarized in Table 1. CLL was diagnosed in all cases according to international CLL workshop criteria and staged according to the Rai system. 4 Patients` peripheral blood samples were obtained by venipuncture, and CLL B lymphocytes were isolated as previously reported. 14 All patients were informed about the objectives and methods of the study and gave their consent according to the Declaration of Helsinki. Approval for this study was obtained from the institutional review board (Comisión Ética, Hospital Universitario Puerta del Mar). The median follow-up time since the first CXCR3 determination was 17. 2 (range 6-31) months, and that since the diagnosis was 45 (range 8-193) months. Fifteen patients had died due to CLL by the time of the last evaluation.
section_name: Patients and samples

section_content: Lymphocyte isolation medium was purchased from ICN (Costa Mesa, CA, USA). Monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC) anti-CD5 (clone L17F12), to phycoerythrin (PE) anti-CD38 (clone HB-7) and anti-CXCR3 (clone IC6), to allophycocyanine (APC) anti-CD5 (clone L17F12) and to peridinin chlorophyll protein (PerCP) anti-CD19 (clone 4G7), and FITC-, PE-, PerCP-and APC-conjugated monoclonal antibodies of appropriate isotype used as negative controls, were obtained from Becton Dickinson (San Jose, CA, USA). The FITC-labeled anti-CXCR3 monoclonal antibody (clone 49801) was from R & D Systems, Inc. (Minneapolis, MN, USA).
section_name: Materials

section_content: Three or four-color labeling experiments were performed as previously reported. 15 Briefly, mononuclear cells were isolated from blood samples by density centrifugation on Ficoll and 0. 5×10 6 peripheral blood mononuclear cells in 100 µL of phosphate-buffered saline (PBS) were incubated with saturating quantities of the appropriate FITC-, PE-, PerCP-and APC-conjugated monoclonal antibodies for 30' in the dark at 4°C. After staining, cells were washed twice in PBS. Isotypematched FITC, PE-, PerCP-and APC-conjugated irrelevant monoclonal antibodies were used as negative controls. Labeled cells were analyzed within 1 hour in a FACScalibur instrument (Becton Dickinson) equipped with an air-cooled argon ion laser that operated at 488 nm and a red diode laser that operated at 635 nm. A minimum of 5000 cells were collected and analyzed by using the CELLQUEST software (Becton Dickinson), and the percentage, and mean fluorescence intensity (MFI) of the cells for each examined molecule were monitored. Lymphocytes were selected in the forward versus side scatter dot plot, and additionally gated as CD19 CD5 positive cells corresponding to either FL3 -FL1 or FL4-FL3 dot plots depending on the monoclonal antibody combination used. The color combination commonly used in the present study was PercP-anti-CD19, PE-anti-CXCR3 and FITC-anti-CD5. The percentage of CD38 CLL B cells was also explored using the same combination with PE-anti-CD38 instead of PEanti-CXCR3. In some experiments, the combination PercP-anti-CD19, APC-anti-CD5 and FITC-anti-CXCR3 was used as an additional control for the fluorochrome and the anti-CXCR3 monoclonal antibody employed, and gave equivalent results. Background fluorescence was set so that less than 1% of the cells were positively stained with the corresponding negative control. The expression of CXCR3 by blood CLL B cells from all patients exhibited a rather homogeneous uni-modal peak (Figure 1A ). Accordingly, the MFI parameter was chosen to compare the differences of expression by the patients` cells.
section_name: Immunofluorescence staining and flow cytometry analysis

section_content: The IgVH gene was amplified as previously described. 16 Briefly, RNA was obtained from purified CLL B cells using a Total RNA Isolation Kit (Roche, Barcelona, Spain), and random-hexamer primed cDNA was generated using a First Strand cDNA Synthesis Kit (Roche). cDNA was amplified by polymerase chain reaction (PCR) using a mixture of complementary 5′ oligonucleotides specific for frame region 1 (FR1) corresponding to all the VH1-VH6 IgVH families, and a consensus 3′ oligonucleotide complementary for the JH region. 16 PCR was performed with the Expand High Fidelity Plus PCR System in 50 µL (Roche). Cycling conditions were 94ºC for 30 second, 60ºC for 30 seconds and 72ºC for 60 seconds up to 35 cycles, followed by a final period of 7 minutes at 72ºC. Products were purified and directly sequenced using the same JH primer in a Genetic Analyzer ABI-310 (Applied Biosystems, Foster City, CA, USA). Nucleotide sequences were analyzed with the IMGT IgVH data base (http://imgt. cines. fr/). 7] [18] Repeated PCR and sequencing reactions from cDNA samples from five CLL patients (two unmutated and three with a large number of mutations) were performed, and identical results were obtained.
section_name: IgVH gene somatic mutations analysis

section_content: Group-wise comparisons of clinical and biological data were performed using the Mann-Whitney U test and the Kruskal-Wallis test (for continuous variables) and the two-tailed Fisher´s exact test or χ 2 -test (for categorical variables). Survival was defined as the time from diagnosis to death or to the last observation. Overall survival was estimated according to the Kaplan-Meier method, and comparisons between curves were performed using the two-sided log rank test. Associations between overall survival and uni-and multivariate analyses were performed according to the Cox´s proportional hazard model.
section_name: Statistical analysis

section_name: Results

section_content: The expression of CXCR3 by CLL B cells was examined in blood samples from 76 consecutive patients. Table 1 summarizes the expression results along with
section_name: Association between CXCR3 expression by CLL B cells and clinical data

section_content: IgVH gene configuration status (<2% or>2% of mutations). In figures 1B, 1C, 1D and 1E results are expressed as the median (thick lines) and interquartile range (boxes) for 76, 41, 63 and 28 patients, respectively. the clinical data of these cases. As previously reported, [9] [10] [11] in all the CLL cases blood tumoral cells expressed this chemokine receptor. Nevertheless, the level of CXCR3 expression (MFI) was rather variable from patient to patient, ranging from 3. 5 to 232. 1 ). The use of a MFI of 20 as a cutoff limit for CXCR3 expression gave a similar patient group distribution and, accordingly, identical results.
section_name: Association between CXCR3 expression (MFI) by peripheral blood CLL B cells and Rai stages. C. Association between CXCR3 expression (MFI) by peripheral blood CLL B cells and the pattern of tumor bone marrow infiltration (diffuse and non-diffuse). D. Association between CXCR3 expression by peripheral blood CLL B cells and CD38 expression (%). E. Association between CXCR3 expression by peripheral blood CLL B cells and

section_content: 1] [22] In the present study, samples from 63 patients were available for analysis of CD38 + percentages. The median percentage of CD38 + CLL B cells was 5 (range, 0 to 90; n=63). When CD38 expression was explored in these cases separated according to their corresponding CXCR3 expression into groups with MFI ≤15 and >15, the CD38 values tended to be significantly higher in the group of patients with low CXCR3 expression (Figure 1D ; p<0. 06). In addition, when the patients were separated into cases with CD38 percentages either higher or lower than 30% (CD38 high and CD38 low, respectively), and their corresponding CXCR3 expression into groups with MFI ≤15 and >15 MFI, the differences observed also tended to be significant (Table 1, bottom part; p<0. 06). Similar results were obtained using 20% as the cut-off. Only one discordant case moved from the CXCR3 low CD38 low group to the CXCR3 low CD38 high group when the the CD38 cut-off was set at 20% instead of 30%. Concordance between the two parameters was observed for 41 patients (65. 1%): 34 CXCR3 high CD38 low cases and seven CXCR3 low CD38 high cases. The number of patients without this concordance was 22 (34. 9%), and these patients could be separated into two groups, one of 18 cases (81. 8%) showing CXCR3 low CD38 low expression and another of four cases (18. 1%) exhibiting CXCR3 high and CD38 high expression. Clinical data from CXCR3/CD38 combined subgroups are presented in Table 2. As can be seen, the CXCR3 high /CD38 low group had more favorable clinical data, whereas the CXCR3 low /CD38 high group showed worse indicators, as expected. Interestingly, the main group of patients with discordant CXCR3/CD38 expression, consisting of CXCR3 low /CD38 low cases, presented intermediate clinical data. Finally, the small CXCR3 high /CD38 high group (four patients) seemed to be older and have more favorable clinical data. 

18] Accordingly, CXCR3 expression by the tumoral cells of CLL patients was correlated with the mutational status of their corresponding IgVH gene. Cells from only 30 cases of those included in the present study were available for this analysis. In two cases the study was not conclusive. The results of the valid 28 cases are given in Table 1 (bottom part). The majority of cases showing low CXCR3 expression (MFI ≤15) on blood tumor cells (9 out of 13) had unmutated IgVH genes; in contrast, most cases with high CXCR3 levels (MFI >15) (10 out of 15) presented somatically mutated IgVH genes (p<0. 003). In addition, Figure 1E shows that the levels of CLL B-cell CXCR3 expression, measured as MFI, observed in the patients separated into two groups according to the number of IgVH mutations were 8. 7 and 35. 6 for those with a germinal line (< 2%) and somatically mutated (> 2%) IgVH configuration, respectively (median, p<0. 04; Mann-Whitney U test). A specific pattern of IgVH-family usage was not observed in these patients (data not shown).
section_name: Relationship of CXCR3 expression with CD38 + CLL B cell percentage and with IgVH gene mutational status

section_content: A Kaplan-Meier analysis was performed to assess the possibility that CXCR3 expression by blood CLL B cells was associated with overall survival. Figure 2A shows that the survival time from diagnosis for CLL cases showing CXCR3 MFI ≤15 was clearly shorter than that of the cases exhibiting MFI >15 (median: 65 months; 95% CI: 40-90; p<0. 0001). In addition, Figure 2B shows that a similar analysis of the 46 patients in Rai stages 0 and I also revealed a significant association of those CLL cases with low CXCR3 values (MFI ≤15) with an unfavorable course (median survival 108 months; 95% CI 41-175; p<0. 002). 1] [22] The effect of the combination of CXCR3 and CD38 parameters was examined in the 63 cases available. Figure 2C shows that the CXCR3 low CD38 high group also had a shorter survival (median: 34 months; 95% CI 10-58; p<0. 0001), the CXCR3 high CD38 low combination appeared to confer a good prognosis (the median survival time was not reached), and an intermediate prospect for survival was obtained for the CXCR3 low CD38 low group (median: 84; 95% CI: 30-138; p<0. 0001). The small number of cases showing CXCR3 high CD38 high expression apparently had a favorable prognosis. 

The fact that a considerable proportion of the patients in Rai stages 0 and I exhibited a low level of CXCR3 expression (MFI ≤15) led us to explore the possible role of CXCR3 expression as a predictor of the clinical outcome for these cases. To confirm the possibility that CXCR3 expression by CLL B cells had a predictive value, a Cox proportional regression model analysis was performed. All variables analyzed were individually evaluated, with the exception of IgVH mutational status, which was not included due to the limited number of cases available for the assessment of this parameter. As shown in Table 3 (upper part), hemoglobin concentration, CD38 (%) and CXCR3 (MFI) expression by blood CLL B cells were significant factors in determining overall survival. In addition, Rai stages III-IV and a diffuse pattern of bone marrow infiltration also tended to be significant. Variables significantly related to survival were then included in the multivariate Cox proportional hazard regression model. As can be seen (Table 3, bottom part), this analysis revealed that low CXCR3 expression (MFI ≤15) by blood CLL B cells was the only parameter that independently and strongly predicted a poor clinical evolution (HR 24. 52).
section_name: CXCR3 expression level by blood CLL B cells and survival

section_content: It is now clear that CLL is a complex condition that presents with different clinical outcomes and survival rates. As a result, increasing efforts are being made to find parameters that are readily detectable at reasonable cost for general use as prognostic factors. The present study shows that CXCR3 expression is a stable parameter easily detectable on blood CLL B cells. Furthermore, low CXCR3 expression by blood CLL B cells is associated with clinical features clearly indicative of disease progression, such as diffuse bone marrow tumor infiltration, as well as with higher disease stage categories. Kaplan-Meier analysis of the patients separated into two groups according to whether their blood CLL B cells showed low (MFI ≤15) or high (MFI >15) expression of CXCR3 revealed that the former group had a significantly shorter survival. The question arises as to whether these findings only indicate an association between low CXCR3 expression and advanced stages of the disease, or whether this parameter can actually predict a poor prognosis at the onset of disease. In favor of the latter assumption is the fact that, during the period of this study (31 months), and after repeated evaluation of a large number of these patients at different times, CXCR3 expression was stable and no transition 2, 6, 7, [16] [17] [18] 20, 21 Data obtained from the available 28 samples in this study revealed a good correlation between low CXCR3 expression and germline IgVH gene configuration, although the association between these two parameters was partial, and the overall concordance was 78% (22 of 28 samples analyzed). There ore, mutational IgVH gene status and CXCR3 expression appear to be widely overlapping parameters but not totally coincidental phenomena. The present results also show that low CXCR3 expression tends to correlate with high CD38 expression. Examined in combination, the group of patients exhibiting low CXCR3 and high CD38 expression had short survival times, while the group with high CXCR3 and low CD38 expression had a favorable prognosis. However, the proportion of CXCR3/CD38 discordant cases (34. 9%) was considerable, and most of these (81. 8%) corresponded to patients with low CXCR3 and low CD38 expression. Moreover, this latter group had intermediate clinical data and survival probabilities (Table 2 and Figure 2C ), a finding that allowed the identification of a sub-group of patients in whom low expression of CXCR3 appeared to indicate a poor prognosis. This observation is similar to those previously reported for CLL cases without concordance between CD38 expression and either ZAP-70 expression or IgVH mutational status. [21] [22] [23] More importantly, multivariate analysis revealed that low expression (MFI ≤15) of CXCR3 by blood CLL B cells was a clear, independent predictor of death. Collectively, these findings lead us to propose that CXCR3 expression by CLL B cells could be considered a predictive factor for prognosis. 

It is well established that chemokine receptors participate in tumor invasion. 8 Nevertheless, the mechanisms by which CXCR3 might act in the pathogenesis of CLL remain unknown. 0] [11] In addition, the chemokine CXCL12 (SDF-1), the ligand of CXCR4, is produced by stromal cells of many organs, included the bone marrow, and it exerts not only chemotactic, but also anti-apoptotic effects on CLL B cells in in vitro systems. 24 Furthermore, CCR7 expression on the tumor cells of certain CLL patients has been associated with lymph node infiltration. 25 In the light of this, it has been proposed that these chemokine receptors participate in the invasion the bone marrow and lymph nodes by CLL B cells. Hence, the prognostic value of CXCR4 and CCR7 expression in CLL has been examined, and conflicting results have been reported. Although low CXCR4 expression has been associated with enhanced survival, 26 and high CXCR4 expression with advanced disease stages, 27 two studies including larger numbers of patients 28, 29 did not provide evidence of any relationship between these parameters and either disease stage or a diffuse pattern of bone marrow infiltration, and only showed a correlation with the number of circulating CLL B cells. Moreover, it has recently been found that tumor cells from a variety of non-Hodgkin's B-cell lymphomas, including CLL, express an intricate profile of CC and CXC chemokine receptors that exhibit marked functional heterogeneity. 30 Therefore, the mechanisms underlying the invasive capacity of CLL B cells still remain largely unknown, and factors additional to CXCR4 and CCR7 expression are likely to be implicated in the progression of the disease. Further work will be required to ascertain whether low CXCR3 expression has a role in the invasive capacity and progression of this tumor.
section_name: Discussion

section_content: this work was supported by grants 225/03 and 91/04 from the

section_name: Author Contributions

section_content: JAB acted as principal investigator: he designed the protocols and wrote the report; EO: conducted most laboratory work and helped in manuscript preparation; LD-P: conducted laboratory genetic work; JM, AP, RF participated in the data collection; AC-C: analysis and interpretation of the genetic results.
section_name: Author Contributions

section_content: The authors reported no potential conflicts of interest.
section_name: Conflict of Interest