Serum YKL‐40 and bone marrow angiogenesis in multiple myeloma (2008)

In a recently published study, Saidi et al. showed that elevated levels of CHI3L1 mRNA, encoding the secreted glycoprotein YKL-40, are associated with poor survival in glioblastoma, probably by promoting angiogenesis.1 A role for YKL-40 in angiogenesis is supported by a few in vitro studies on the porcine YKL-40 analogue, gp38k, showing 84% sequence homology with human YKL-40.2 Gp38k is secreted by differentiating vascular smooth muscle cells2 and besides modulating adhesion and migration of these cells,3 gp38k stimulates both directional migration of human umbilical vein endothelial cells (HUVEC), at a level comparable to that achieved with the endothelial cell chemoattractant basic fibroblast growth factor (bFGF),4 and reorganisation of HUVEC with the formation of branching tubules.4 Angiogenesis plays a central role in the pathogenesis and progression of solid tumours. In recent years, there has been growing evidence that bone marrow (BM) angiogenesis exerts a similar role in certain hematological malignancies, including multiple myeloma (MM).5 As seen in glioblastoma and several other types of solid tumours,6, 7 a subgroup of patients with MM display elevated levels of serum YKL-40 associated with a more aggressive course of the disease,8 but a possible role for YKL-40 in the biology of MM remains to be established. Inspired by the study from Saidi et al., we have investigated the association between serum YKL-40 and the degree of BM angiogenesis in 45 patients with newly diagnosed MM. Patient characteristics included age (median 67 years; range, 44–83 years), gender (49% male; 51% female), M-protein isotype (69% IgG; 20% IgA; 9% light chains only; 2% nonsecretory), and stage according to the International Staging System ISS (19% stage I; 49% stage II; 32% stage III). Thirty-six patients received conventional treatment, and 9 patients received high-dose chemotherapy followed by autologous stem cell transplantation. Serum concentrations of YKL-40 were determined in duplicates using a sandwich-type enzyme-linked immunosorbent assay (ELISA) (Quidel®, Santa Clara, CA).8 BM angiogenesis was estimated as microvessel density (MVD), as previously described.9 The angiogenic cytokines bFGF, hepatocyte growth factor (HGF) and interleukin-6 (IL-6) were measured in serum, also as previously described.9 The study was approved by the local ethical committee and performed in accordance with the Helsinki II declaration. The median serum YKL-40 for all 45 MM patients was 123 μg/l (range, 26–1,828 μg/l). Using the upper limit in the age-specific 90% reference range for healthy adults,8 the study population was divided into one group of 21 MM patients with a normal serum YKL-40, i.e., below the limit, and one group of 24 MM patients with an elevated serum YKL-40, i.e., above the limit. In this study population, the serum YKL-40 level lacked association with the degree of BM angiogenesis estimated as MVD (Fig. 1a). The median MVD was 109 vessel profiles per mm2 (range, 15–238 vessel profiles per mm2). The degree of BM angiogenesis was reflected in serum concentrations of the known proangiogenic mediator HGF (Spearman's rho = 0.46; p = 0.002), whereas MVD lacked association with serum concentrations of two other proangiogenic factors, bFGF (Spearman's rho = −0.29; p = 0.05) and IL-6 (Spearman's rho = −0.04; p = 0.8). Furthermore, the serum YKL-40 level lacked association with serum bFGF and serum HGF (Figs. 1B and 1C), but patients with an elevated serum YKL-40 showed significantly higher serum concentrations of IL-6 compared to patients with a normal serum YKL-40 (Fig. 1D). The association between serum YKL-40 and serum IL-6 was further analysed using serum YKL-40 as a continuous variable, and a strong positive correlation between serum YKL-40 and serum IL-6 was demonstrated (Spearman's rho = 0.57; p < 0.0001). Serum YKL-40 and bone marrow angiogenesis in multiple myeloma (MM). Box-plots displaying median, interquartile range and total range of microvessel density (MVD) (a), serum basic fibroblast growth factor (bFGF) (b), serum hepatocyte growth factor (HGF) (c) and serum interleukin-6 (IL-6) (d) in newly diagnosed MM patients with either normal (n = 21) or elevated (n = 24) levels of serum YKL-40. Variables were compared between groups using the Mann-Whitney test. The association between the serum YKL-40 level and overall survival was