Med Oncol (2009) 26:10–15 DOI 10.1007/s12032-008-9070-2
ORIGINAL PAPER
Serum interleukin-6 in patients with metastatic bone disease: correlation with cystatin C Francesca M. Tumminello Æ Giuseppe Badalamenti Æ Lorena Incorvaia Æ Fabio Fulfaro Æ Calogero D’Amico Æ Gaetano Leto
Received: 25 January 2008 / Accepted: 17 April 2008 / Published online: 7 May 2008 Ó Humana Press Inc. 2008
Abstract The clinical significance of serum interleukin-6 (IL-6) and its correlation with cystatin C (Cyst C), an endogenous inhibitor of cysteine proteinase cathepsin K, was investigated by immunoassays in patients with bone metastasis from breast cancer (BCa) or prostate cancer (PCa). Additional studies were also performed in these patients to assess the effects of zoledronic acid (ZA) administration on the circulating levels of these molecules. Mean IL-6 and Cyst C serum concentrations were significantly increased in BCa patients and in patients with primary osteoporosis (PO) compared to healthy subjects (HS). However, Cyst C, but not IL-6, resulted significantly more elevated in BCa patients than in PO patients. Furthermore, in BCa patients no correlation was highlighted between IL-6 and Cyst C or between these molecules and some clinicobiological parameters of malignant progression. Mean IL-6 levels were also higher in PCa patients and in patients with benign prostatic hyperplasia (BPH) than in HS while Cyst C resulted significantly higher in PCa but not in BPH patients as compared to HS. In PCa patients, a positive correlation was highlighted between IL-6 and number of bone metastases or serum prostate-specific antigen but not with the Gleason score. Conversely, Cyst C levels did not correlate with any of the parameters considered above or with IL-6. Receiver operating characteristic
F. M. Tumminello C. D’Amico G. Leto (&) Department of Surgery and Oncology, Laboratory of Experimental Chemotherapy and Tumor Markers, Policlinico Universitario P. Giaccone, 90127 Palermo, Italy e-mail:
[email protected] G. Badalamenti L. Incorvaia F. Fulfaro Section of Medical Oncology, Department of Surgery and Oncology, Policlinico Universitario P. Giaccone, 90127 Palermo, Italy
(ROC) curve analysis showed a poor diagnostic accuracy of IL-6 and Cyst C to detect BCa patients with skeletal metastases while, in PCa patients, only IL-6 showed a fair diagnostic performance in this respect. Finally, the administration of ZA to patients with bone metastases induced a statistically significant increase of serum IL-6 and Cyst C only PCa patients with bone metastasis. These data indicate that IL-6 and Cyst C may be regarded as novel targets for cancer treatment and as markers of increased osteoblastic activity associated to bisphosphonate treatments in PCa patients with bone metastases. Keywords Breast cancer Bone metastasis Cystatin C Interleukin-6 Prostate cancer Tumor markers Zoledronic acid
Introduction Interleukin-6 (IL-6) is a multifunctional cytokine implicated in a variety of important physiological and pathological processes including bone remodeling [1–3]. In particular, in this latter process, this cytokine has been shown to stimulate osteoclast and osteoblast differentiation, osteoclast-mediated bone resorption and to facilitate osteoblast proliferation [1–3]. Additionally, increased levels of this molecule can be observed in several pathological conditions associated with an altered bone resorption [1, 4– 7]. The activity of IL-6 during bone remodeling processes appears to be regulated by several factors [1–3]. Interestingly, recent in vitro studies show that cystatin C (Cyst C), a natural occurring inhibitor of osteoclast cysteine proteinase Cathepsin K, modulates IL-6 activity during bone resorption by interfering with late steps of osteoclast differentiation [8, 9]. Therefore, any dysregulation in the
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expression levels of IL-6 and/or Cyst C may affect the normal bone turnover and, eventually, may lead to the onset of a number of pathological conditions associated with an altered bone resorption including tumor-related bone disease [10]. On the basis of these considerations, preliminary investigations were undertaken to assess the distribution and the clinical significance of serum IL-6 in patients with bone metastasis from breast cancer (BCa) or prostate cancer (PCa) and its relationship with circulating Cyst C. These tumors were chosen as they induce different types of bone lesions, namely, lytic (breast cancer) or blastic (prostate cancer) [10]. Furthermore, parallel studies were undertaken to evaluate the effects of zoledronic acid (ZA), an aminobisphosphonate derivative endowed with antiosteoclastic and analgesic activity [11–13], on the serum concentrations of IL-6 and Cyst C in patients with skeletal metastases. These studies were aimed at evaluating the clinical usefulness of these molecules as additional markers for the clinical and therapeutic management of patients with cancer-related bone diseases undergoing palliative treatments with bisphoshonates.
Patients and methods The study included 46 registered healthy blood donors (HS) of both sexes (21 females and 25 males); 10 female patients with age-related osteoporosis (PO) (mean age 74.1 ± 10.5 years), 14 patients with histologically confirmed benign prostatic hyperplasia (BPH) (mean age 62.8 ± 6.2 years), 26 patients with breast cancer (mean age 59.3 ± 9.8), and 34 patients with prostate cancer (mean age 72.4 ± 7.8). Both BCa and PCa groups comprised patients with localized disease (M0) and patients with clinically documented bone metastases (BM) and no clinical evidence of extraskeletal involvement [12]. BM patients who were eligible for treatment with ZA (ZometaÒ Novartis, Basel, Switzerland), according to the criteria described previously [12, 13], underwent serial administrations with the drug (4 mg i.v. by a 15 min infusion every 4 weeks). The degree of pain severity in these patients and the symptomatic response to ZA treatment were assessed by using a visual analogue scale (VAS) score as reported [12]. A VAS score B4 was considered as acceptable analgesic response [12, 13]. The study was approved by the local ethical committee and carried out in accordance with the Declaration of Helsinki [14]. IL-6 and cystatin C assay Peripheral venous blood specimen from cancer patients or patients with nonmalignant diseases were collected before starting any clinical treatment. Additional blood samples
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were collected from patients treated with ZA, prior to a second drug administration, namely on day 28. The blood was drawn into polycarbonate tubes, allowed to clot at room temperature, and then centrifuged at 3500 rpm for 15 min (Hereus Omnifuge 2.0 RS, Hereus Sepatech). Serum aliquots were stored at -80°C until assays. IL-6 and Cyst C concentrations were determined by commercially available two-step sandwich enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers’ instruction (Amersham, Interleukin-6 Human Biotrak ELISA System, Little Chalfont, UK; Cystatin C, Biovendor, Modrice, Czech Republic). The detection limits reported by the manufacturers were \1.0 pg/ml for IL-6 and 80 ng/ml for Cyst C. The timing of IL-6 and Cyst C assays (i.e., day 0 and day 28) was chosen on the basis of pharmacokinetics studies of ZA in patients with bone metastasis which showed that the antiresorptive effects of the drug, assessed by the trend of the decline of bone resorption markers, were maintained over 4 weeks [15]. Statistical analysis The normal distribution of the data in the various groups was assessed by the D’Agostino-Pearson test. Therefore, statistical analysis was performed, where required, by the nonparametric Mann–Whitney U test, Wilcoxon signed rank test, Kruskall–Wallis test and the Spearman rank correlation test or by the parametric Student t test for unpaired or paired samples. The diagnostic performance of IL-6 or Cyst C to discriminate between M0 and BM patients was determined by the receiver operating characteristic (ROC) curve [16]. The significance of the difference between the areas under the ROC curves (AUCs) was assessed according to Hanley and McNeil [17]. The relationship between degree of pain severity (VAS score) and IL-6 or Cyst C serum content was evaluated by the Pearson’s correlation coefficient. The correlation between symptomatic response to ZA treatments and time course variation of IL-6 or Cyst C serum concentrations was assessed by linear regression analysis. P values \0.05 were considered statistically significant. Data analysis was performed by using the Medcalc version 7.4.4 statistical software package (MEDCALC Mariakerke, Belgium).
Results In the BCa group, mean IL-6 serum levels were significantly higher in cancer patients and in PO patients compared to healthy subjects (Table 1). However, no statistically significant difference in the serum concentrations of this cytokine was observed between BCa patients and
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Table 1 Interleukin-6 (IL-6) and cystatin C serum distribution in healthy subjects (HS), in patients with primary osteoporosis (PO) or benign prostate hyperplasia (BPH) and in patients with breast cancer (BCa) or prostate cancer (PCa) HS (n = 21)
PO (n = 10)
BCa (n = 26)
Breast cancer group IL-6 (pg/ml) Cystatin C (ng/ml)
0.03 ± 0.03
1.98 ± 0.63*
846.7 ± 404.0
1126.7 ± 704.0***
2.3 ± 1.1** 1457.6 ± 1110.8****
*P = 0.016, **P = 0.05, ***P = 0.0027 and ****P \ 0.0001 vs. HS; P = 0.014 vs. PO HS (n = 25)
BPH (n = 14)
PCa (n = 34)
Prostate cancer group IL-6 (pg/ml) Cystatin C (ng/ml)
0.43 ± 0.18 1092.8 ± 568.0
3.3 ± 0.55* 1274.1 ± 832.0
2.3 ± 0.44** 1514.7 ± 1112.4***
*P = 0.03, **P = 0.013 and ***P = 0.0002 vs. HS; P = 0.024 vs. BPH Note: Results are expressed as mean ± standard error. Statistical analysis of the data was performed by the nonparametric Mann–Whitney U test. Number of evaluable subjects is shown in parenthesis
PO patients. Conversely, Cyst C serum levels were significantly more elevated in BCa patients than in PO patients (Table 1). In BCa patients, no correlation was highlighted between IL-6 and Cyst C (Spearman rank correlation coefficient rs = 0.067; P = 0.73) or between the serum concentrations of these molecules and some clinicobiological parameters of progression of this tumor, namely, number of bone metastases, tumor size, tumor grade, hormone receptor status, and serum carbohydrate antigen Ca15.3 (Table 2). In the prostate cancer group, mean IL-6 levels were significantly increased in PCa patients or BPH patients as compared to normal subjects whereas Cyst C levels resulted more elevated only in PCa patients as compared to HS (Table 1). However, unlike IL-6, Cyst C levels were significantly higher in PCa patients than in BPH patients (Table 1). Interestingly, in cancer patients a positive correlation was highlighted between IL-6 levels and number of bone metastases (P = 0.01) or serum prostate-specific antigen (PSA) levels (P = 0.014) but not with the Gleason score (P = 0.54) (Table 2). On the contrary, Cyst C levels did not correlate with any of the parameters considered above (Table 2) or IL-6 (Spearman rank correlation coefficient rs = 0.051; P = 0.78). ROC curve analysis generated to assess the effectiveness of IL-6 or Cyst C to discriminate BCa patients with and without bone metastasis showed a poor diagnostic accuracy of these molecules (Fig. 1). On the other hand, in PCa patients, IL-6 showed a fair diagnostic performance to detect patients with bone metastasis (P = 0.014) (Fig. 1). However, the AUC of IL-6 did not result significantly different from that of Cyst C (P = 0.21). Interestingly, the administration of ZA to patients with skeletal metastases who were eligible for drug treatment (8/10 BCa patients and 12/18 PCa patients, respectively) induced an evident increase of both IL-6 or
Cyst C serum levels (Table 3). However, statistically significant differences between pre- and post-treatment values of these molecules were observed only in PCa patients (Table 3). Furthermore, ZA administration was associated with a significant symptomatic response (VAS score B4) in 6/8 (75%) of BCa patients and in 10/12 (83.3%) of PCa patients. Nevertheless, in these patients, no significant relationship was highlighted between IL-6 or Cyst C levels and degree of pain severity (VAS score) or between time course variation of the serum concentrations of these molecules and analgesic response to ZA treatment (data not reported).
Discussion The present studies show that serum IL-6 and Cyst C are increased in cancer patients. In particular, these data show that, at least in prostate cancer, IL-6 serum levels positively correlate with the number of bone lesions and with the PSA levels, while cystatin C levels are significantly more elevated in BCa or PCa patients than in patients with nonmalignant conditions. The elevated levels of Cyst C in cancer patients or in PO patients do not appear to be related to an altered renal function as none of these subjects exhibited clinical evident sign of an impaired kidney function [18]. Therefore, these findings are consistent, in part, with other clinical studies which show that a number of human tumors including breast cancer and prostate cancer present altered expression levels of IL-6 or Cyst C and that these phenomena are associated with a poor clinical outcome [1, 4–7, 19–24]. However, these data also show that the serum concentrations of IL-6 are significantly increased in patients with PO or BPH as compared to normal subjects and that these concentrations do not
Med Oncol (2009) 26:10–15 Table 2 Interleukin-6 (IL-6) and cystatin C serum distribution in patients with breast cancer or prostate cancer
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Parameter
IL-6 (pg/ml)
Cyst C (ng/ml)
T1 (n = 7)
0.0 (0–0)
1175 (600–1600)
T2 (n = 11)
0.71 (0–26.8)
1200 (1050–2100)
T3 (n = 8)
0.75 (0–3.1)
Breast cancer Tumor size
1375 (900–3800) a
P = 0.074 (n.s.)
P = 0.55 (n.s.)a
G1 (n = 6)
0.0 (0–26.8)
1511 (1100–2800)
G2 (n = 12)
0.0 (0–16.7)
1200 (600–2100)
G3 (n = 7)
0.0 (0–2.8)
1300 (1100–2700)
P = 0.89 (n.s.)a
P = 0.23 (n..s.)a
Estrogen negative (n = 6)
0.0 (0–26.8)
1400 (1250–1590)
Estrogen positive (n = 19)
0.7 (0–3.7)
1200 (600–3800)
Progesterone negative (n = 6)
P = 0.94 (n..s.)b 1.6 (0–16.7)
P = 0.14 (n.s.)b 1400 (1050–1900)
Tumor grade
Receptor status
Progesterone positive (n = 20)
0.0 (0–26.8)
1200 (600–3800)
P = 0.13 (n.s.)b
P = 0.14 (n.s.)b
M0 (n = 16)
0.0 (0–26.8)
1200 (600–2700)
BM (n = 10)
0.6 (0–3.7)
Bone metastasis
P = 0.1 (n..s.) Ca15.3
1400 (900–3800) b
P = 0.81 (n.s.)b
c
\30 U/ml (n = 8)
0.0 (0–26.8)
1200 (600–3800)
[30 U/ml (n = 18)
2.1 (0–3.7)
1400 (1180–1900)
P = 0.34 (n.s.)b
P = 0.1 9 (n.s.)b
2–4 (n = 12)
3.4 (0–6.8)
1413 (1169–2675)
5–7 (n = 9)
0.0 (0–5.97)
1413 (1128–2691)
8–10 (n = 9)
1.8 (0–8.1) P = 0.54 (n.s.)a
1476 (1119–3940) P = 0.88 (n.s.)a
M0 (n = 16)
0.0 (0–4.6)
1325 (930–3940)
BM (n = 18)
3.8 (0–8.6)
1410 (260–2675)
P = 0.010b
P = 0.30 (n.s.)b
Prostate cancer Gleason score Note: Data are reported as median and (range); n = number of evaluable subjects. Due to some missing sample, number of subjects in each group may vary a
Kruskall–Wallis test
b
Mann-Whitney U test; n.s. = not statistically significant
c
Cut-off reported by the manufacturer
Bone metastasis
PSAd \17 ng/ml (n = 14)
0.0 (0–4.6)
1406 (930–3940)
[17 ng/ml (n = 16)
3.9 (0–6.8)
1416 (1119–2691)
P = 0.014b
P = 0.26 (n.s.)b
d
Cut-off determined by ROC curve analysis
significantly differ from those measured in breast cancer patients. These results are in agreement with those studies which highlight that PO and BPH patients present increased levels of IL-6 and that this phenomenon appears to be correlated with the specific involvement of this pleiotropic cytokine in the pathogenesis of these inflammatory diseases [1, 19, 25, 26]. On the other hand, the altered levels of cystatin C observed in PO patients further support the hypothesis that this inhibitor appears to play an
active role in bone remodeling processes [8, 27–29]. However, these observations and the results obtained by the ROC curve analysis seem to rule out that, at least in breast cancer, IL-6 and Cyst C may be of clinical usefulness as specific markers of metastatic bone disease. Conversely, in prostate cancer, according to the data obtained by the ROC analysis, IL-6 appears to be of interest as potential gauge of metastatic spread to the bone. Interestingly, the administration of ZA to BCa or PCa
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Fig. 1 ROC curve analysis for IL-6 and cystatin C to detect patients with bone metastasis from breast cancer or prostate cancer. The significance of the difference between the areas under the ROC curves (AUCs) was assessed according to Hanley and McNeil [17]
Prostate cancer 100
80
80
60
60
Sensitivity
Sensitivity
Breast cancer 100
40
40
____ IL-6 : AUC 0.77±0.08 p=0.014 _ _ _ IL-6 : AUC 0.54±0.1 p=0.087 n.s
20
20 _ _ _ _Cyst C: AUC 0.62±0.1 p=0.29 n.s
____Cyst C:AUC 0.68±0.1 p=0.86 n.s
0
0 0
20
40
60
100-Specificity
Table 3 Effects of zoledronic acid administration (4 mg i.v. by a 15’ infusion) on IL-6 or Cystatin C serum levels and on bone pain intensity (VAS score), in patients with skeletal metastasis from breast cancer patients or prostate cancer Baseline
Day 28
P value
IL-6 (pg/ml)
1.72 ± 1.53
2.58 ± 2.37
0.10 (n.s.)a
Cystatin C (ng/ml)
1570 ± 910
1980 ± 510
0.11 ( n.s.)b
6.6 ± 1.5
3.7 ± 2.4
0.016a
IL-6 (pg/ml)
2.14 ± 2.39
4.78 ± 5.4
0.05a
Cystatin C (ng/ml)
1680 ± 530
2200 ± 940
0.005a
6.0 ± 1.7
3.6 ± 0.8
0.0002a
Breast cancer (n = 8)
VAS score Prostate cancer (n = 12)
VAS score
c
Note: Results are reported as mean ± standard deviation. Number of evaluable subjects is shown in parenthesis. n.s. = not significant a
Student t test for paired samples
b
Wilcoxon signed rank test
c
Visual analogue scale (VAS) score was determined as reported in ref. [12]
patients with bone metastases induced an evident increase of IL-6 or Cyst C serum levels compared to pre-treatment values. However, statistically significant differences between pre- and post-treatment values were observed for both these molecules in PCa patients. These data further confirm and extent previous observations from other studies which show that the infusion of ZA to patients with metastatic breast cancer induces a not statistically significant increase of the circulating levels of IL-6 up to 7 days after drug administration [30]. On the other hand, as IL-6 and Cyst C have been shown to stimulate osteoblast production
80
100
0
20
40
60
80
100
100-Specificity
and differentiation and to inhibit osteoclastogenesis [2, 3, 8, 27–29] it is conceivable to speculate that the increased levels of these molecules induced by ZA in PCa patients with bone metastases may likely reflect an enhanced osteoblastic activity, which in PCa is predominant [31, 32], elicited by the drug. This hypothesis is further corroborated by some in vitro studies which highlight that ZA stimulate the proliferation and differentiation of normal human osteoblasts [33]. Furthermore, the administration of ZA in BM patients was associated with a significant symptomatic response which paralleled the changes of IL-6 and Cyst serum levels induced by the drug. The analgesic effect of ZA in patients with bone metastasis from breast cancer or prostate cancer is well documented [11, 12, 34–36]. In this context, our findings are suggestive for an involvement of these molecules in the pathogenesis of pain associated with bone metastasis. However, in our small series, the lack of a significant relationship between analgesic response and time course variation of serum IL-6 or Cyst C induced by ZA treatment seems to rule out a direct role of these molecules in the pathogenesis of pain associated with metastatic bone disease. In summary, IL-6 and Cyst C appear to be of clinical interest as potential targets for new therapeutic approaches in cancer treatment while their clinical role as gauges of metastatic bone diseases remains to be better assessed. Nevertheless, these molecules may be regarded as possible, novel markers of enhanced osteoblastic activity associated with the therapeutic response of patients with skeletal metastases to ZA treatments. Further investigations with a wide number of subjects may better define the role of these molecules in the clinical and therapeutic management of patients with tumor-related bone disease.
Med Oncol (2009) 26:10–15 Acknowledgments This work was supported by funds from Ministero della Universita` e Ricerca (MIUR ex quota 60%).
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