YKL-40, also known as human cartilage glycoprotein 39, is a member of the “mammalian chitinase-like proteins”, but lacking of chitinase activity. Increased variations in serum concentrations are associated with inflammatory processes and several types of cancer.In this study we evaluated serum YKL-40 levels in healthy controls and in women with benign and malignant breast tumors.            YKL-40 serum levels were measured by enzyme-linked immunosorbent assay – ELISA in 32 patients. The effect of the various factors was analyzed using correlation and regression analyses simultaneously determining the size and direction of correlation. The level of statistical significance of null hypothesis was P<0.05.  Our study showed that serum YKL-40 level in breast carcinoma was significantly higher than the concentration in healthy controls (p<0.01). The changes in protein levels were higher than 25%. Serum YKL-40 increased with age (rxy=0.46). The correlation between glycoprotein quantity and age was positive, but feeble.This investigation is first in Bulgaria to demonstrate significantly elevated serum YKL-40 level in breast carcinoma compared to women with benign breast tumors and healthy controls. Longitudinal studies are needed to confirm YKL-40 as a potential and reliable biomarker

Maria Kazakova

Tanya Deneva

Vanya Uzunova

Victoria Sarafian

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  / JofIMAB; Issue: vol. 16, book 3, 2010 /  3YKL-40 IN SERA OF BREAST TUMOR PATIENTSMaria Kazakova1, Tanya Deneva2, Vanya Uzunova3, Victoria Sarafian11) Department of Medical Biology2) Department of Clinical Laboratory3) Clinic of Thoraco-Abdominal SurgeryMedical University - Plovdiv, BulgariaJournal of IMAB - Annual Proceeding (Scientific Papers) vol. 16, book 3, 2010SUMMARYYKL-40, also known as human cartilage glycoprotein39, is a member of the “mammalian chitinase-like proteins”,but lacking of chitinase activity. Increased variations in serumconcentrations are associated with inflammatory processesand several types of cancer.In this study we evaluated serum YKL-40 levels inhealthy controls and in women with benign and malignantbreast tumors.YKL-40 serum levels were measured by enzyme-linkedimmunosorbent assay – ELISA in 32 patients. The effect ofthe various factors was analyzed using correlation andregression analyses simultaneously determining the size anddirection of correlation. The level of statistical significanceof null hypothesis was P<0.05.Our study showed that serum YKL-40 level in breastcarcinoma was significantly higher than the concentration inhealthy controls (p<0.01). The changes in protein levels werehigher than 25%. Serum YKL-40 increased with age (rxy=0.46).The correlation between glycoprotein quantity and age waspositive, but feeble.This investigation is first in Bulgaria to demonstratesignificantly elevated serum YKL-40 level in breast carcinomacompared to women with benign breast tumors and healthycontrols. Longitudinal studies are needed to confirm YKL-40as a potential and reliable biomarker.Key words: YKL-40, ELISA, breast tumorINTRODUCTIONCancer research is focused on identifying themolecular and genetic changes that cause malignanttransformation. Molecular targeted therapy for advanced solidtumors directed towards receptors for growth factors orsignaling and regulatory molecules has been proven  to besuccessful (1).According to the American Cancer Society estimatedcancer death in women for 2009 is 269,800. Breast canceraccounts for 15% of all cases of death (National CancerSociety). Currently available breast cancer screening toolssuch as mammography and breast examination miss 10–40%of early breast cancers (2).The development of noninvasive techniques thatcould distinguish women with or without breast cancer is ofvast importance. The advantages of the markers include earlydetection or screening, discriminating benign from malignantdisease, histological differentiation, and early prognosis (3).YKL-40 is an extracellular matrix glycoprotein with ahigh sequence homology to the proteins of the chitinase likefamily, but without chitinase activity (4). The plasma proteinis secreted by macrophages, chondrocytes, activatedneutrophils, differentiated monocytes, vascular smoothmuscle cell and cancer cells. Increased serum concentrationsare associated with inflammatory processes and several typesof cancer.The binding site of YKL-40 to receptors on the cellsurface is not known yet and the biological function of theprotein is only poorly understood.Elevated serum level of YKL-40 has a potentialprognostic value in several cancers including breast (1), ovary(5, 6, 7), colorectal (8), glioblastoma (9, 10), melanoma (11). Inthese studies, increased variations in serum protein areassociated with short recurrence and short overall survival.It is assumed that YKL-40 has a role in cancerproliferation, differentiation, metastatic potential,angiogenesis, however, these effects are not obtained yetin vivo.On the other hand, serum YKL-40 is not specific forcancer only. Elevated values are found in processescharacterized by acute or chronic inflammation and intensiveextracellular tissue remodeling – rheumatoid arthritis (12, 13),osteoarthritis (14, 15), inflammatory bowel disease (16), giantcell arthritis (17), pulmonary sarcoidosis (18), liver fibrosis (19).The aim of the present study was to determine serumYKL-40 levels in women with benign and malignant breasttumors and healthy controls. This is the first study in Bulgariainvestigating YKL-40 expression.MATERIALS AND METHODSThe control group consisted of 10 healthy femalevolunteers aged (18-50) without cancer or joint, liver,metabolic, or endocrine diseases. Clinical and routinehematological, biochemical and coagulation tests wereperformed to assess their health status.DOI: 10.5272/jimab.1632010_3-74  / JofIMAB; Issue: vol. 16, book 3, 2010 /The target group consisted of 14 women withfibroadenoma and 8 women with  breast carcinoma. The studywas approved by the University Ethics Committee. Informedcontent was asked and achieved from all examined individualsaccording to the Helsinki Declaration.Venous blood samples were collected from patientsbefore surgery or chemoradiation in the morning (4.5 mmol/lblood, Monovette, Sarstedt) as atraumatically as possible aftera 12-hour fasting and 30-minute rest immediately prior totesting. The samples were centrifuged at 2500 rpm for 10minutes. The serum was kept at -20°C for no more than amonth before analysis.Serum YKL-40 concentrations were determined by atwo-site, sandwich-type, enzyme-linked immunosorbent assay(ELISA) (Quidel Corporation, Cat. No. 8020) according to themanufacturer’s instructions. The validation of the method wasperformed in compliance with the international standard ofquality and competence of medical laboratories (BDS/EN/ISO15189). The method shows high precision; the results areconsistent with the recommended minimal non-reproducibility(intra-assay CV<10%; inter-assay CV<12%) for ELISA instudying YKL-40 as given by the manufacturer. The detectionlimit of the YKL-40 assay was 20.0 ng/ml. All samples wereanalysed in duplicates.Microsoft Excel 2000 and SPS 12.0 (Windows XP) wereused to analyze the results.  The level of statisticalsignificance of hull hypothesis was P<0.05.  The effect of thevarious factors was analyzed using correlation and regressionanalyses simultaneously determining the size and directionof correlation.RESULTSOur investigation revealed a mean value 41,11 (20-59)ng/ml of serum YKL-40 in healthy subjects. The median serumconcentration of YKL-40 in patients with breast carcinomawas 100,27 (36-169) ng/ml  and it was significantly highercompared to the level in women with fibroadenoma 44,58 (22-87) ng/ml (P<0.001).Obviously, the results show that serum YKL-40 levelin breast carcinoma is significantly higher than theconcentration in healthy controls. The results are presentedon Fig. 1.Next, we determined the correlation between age andserum YKL-40 level in all patients aged 18-75. It wasconstructed by linear regression with the glycoprotein as thedependent variable and age as the explanatory variable.Serum YKL-40 increased with age (rxy=0.46). The correlationwas positive but feeble.DISCUSSIONDifferent research groups showed that in healthyadults (aged 18-79), the median level of serum YKL-40determined by ELISA was 43 ìg/l (range 20-184 ìg/ l) (20).At the cellular level YKL-40 protein expression is highin tissues characterized by rapid proliferation, differentiation,and undergoing morphogenetic changes. YKL-40 proteinexpression is widely distributed at specific time points in earlydeveloping human musculoskeletal system. (21). Recentstudies show that the expression of YKL-40 in normal adulttissues is feeble and show positive relation with metabolicactivity (22).Our results revealed that serum protein level in healthyadults was 41,11 ng/ml - values close to the ones observedin the Danish population. We have found that serum YKL-40increased with age (rxy=0.46), but the correlation was feeble.According to Johansen et al.  (2008), serum YKL-40 increasedwith age but in the normal reference interval. Aging isassociated with enhanced inflammatory activity reflected byincreased circulating levels of TNF-β, IL-6, cytokineantagonists and acute phase proteins (23).It is assumed that YKL-40 could participate not onlyin normal cell functions but also in inflammation andneoplastic processes. The complete biological function ofYKL-40 in cancer is not clear. It has been hypothesized thatthe protein exerts growth properties on chondrocytes,synovial cells and fibroblasts where it works synergisticallywith insulin-like growth factor (24, 25). It could not be excludedthat YKL-40 might act as a growth factor for cancer cells, too.Another possibility is that YKL-40 protects cancercells from undergoing apoptosis. It is also called “breastregression protein” (Brp-39), because it induces mammaryinvolution in mice a few days after weaning (26).It is well known that the programmed cell death is amajor process in mammary involution.  In fact, YKL-40determines which cells to survive during the drastic tissueremodeling and acts as a protective molecule (14).Obviously, our study showed that serum YKL-40 levelin breast carcinoma is significantly higher than theconcentration in women with fibroadenoma (p<0.01). Theresults are presented in Table 1.The changes in serum YKL-40 levels in our study arehigher than 25%. Johansen et al., (2006) suggested thatchanges in serum YKL-40 concentration higher than 20%should be indicative of elevated serum range.It is believed that increased expression of YKL-40 mayimprove the identification of women at increased breastcancer risk. The protein was determined in tissue, nippleaspirated fluid and serum of 118 women - 61 healthy subjects,10 with precancer and 47 with breast cancer. It was assessedthat YKL-40 was concentrated in nipple aspirated fluidrelative to serum. Nipple aspirated fluid levels of YKL-40 werehigher in precancers than either levels from healthy women.It was observed that YKL-40 expression in precanceroustissue which trended higher than in normal or canceroustissue (2).Cancer development depends on the interactionbetween cancer cells and the extracellular matrix components.The immunohistochemical analysis of YKL-40 expression in  / JofIMAB; Issue: vol. 16, book 3, 2010 /  5biopsies from breast cancer (27) and colorectal cancer (28)are positive for protein expression. Furthermore, themacrophages and neutrophils surrounding the breast andcolorectal cancer cells also express YKL-40.YKL-40 is not specific for all types of cancer disease.It was shown that at the time of the first cancer diagnosis16-74% of the patients had elevated serum YKL-40 levels.However, in patients with metastatic cancer, serum YKL-40levels increased 39-83% at the time of diagnosis (29).It is considered that serum YKL-40 could be moreuseful for monitoring disease and progression in cancerpatients after treatment, providing independent informationof survival (28, 10, 21).A study by Yamac et al., (2008) demonstrated that themedian serum YKL-40 concentration in patients with locallyadvanced breast cancer was 149.5 ìg/ml and it was higherthan levels observed in healthy female controls. It wassuggested that YKL-40 may be a useful prognostic indicatorof outcome for patients with locally advanced breast cancer(31).The serum levels of an epithelial growth factor andYKL-40 in relation to outcome in 100 patients with their firstdiagnosis of recurrent breast cancer was assessed.  Elevatedserum YKL-40 concentrations were found in 30%  of thepatients and epithelial growth factor in 32 % of the subjects.It was found that high serum levels of epithelial growth factorand YKL-40 reflected increased aggressiveness anddecreased response to anthracycline-based therapy. It wassuggested that high serum YKL-40 and epithelial growthfactor independently identified patients with metastatic breastcancer with a poor prognosis (1).YKL-40 facilitates cell attachment and migration ofvascular endothelial cells, which is an indication that theprotein may function in angiogenesis (13).Immunohistochemical analysis of human breast cancershowed a correlation between YKL-40 expression and bloodvessel density. These results are another evidence for theangiogenic activities of YKL-40 in cancer development (32).CONCLUSIONIn conclusion, our study is the first in Bulgaria to showsignificantly elevated serum YKL-40 level in breast carcinomacompared to women with benign breast tumors and healthycontrols. Prospective investigations are aimed at evaluationof YKL-40 as a reliable biomarker and an appropriate targetfor development of anticancer therapy.Figure 1. Serum levels of YKL-40 in healthy subjectsand patients with breast tumors020406080100120140concentration (ng/ml)healthy subjects fibroadenomacarcinomaTable 1.  Comparative data on YKL-40 serum levels inbreast fibroadenoma and carcinomaCriteria Fibroadenoma CarcinomaNumber of patients 14 8Concentrationof YKL-40 (ng/ml) 41,11 100,27SD 12,61 80,33P-value <0,02 <0,026  / JofIMAB; Issue: vol. 16, book 3, 2010 /Acknowledgments:The study is supported by grants No.-1/2009 and No.-5/2008 from Medical University - Plovdiv.Fig. 2. Summarized biological functions of YKL-40YKL-40Inflammation TumorogenesisGlioblastomaBreast cancerOvarian cancerColorectal cancerMelanomaRheumatoid arthritisOsteoarthritisInflammatory bowel diseaseGiant cell arteritisHealthy subjectsMammary involutionGrowth propertiesCell survivalDifferentiationEmbryonic developmentREFERENCES:1. Jensen BV, Johansen JS, Price PA.High levels of serum HER-2/neu and YKL-40 independently reflect aggressiveness ofmetastatic breast cancer. Clin Cancer Res,2003; 9: 4423-4434.2. Qin W, Zhu W, Schalter L., et al.Increased expression of the inflammatoryprotein YKL-40 in precancers of the breast.Int  J  Cancer, 2007; 121: 1536–1542.3. Zhong L, Ge K,  Zu J., et al.Autoantibodies as potential biomarkers forbreast cancer. Breast Cancer Res, 2008; 10:doi:10.1186/bcr2091.4. Hakala BE, White C, Recklies AD.Human cartilage gp-39, a major secretoryproduct of articular chondrocytes andsynovial cells, is a mammalian member of achitinase protein family. J Biol Chem, 1993;268: 25803-25810.5. Hrgdall EVS, Johansen JS, Kjaer SK.,et al. High plasma YKL-40 level in patientswith ovarian cancer stage III is related toshorter survival. Oncol Rep, 2003; 10: 1535-1538.6. Hrgdall E, Ringsholt M, Høgdall C.,et al. YKL-40 tissue expression and plasmalevels in patients with ovarian cancer. BMCCancer, 2009; 9: 8 doi: 10.1186/1471-2407-9-87. Dupont J, Tanwar MK, Thaler HT.,et al. