High serum levels of YKL-40 in patients with squamous cell carcinoma of the head and neck are associated with short survival Anne Roslind 1 * , Julia S. Johansen 2 , Ib J. Christensen 3 , Katalin Kiss 4 , Eva Balslev 4 , Dorte L. Nielsen 1 , Jens Bentzen 1 , Paul A. Price 5 and Elo Andersen 1 1 Department of Oncology, Herlev Hospital, University of Copenhagen, Denmark 2 Department of Rheumatology, Herlev Hospital, University of Copenhagen, Denmark 3 Department of Surgical Gastroenterology, Hvidovre Hospital, University of Copenhagen, Denmark 4 Department of Pathology, Herlev Hospital, University of Copenhagen, Denmark 5 Department of Biology, University of California, San Diego, La Jolla, CA YKL-40 is a glycoprotein secreted by macrophages, neutrophils and malignant tumor cells. Elevated serum levels of YKL-40 are associated with poor prognosis in several malignancies. In this study, we examined the prognostic value of serum YKL-40 before treatment and during follow-up in patients with squamous cell carcinoma of the head and neck (HNSCC). YKL-40 was deter- mined by ELISA retrospectively in serum from 173 patients with primary HNSCC before treatment and up to 2 years after treat- ment. Median follow-up time was 7.9 years. YKL-40 protein expression in tumor biopsies was assessed by immunohistochemis- try in 50 patients. Pretreatment serum YKL-40 was elevated in 53%. Patients with high serum YKL-40 had shorter survival than patients with normal serum YKL-40 (33 vs. 84 months; p 5 0.008). Multivariate Cox analysis including pretreatment serum YKL-40, age, sex, primary tumor site, TNM classification and treatment demonstrated that TNM classification (HR 5 2.61, p 5 0.02) and serum YKL-40 (log-transformed continuous variable: HR 5 1.55, p < 0.0001) were independent prognostic variables of overall survival (OS). Multivariate Cox analysis demonstrated that TNM classification (HR 5 5.77, p 5 0.001) and serum YKL- 40 (dichotomous variable: HR 5 2.75, p 5 0.01) were independent predictors of recurrence-free survival. During follow-up after radiotherapy, a high serum YKL-40 (log-transformed continuous variable) in patients with TNM Stage III and IV disease predicted poorer OS within 6 months (HR 5 1.95, p < 0.0001). Immunohis- tochemical analysis showed YKL-40 expression in the malignant tumor cells. In conclusion, serum YKL-40 was demonstrated to be an independent prognostic biomarker of recurrence-free and overall survival in patients with HNSCC. ' 2007 Wiley-Liss, Inc. Key words: YKL-40; CHI3L1; biomarker; prognosis; head and neck cancer Cancers of the oral cavity, larynx and pharynx, collectively referred to as squamous cell carcinomas of the head and neck (HNSCC), are common types of human cancer. In the United States 31,000 men and women are diagnosed with HNSCC each year, and the 5-year relative survival rate for the patients is about 60%. 1 Although treatment of locoregionally advanced HNSCC with chemoradiotherapy has improved the survival of the patients, 2 combined-modality treatments are often associated with severe acute and late toxicities, and the optimal therapeutic regimen remains to be defined. The choice of treatment strategy is currently based primarily on the clinical prognosticators T stage and nodal disease. To minimize toxicity and improve efficacy of the treat- ment of HNSCC, it is necessary to find appropriate prognostic and predictive features that can be used to choose and modify the treat- ment given. No serum markers have yet been identified in HNSCC, although ongoing research has identified potential candidates. 3,4 YKL-40 is a new biomarker with a prognostic value in cancer. 5 A number of retrospective studies and one prospective study in patients with solid tumors and hematological malignancies 6–21 have shown that an elevated serum YKL-40 is an independent bio- marker of poor prognosis. In glioblastomas, YKL-40 expression assessed by immunohistochemistry is associated with increased resistance to radiotherapy and decreased overall survival, 22,23 and YKL-40 is upregulated at recurrence. 24 YKL-40 is a secreted heparin- and chitin-binding glycoprotein belonging to a group of ‘‘mammalian chitinase-like proteins’’ with an amino acid sequence similar to the 18-glycosyl hydrolase group of bacterial chitinases. 25 However, YKL-40 retains no enzymatic activity. In vitro, YKL-40 is secreted by a variety of cells, such as activated neutrophils, 26 macrophages 27 , 28 and some cancer cell lines. 29–31 In vivo, YKL-40 mRNA has been demonstrated in tumor-associated macrophages (TAM) 32 and in normal and malig- nant epithelial cells of the breast. 33 The human YKL-40 gene (CHI3L1) 28,34 and the crystal structure of the protein 35,36 have been characterized. The serum level and tumor tissue expression of YKL-40 in squamous cell carcinomas are unknown. We hypothesized that YKL-40 serum levels in patients with HNSCC might be associated with prognosis. Thus, in the present retrospective study we investi- gated the prognostic value of serum YKL-40 in pretreatment serum samples and in serial samples collected during the follow-up period in patients with HNSCC. Furthermore, the YKL-40 protein expression in pretreatment tumor samples from a subset of the patients was assessed by immunohistochemistry. Material and methods Study population From June 1997 to September 1998, 173 consecutive patients (median age 59, range 41–91) diagnosed with HNSCC and admit- ted to the Department of Oncology, Herlev Hospital, Denmark for primary radiotherapy with curative intent were included in a study comprising the collection of serial blood samples. Eligibility crite- ria were primary invasive HNSCC of the larynx, pharynx or oral cavity (Stage I–IV, TNM classification, UICC Geneva 1982) with- out evidence of distant metastases. For the present retrospective study, patients were ineligible if they had prior malignancy (except for nonmelanoma skin cancer or in situ cervical cancer), acute infection or rheumatic disease (confounding conditions causing elevated serum YKL-40). Abbreviations: CI, confidence interval; CV, coefficient of variation; HNSCC, squamous cell carcinoma of the head and neck; HR, hazard ratio; OS, overall survival; RFS, recurrence-free survival; TAM, tumor-associ- ated macrophages. Grant sponsors: Ingeniør August Frederik Wedell Erichsen Foundation, The Hørslev Foundation, Else and Mogens Wedell-Wedellsborg Foundation, Aase and Ejnar Danielsen Foundation, Desir ee and Niels Yde Foundation. *Correspondence to: Department of Oncology, Herlev Hospital, Herlev Ringvej 75, DK-2730, Denmark. Fax: 145-4488-3094. E-mail: anne.roslind@dadlnet.dk Received 27 April 2007; Accepted after revision 23 July 2007 DOI 10.1002/ijc.23152 Published online 23 October 2007 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 122, 857–863 (2008) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer