Prognostic Value of Plasma Interleukin-6 Levels in Patients with Chronic Lymphocytic Leukemia

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1 1071 Prognostic Value of Plasma Interleukin-6 Levels in Patients with Chronic Lymphocytic Leukemia Raymond Lai, M.D, PhD. 1 Susan O Brien, M.D. 2 Taghi Maushouri, M.S. 1 Anna Rogers, 1 Hagop Kantarjian, M.D. 2 Micheal Keating, M.D. 2 Maher Albitar, M.D. 1 1 Department of Hematopathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas. 2 Department of Leukemia, The University of Texas M. D. Anderson Cancer Center, Houston, Texas. BACKGROUND. Interleukin 6 (IL-6) is a B-cell growth and differentiation factor, which may promote the growth of B-cell neoplasms. In chronic lymphocytic leukemia (CLL) patients, IL-6 plasma levels increased in a stage-dependent manner, suggesting that IL-6 may be a useful prognostic marker. The purpose of this study is to fully assess the prognostic value of IL-6 in CLL patients. METHODS. We measured the plasma levels of IL-6 in 100 CLL patients using an enzyme-linked immunoassay method. RESULTS. Increasing levels of IL-6 significantly correlated with patient age, severity of anemia, Rai stage, white cell count, and -2-microglobulin ( -2M). Although CLL patients did not differ significantly from the normal controls in the median IL-6 plasma level (P 0.38), patients with advanced diseases (defined by Rai stage III/IV or -2M 3.5) had a significantly higher median IL-6 plasma level than the normal controls (P 0.05). Furthermore, in patients with advanced diseases, Cox regression hazards model showed that a higher IL-6 level correlated with shorter survival (P ). Using IL-6 level of 3 pg/ml as a cutoff, patients with low IL-6 levels had a significantly longer overall survival than those with high IL-6 levels (log rank test, P 0.002). In patients with CD38-positive CLL, patients with high IL-6 levels ( 3 pg/ml) had significantly shorter survival (P 0.03). To conclude, IL-6 is a particularly useful predictor for survival in CLL patients with advanced diseases. CONCLUSIONS. Our findings suggest that patients with advanced-stage CLL as well as high IL-6 plasma levels may require aggressive therapeutic approaches and special consideration for experimental therapy. Cancer 2002;95: American Cancer Society. DOI /cncr KEYWORDS: interleukin-6, chronic lymphocytic leukemia, prognosis, CD38 positivity. Presented at the Annual Meeting of the American Society of Hematology, San Franscico, December Address for reprints: Maher Albitar, M.D., Department of Hematopathology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 72, Houston, TX ; Fax: (713) ; malbitar@mdanderson.org Received 16 February 2002; revision received February 20, 2002; accepted April 12, Chronic lymphocytic leukemis (CLL) is characterized by the expansion of mature-appearing B lymphocytes. Impaired apoptosis and extended survival of neoplastic cells are important factors in the pathogenesis of this disease. 1 The mechanisms underlying these defects remain to be defined, but autocrine secretion and aberrant levels of certain cytokines have been implicated. 2 4 Interleukin 6 (IL-6) is a B-cell growth and differentiation factor. High levels of IL-6 are commonly found in the plasma from patients with autoimmune diseases. 5 IL-6 may play a role in the autocrine pathway by promoting the growth of some B-cell lymphomas and myelomas. 6,7 Initially, IL-6 production was found to be lower in B-CLL cells of patients with progressive disease. 8 However, several more recent studies found that IL-6 plasma levels were higher in CLL patients than in normal controls and that the levels are elevated in a 2002 American Cancer Society

2 1072 CANCER September 1, 2002 / Volume 95 / Number 5 TABLE 1 Correlation of Interleukin-6 Plasma Levels and Some of the Clinical and Laboratory Parameters Clinical parameters Mean (range) R value P value 2-microglobulin 3.1 ( ) Hemoglobin (g/dl) 12.1 (4 17.4) Age 60 (32 82) White cell count 62.1 (3 652) Platelet (4 355) disease stage-dependent manner. 9,10 These findings suggest that IL-6 may be a prognostic marker for CLL. Nevertheless, this possibility has not been examined extensively. To assess fully the prognostic value of IL-6 in CLL patients, we measured IL-6 plasma levels in a cohort of 100 patients and correlated the levels with overall survival in these patients. In addition, we attempted to determine whether IL-6 levels correlate with other known prognostic factors, including CD38 expression and -2-microglobulin. MATERIALS AND METHODS Patients One hundred CLL patients were included in this study (Table 1). There were 67 males and 33 females, of whom 56 were newly diagnosed and previously untreated patients (from 1994 to 1995). In each case, the diagnosis was made based on a combination of morphology, immunophenotypic analysis, and molecular studies (if indicated). Cytogenetic data were available for the majority of patients. Patients were treated according to the Institutional Review Board-approved clinical research protocols at M. D. Anderson Cancer Center (MDACC) after signing informed consent. All patients were regularly followed up in the outpatient clinic of the Department of Leukemia (MDACC). Blood Samples Platelet-poor ethyldiaminetetraacetic acid plasma samples were collected from 10 ml of peripheral venous blood from each patient at the time of their initial presentation and evaluation at the MDACC. Specimens were quickly frozen at 80 C soon after collection. Plasma samples from 11 normal individuals were used to determine control plasma values for IL-6. Plasma IL-6 Enzyme-Linked Immunoassay (ELISA) The IL-6 plasma levels were measured by an immunoassay method using a standard IL-6 ELISA kit purchased from R & D (Minneapolis, MN), following the manufacturer s instructions. Briefly, 100 L of plasma and 100 L assay diluent were added to each well in a 96-well polystyrene microplate. Each of these wells was coated with a murine monoclonal antibody specific for IL-6. The mixtures were then incubated at room temperature for 2 hours. The plates were washed four times with wash buffer (400 L) supplied with the kit. Enzyme-linked polyclonal antibody specific for IL-6 was added to the wells and the mixtures were incubated at room temperature for 2 hours followed by another washing. A substrate solution was added to the wells and a blue color developed. The intensity of the blue color was proportional to the amount of IL-6 in the patient s plasma. The color development was stopped with a stop solution provided in the kit. The intensity of the color was measured and compared with a standard curve. Reading was done at the 540-nm wavelength as recommended by the manufacturer. Results were expressed in picograms per milliliter. We compared the standard IL-6 ELISA assay with that of the high sensitivity (HS) assay by reanalyzing 40 samples using the HS assay. There was no significant difference between the two assays (P 0.5) in these samples. The sensitivity of both assays is (0.094 pg/ml) as reported by the manufacturer. In a separate experiment, 11 samples were analyzed fresh before and after freezing. There was no significant difference in IL-6 levels between the two measurements. Statistical Analysis Associations between patient characteristics (covariates) were assessed for pairs of numerical variables by Spearman correlation and associations between categorical and continuous variables were assessed by Kruskall Wallis by ranks and by the Cox proportional hazards model. The clinical outcome evaluated was overall survival, which was analyzed statistically by performing Kaplan Meier plots and log rank tests to calculate differences in outcome between groups. RESULTS Normal controls had a median IL-6 plasma level of 2.6 pg/ml (range, pg/ml) and CLL patients had a median IL-6 plasma level of 6.8 pg/ml (range, pg/ml). This difference in the IL-6 medians between normal controls and CLL patients was not statistically significant (P 0.38). However, CLL patients with Rai Stage III or IV had significantly higher levels of IL-6 than those with Rai Stage 0, I, or II (P 0.006, Kruskal Wallis test). Similarly, CLL patients with a -2M level higher than 3.5 also had a higher median IL-6 plasma level compared with those with levels of -2M below 3.5 (P 0.02, Kruskal Wallis