investigated in the 36 patients receiving conventional treatment. During the time of observation, 34 patients died. Patients with an elevated serum YKL-40 tended to have a shorter overall survival (median 24 months; 95% confidence limits (CI) 15–35 months) compared to patients with a normal serum YKL-40 (median 34 months; 95% CI 25–43 months) (Fig. 2). Elevated serum YKL-40 (HR = 2.71; 95% CI 1.24–5.91; p = 0.01) and high MVD (HR = 1.008; 95% CI 1.002–1.014; p = 0.007) were independent predictors of poor overall survival when included in a Cox proportional hazards model with backward stepwise elimination based on likelihood ratio. MVD was entered as a continuous variable. We then tested if serum concentrations of one of the angiogenic cytokines contributed to the model. Serum IL-6 (log) eliminated serum YKL-40 level from the multivariate model, whereas including serum bFGF (log) or serum HGF (log) had no influence. For the multivariate analyses assumptions were tested and skewed variables were log-transformed. Serum YKL-40 and overall survival in multiple myeloma (MM). Kaplan-Meier plot of overall survival in 17 multiple myeloma patients with normal serum YKL-40 (---) and 19 multiple myeloma patients with elevated serum YKL-40 (—), all treated with conventional chemotherapy. Censoring is marked (|). Serum YKL-40 level as a prognostic factor for overall survival in MM patients treated with conventional chemotherapy is consistent with a similar finding in MM patients treated with high-dose chemotherapy followed by autologous stem cell transplantation.8 Also, MM patients with elevated serum concentrations of YKL-40 have more severe bone destruction including increased bone resorptive activity and early progression of myeloma-related bone disease.10 These findings suggest a role for YKL-40 in the disease process. The results presented here do not support a causal relationship between YKL-40 and BM angiogenesis in MM, but functional studies are needed to fully exclude proangiogenic effects of YKL-40 in MM. Neither do our results contradict proangiogenic effects of YKL-40 in glioblastoma, since Saidi et al. used different biological parameters in their study.1 It may well be that angiogenesis induced by solid tumours, like glioblastoma, requires YKL-40, whereas BM angiogenesis in haematological malignancies, like MM, relies on other pathways, but this remains hypothetical at this point. The strong association between serum YKL-40 and serum IL-6 is interesting. IL-6 is a central cytokine in the pathogenesis of MM, and besides promoting angiogenesis in the BM,5 IL-6 is known to induce myeloma cells proliferation,11 to inhibit apoptosis in myeloma cells,11 and to stimulate osteoclastogenesis contributing to the myeloma-related bone destruction.12 It is possible that the association between YKL-40 and IL-6 reflects one of these other pathological processes in MM. Another possible explanation is an IL-6 mediated regulation of YKL-40 secretion. IL-6 has been demonstrated to stimulate YKL-40 secretion by freshly isolated human chondrocytes,13 and correlations between concentrations of YKL-40 and IL-6 have previously been shown in both synovial fluid and serum from patients with rheumatoid arthritis.14, 15 A recent study of IL-6 wild-type and knockout mice further supports an IL-6 mediated regulation of YKL-40 secretion (JS Johansen, personal communication). In MM, YKL-40 originates from cells in the BM microenvironment surrounding the myeloma cells,8 but the exact regulation as well as the biological role of YKL-40 remain to be established. In conclusion, elevated serum YKL-40 concentrations are associated with poor survival in MM - as are elevated serum YKL-40 and high transcript levels of CHI3L1 in glioblastoma — but it lacks association with BM angiogenesis estimated as MVD. Interestingly, YKL-40 shows a strong association with serum IL-6 supporting the hypothesis, that IL-6 is involved in the regulation of YKL-40 secretion in MM. YKL-40 may act as a general modulator of different protumour biological phenomena, depending on the disease and the tissue context. We thank Ms. Tonni Loeve Hansen and Ms. Debbie Nadelmann for technical assistance. The Faculty of Health Sciences, Copenhagen University, supported the study. Quidel provided the YKL-40 ELISA kits®. Yours sincerely, Anne K. Mylin, Niels F. Andersen, Julia S. Johansen, Niels Abildgaard, Lene Heickendorff, Therese Standal, Peter Gimsing, Lene M. Knudsen.