Plasma YKL-40 as a biomarker in patients with nonmetastatic bone and soft tissue sarcomas: a prospective exploratory clinical study

Purpose: YKL-40 is a glycoprotein with a role in inflammation, tissue remodeling, tumor angiogenesis, and protection against apoptosis. We hypothesized that high preoperative plasma YKL-40 in patients with nonmetastatic bone and soft tissue sarcoma (STS) is associated with short overall survival (OS), and that plasma YKL-40 is an independent predictor for OS. Materials and methods: Plasma was collected preoperatively from 65 patients with nonmetastatic bone (n=14) or STS (n=51) in the lower extremities (n=43), the upper extremities (n=16) or the trunk wall/spine (n=6). All patients underwent surgical cancer treatment. Twenty patients developed metastases during the follow-up period (minimum 5 y). The plasma concentration of YKL-40 was determined by enzyme-linked immunosorbent assay. Results: Twenty-seven patients died [mean: 3.2 (range: 0.2–7.3) y postoperatively] and 38 patients were still alive after a follow-up of mean 6.9 (5.8–8.2) years postoperatively. Plasma YKL-40 was higher in patients who died during follow-up (P=0.008), in males (P=0.007) and in patients 61 years of age and above (P=0.001). The 5-year OS was 68% and OS was lower in patients with high (≥95% percentile age-corrected) plasma YKL-40 (P=0.021), age 61 years and above (P=0.013), high histologic malignancy grade (P=0.047) and male sex (P=0.051). Multivariable analysis showed that only plasma YKL-40 (age-corrected (hazard ratio=2.80, 95% confidence interval: 1.13–6.91, P=0.026) and malignancy grade (hazard ratio=9.9×107, 95% confidence interval: 0–∞, P=0.007) remained independent prognostic factors for OS. Conclusions: High preoperative plasma YKL-40 was related to short OS in patients with nonmetastatic bone and STS and plasma YKL-40 (age-corrected) was an independent prognostic risk factor for OS.

Background YKL-40 (also named chitinase 3 like protein 1) is a 40 KDa glycoprotein, and it was first described in 1992, where it was found to be secreted in vitro by the human osteosarcoma cell line MG-63 [1] . Later studies demonstrated YKL-40 expression in soft tissue sarcomas (STS), chondrosarcomas, and osteosarcomas [2] , and that high YKL-40 expression was related to short survival in STS [3] . It has also been found that YKL-40 expression is related to malignancy grade in STS, but not in chondrosarcomas [3,4] . Furthermore, YKL-40 is a growth factor for fibroblasts, chondrocytes, bone cells, and connective tissue [5][6][7][8] .

Objectives
In this prospective biomarker study, we examined the prognostic value of preoperative plasma YKL-40 in patients with nonmetastatic bone and STS. We hypothesized that high preoperative plasma YKL-40 in these patients is associated with short overall survival (OS), and that plasma YKL-40 is an independent predictor for OS.

Patients
Sixty-five patients operated on for the first time for a biopsy verified STS (n = 51) or bone sarcoma (n = 14) at the Musculoskeletal Tumor Section, Rigshospitalet, University of Copenhagen were included in the study. The patients were included during the period December 2009 to April 2012, and in that period the department operated ∼60-80 newly diagnosed sarcoma cases each year. Seven patients initially included in the study were excluded because the diagnosis was later changed to malignant melanoma metastasis (n = 1), lipoma (n = 1), aneurysmal bone cyst (n = 1), and atypical lipomatous tumors (n = 4). One patient was excluded due to metastases present at the time of diagnosis. The number of patients included in the present study was therefore 65. The clinical characteristics of patients and tumor characteristics are summarized in Table 1.
From the electronic patient files information regarding metastasis status at the time of diagnosis or later, chemotherapy treatment, surgical margin, local recurrence, tumor type, and subtype were collected and updated by January 1, 2018. This resulted in a minimum follow-up period of 5 years for patients still alive at the time of follow-up, and a maximum follow-up of 8 years (range: 5.8-8.2 y). Data from The Danish Civil Registration system [26] was used for information regarding vital status, thus no patients were lost to follow-up for survival status.
The majority of tumors were located at the lower extremities (n = 43) or upper extremities (n = 16), and a small number was located in the trunk wall or spine (n = 6). Using the FNCLCC (French Fédération Nationale des Centres de Lutte Contre le Cancer) Trojani grading system [27] the tumors were classified as low malignant [Trojani grade I (n = 6)], or high malignant (n = 46) [Trojani grade II (n = 23) or III (n = 23)]. A total of 22 different histologic types of sarcomas were included in the study, and the most common types were myxofibrosarcomas (n = 11), sarcomas not otherwise specified (n = 9), and chondrosarcomas (n = 8) ( Table 1).
All 65 patients underwent surgical treatment for their cancer. In 34 patients, a wide margin was obtained, while 23 patients had the tumor removed with a marginal margin. In 3 patients, the tumor was removed with a positive margin and in 5 patients the surgical margin was not described. Thirteen patients received chemotherapy [postsurgery (n = 10), presurgery and postsurgery (n = 3)], and 27 patients were treated with radiotherapy [postsurgery (n = 20), perioperative brachytherapy combined with later external radiation therapy (n = 2), perioperative brachytherapy alone (n = 3), and as a part of palliative treatment (n = 2) because of a local recurrence during the follow-up period].