3 IL-6 in CLL Patients/Lai et al test). We also analyzed the IL-6 level in previously untreated patients (n 56) separately. We found no significant difference in survival between patients with high expression of IL-6 and patients with low expression (P 0.4). Increasing levels of IL-6 in our cohort of patients positively correlated with a number of clinical and biologic parameters including patient age, degree of anemia, -2M, and white cell count (Table 1). No significant correlation was found between IL-6 level and platelet counts. We also correlated IL-6 levels with karyotypic analysis at the time of testing. Karyotypic analysis was available for 54 patients. The patients were assigned to two groups according to this analysis. The first group consisted of patients who had one or more of the following: 11q deletion, trisomy 12, any chromosome 17 aberration; there were 9 patients (17%) in this group. There was no significant difference in IL-6 level between the two groups (P 0.23, analysis of variance test). When statistical analysis for correlation with survival was performed using the Cox proportional hazards model and IL-6 as a continuous variable, we found significant correlation between increasing levels of IL-6 and shorter survival (P ). Using a multivariate model incorporating -2M and IL-6, only -2M was identified as a significant predictor of survival. Because we previously identified that relatively high -2M levels ( 3.5) were associated with higher levels of IL-6, we used the Cox proportional hazards model to predict survival in the group of patients with high levels of -2M. In patients (n 48) who had high levels of -2M, IL-6 expression was a strong predictor of survival (P ). By contrast, IL-6 expression was a not a significant predictor of survival in patients with low levels of -2M (P 0.7). Similarly, in view of the correlation between IL-6 levels and Rai stage shown previously, we evaluated the prognostic value of IL-6 as a continuous variable in patients with advanced and early Rai stage disease. We found that IL-6 expression was a significant predictor of survival in the group of patients with an advanced Rai stage (III or IV), but not in the group of patients with with an early Rai stage (0 II; P 0.029). We then analyzed the relationship between overall survival and IL-6 expression as a discrete variable. Using 3 pg/ml (which was equivalent to the sum of the median and SD of the normal controls) as a cutoff, we divided our cohort into two groups: the low IL-6 group (n 39) and the high IL-6 group (n 61). The low IL-6 group had a median IL-6 level of 2.3 pg/ml, which was significantly lower than that of normal controls (P ). The high IL-6 group had a median FIGURE 1. Prognostic significance of interleukin-6 in patients with chronic lymphocytic leukemia. FIGURE 2. Prognostic significance of interleukin-6 in CD38-positive chronic lymphocytic leukemia patients. IL-6 level of 4.4 pg/ml, which was significantly higher than that of normal controls (P ). As shown in Figure 1, the low IL-6 group had a significantly longer overall survival than the high IL-6 group (P 0.002). The predictive value of IL-6 in CD38-positive and CD38-negative patients was also examined. There were 54 patients in this cohort who were CD38 positive and 46 patients who were CD38 negative. IL-6 levels did not correlate with CD38 expression and IL-6 was not a significant predictor of survival in CD38- negative cases. However, using the cutoff of 3 pg/ml, IL-6 expression stratified CD38-positive cases into two prognostic groups. Only 1 of 32 patients with CD38 positivity and a low level of IL-6 died in a 50-month follow-up period (Fig. 2). In contrast, in the same follow-up period, 17 of 22 patients with CD38 positivity and high expression of IL-6 died of disease. Therefore, IL-6 was a predictor of survival in CD38-positive

4 1074 CANCER September 1, 2002 / Volume 95 / Number 5 CLL patients (P 0.03). A similar significant correlation between IL-6 expression and survival in CD38- positive cases was also obtained when statistical analysis was performed using the Cox proportional hazards model and IL-6 expression as a continuous variable. DISCUSSION In this study, we identified that increasing levels of IL-6 correlate with overall survival as well as a number of established prognostic factors for CLL, including patient age, degree of anemia, -2M level, and Rai stage. Although CLL patients as a group do not have a significantly higher median level of IL-6 than normal controls, those with advanced diseases as defined by Rai stage III/IV or -2M level higher than 3.5 have significantly elevated IL-6 levels compared with normal controls. More importantly, in the group of patients with advanced diseases (i.e., high -2M and/or Rai III/IV), increasing levels of IL-6 significantly correlate with shorter survival. This impression is confirmed further by multivariate analysis, which showed IL-6 to be an independent prognostic factor in these patients. Based on these observations, we conclude that IL-6 expression is an independent predictor of survival in patients with advanced-stage CLL. This is supported by the fact that when we analyzed previously untreated patients (56 patients), we found no correlation between IL-6 levels and survival. We also found that IL-6 as a discrete variable (i.e., higher or lower than 3 pg/ml) is useful prognostically. Not only did the low IL-6 group have a significantly longer overall survival than the high IL-6 group, IL-6 also stratified CD38-positive cases into two prognostic groups and may be able to improve significantly the prognostic value of CD38 positivity in these patients. This improvement on the prognostic value of CD38 may be related to the fact CD38 and IL-6 are associated with different biologic parameters. The prognostic value of IL-6 in this setting is probably reflective of the functional status of the immune system of the patients. In contrast, the prognostic value of CD38 expression may be related to the developmental state of the malignant B-cell clone (i.e., mutation status of the immunoglobulin variable region and stage of differentiation). However, this concept is controversial and further study is needed to investigate this possibility. IL-6, either as a discrete or continuous variable, predicts poor outcome in CLL patients with advanced diseases. The biologic basis of this finding requires further studies. Nevertheless, one possible explanation is that high IL-6 levels in some of these patients are a consequence of a severely defective immune system, such that the biologic refractoriness to IL-6 is a prominent feature or the negative feedback on IL-6 production is impaired. Hulkkonen et al. 11 reported that in vitro IL-6 production is lower in CLL cells of patients with advanced stage of disease (Binet C) after mitogenic stimulation. We observed that IL-6 plasma levels increase in patients with advanced stage of disease. This discrepancy may be explained by the possibility that IL-6 in the plasma of patients with advanced diseases is produced by cell types other than the neoplastic B cells, such as macrophages and T cells. The other possible explanation is that the in vitro experiments are not reflective of the in vivo IL-6 production. We also identified significant correlation between IL-6 and the levels of a number of other cytokines in the plasma, including IL-1, interleukin-1 receptor antagonist (IL-1Ra), and tumor necrosis factor-alpha (TNF- ; data not shown). Some of these are not unexpected, but they are potent inducers of IL-6. Using multivariate analysis, a model incorporating IL-6, IL-1, TNF-, and IL-1Ra showed that only TNF- and IL-6 remained predictors of survival, whereas each of these cytokines was predictive of survival when evaluated in univariate analysis. If we incorporate -2M in this model, only TNF- and -2M were predictive of survival but not IL-6, IL-1, or IL-1Ra. Further studies incorporating various cytokines are necessary to understand better the interaction of these cytokines and their role in CLL patients. It has been hypothesized that the defect in IL-6 production may explain the frequent presence of hypogammaglobulinemia and thrombocytopenia in patients with advanced- stage CLL disease because IL-6 is essential for B-cell development 12 and for normal megakaryocyte maturation. 13 However, we did not observe any significant correlation between platelet count and IL-6 in this study. In this regard, it is likely that aberrant levels of IL-6 alone cannot explain thrombocytopenia in patients with advanced CLL. Other abnormalities involving other cytokines may also be responsible. In conclusion, we found that IL-6 plasma levels are elevated significantly in CLL patients with advanced disease and that IL-6 is an independent prognostic factor in predicting overall survival in these patients. IL-6 also can improve the prognostic value of CD38 in CLL patients. Our findings suggest that patients with advanced CLL in conjunction with high IL-6 plasma levels may require aggressive therapeutic approaches and special consideration for experimental therapy.

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