Plasma YKL-40 analysis
The blood sample was taken before a patient's first surgery either the day before surgery or on the day of surgery. Blood was drawn into a 6 mL ethylene-diamine-tetra-acetic acid (EDTA) tube and stored between 1/2 −2 hours at room temperature, and then centrifuged for 10 minutes at 3000 rounds per minute. Thereafter plasma was isolated and then stored at −80°C. YKL-40 was determined in duplicate in the preoperative plasma samples using an enzyme-linked immunosorbent assay (ELISA) (Quidel Corporation, San Diego, CA) according to the manufacturer's instructions. The marker assessments were made blinded to clinical outcome. YKL-40 ELISA characteristics were: detection limit 20 ng/mL and intra-assay and interassay coefficients of variation (CVs) <5% and <6% [28] . An age-corrected 95th percentile for YKL-40 in healthy individuals served as a cut-off for elevated values and was calculated using the formula proposed by Bojesen et al [28] :

Statistics
Statistical analyses were performed using SPSS version 22 for Mac and the software R (R Foundation, Vienna, Austria). P-values <0.05 were considered statistically significant, and results are given as a median or mean and total range (if no other information is provided). Nonparametric test for unpaired data (Mann-Whitney) was used to determine potential differences between plasma YKL-40 concentrations in various subgroups. Kaplan-Meier survival analysis was used to determine the probability of 5-and 8-year OS, and a 95% confidence interval (95% CI) for OS. The log-rank test was performed to compare the OS between subgroups. To make allowance for death as a competing risk factor, the cumulated incidence of local recurrence was calculated by Aalen-Johansen's analysis with death as a competing risk for local recurrence, and the difference in risk between subgroups was assessed by the Gray test. Finally, Cox-regression analysis (univariate/multivariable) with the calculation of hazard ratio (HR) with 95% CI was performed to identify possible independent prognostic factors available at the time of operation.
Ethics and study registration The study was conducted in accordance with the Helsinki declaration with informed consent obtained (after written and oral information) from all study participants prior to inclusion in the study.

Reporting recommendations for tumor marker prognostic studies (REMARK)
The work in this study have been reported in line the with the REMARK [30] .

OS and local recurrence after operation
Eleven patients experienced a local recurrence within the first 5 years postoperatively and the cumulated incidence of local recurrence (calculated by a competing risk regression model with death as a competing risk) was 7.7% (95% CI: 1.2%-14%) and 17% (95% CI: 7.8%-26%) after, respectively, 1 and 5 years of follow-up (Fig. 3). Age-corrected plasma YKL-40 was not a statistically significant factor for development of local recurrence (P = 0.325) (Fig. 3).

Discussion
We found that the age-corrected plasma YKL-40 measured at the time of surgery and malignancy grade are independent prognostic risk factors for OS in patients operated on for nonmetastatic bone and STS. The influence of plasma YKL-40 is a new observation, but the result is in good agreement with another study investigating the prognostic value of YKL-40 protein expression in sarcoma tissue [3] . The influence of the histologic malignancy grade on patient survival has previously been established, and our results confirm this well-known prognostic effect of this parameter [27,29] .
The exact function of YKL-40 in nonmetastatic bone and STS is unknown. Many studies have focused on the function of YKL-40 in other cancer types and have found that neutrophils and tumor-associated macrophages in tumor cells or their surrounding microenvironment can secrete YKL-40 into the extracellular space. Here YKL-40 can induce tumor growth via cancer cell proliferation, inhibition of epithelial cell apoptosis, and promotion of angiogenesis. Furthermore, YKL-40 can increase the invasion of cancer cells and thereby induce tumor progression and metastasis [9] . YKL-40 is not cancer specific, and plasma YKL-40 is elevated in patients with diseases characterized by inflammation such as rheumatoid arthritis, bacterial infection, lung diseases, and inflammatory bowel disease, and elevated plasma YKL-40 is associated with more active disease [10,12,31] .
The inflammatory process around the tumor is critical for tumor progression in many tumors, and this process is also upregulated by YKL-40 [19] . This raises the question, how much plasma YKL-40 is due to underlying inflammatory disease in the body, and how much is a reflection of the tumor cells, their surrounding microenvironment, inflammatory response, and the malignity level of the tumor. Further studies are needed to evaluate this, perhaps studies that compare YKL-40 expression in tumor tissue and the circulating plasma YKL-40 in the same cancer patients.
We found lower plasma YKL-40 concentrations in patients under the age of 61 years, and lower levels in females compared with males. Age and sex are well-known prognostic markers in sarcoma patients and confirmed in studies with age cut-offs ranging from above 45 to above 64 years [32][33][34][35] . Circulating YKL-40 increases with age in healthy subjects. Bojesen et al [28] described an age-adjusted reference level for plasma YKL-40, and proposed that elevated plasma YKL-40 levels should be defined as higher than the 95th or the 97.5th percentile. In the present study, we defined an elevated preoperative plasma YKL-40 level as higher than the age-adjusted 95% percentile [28] and found that     this cut-off was useful to identify a subgroup of sarcoma patients with at very poor prognosis after tumor resection. In general, the 5-year OS in patients with STS is ∼49%-76% and for bone sarcoma 48%-69% [33,[36][37][38][39][40] . We found a relatively high 5-year OS of 67% of the patients included in the present study. This could be explained by the fact that the patients included in the present study were all nonmetastatic at the time of surgery, and only represent a part of the subgroup of nonmetastatic bone and STS operated at our orthopedic oncology unit.
We included patients with 22 different histologic types of nonmetastatic bone and STS but did not have patients with all types of sarcomas. There were too few patients of each sarcoma type to make a comparison regarding plasma YKL-40 levels according to sarcoma type. Thus, future larger studies focusing on plasma YKL-40 and different histologic types of sarcomas are needed to determine, if plasma YKL-40 is a type-specific prognostic biomarker in patients with sarcomas.
YKL-40 is not specific for cancer, and we found no difference in plasma YKL-40 between patients who developed metastases during follow-up, and those that did not, thus indicating that comorbidity could also be of importance. We consider it a limitation of our study that comorbidity was not evaluated. We found that plasma YKL-40 was an independent prognostic factor for survival in patients with sarcoma, but this result should be interpreted with caution as the regression analysis does not oblige to the rule of 10 events per included variable [41] . It has been suggested that this rule is very conservative [42] , and our analysis is not overfitted as the CI and the odds ratio does not change drastically between univariate and multivariable analysis. Thus, we do not think that we have violated the assumption for multivariable analysis. As we did not include a validation cohort, we underline that our findings should be interpreted more as a hypothesis-generating and not definitive, as we have not validated our findings in an independent cohort.

Conclusions
We found the following new observations not previously published: • High preoperative plasma YKL-40 is related to short OS in patients with nonmetastatic bone and STS. • Age-corrected plasma YKL-40 level measured preoperatively is an independent prognostic risk factor for OS in patients with nonmetastatic bone and STS.
Plasma YKL-40 is probably not sarcoma specific, but more likely a general prognostic factor for survival and further studies also including information on comorbidity in larger study populations are warranted.

Sources of Funding
A.P.T. was funded by grants from Det Medicinske Selskab i København and Rigshospitalet (udviklingspuljen). The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data or in writing the manuscript. Hospital, Herlev, Denmark, for excellent technical assistance with the YKL-40 ELISA measurements. The patients are thanked for their willingness to participate.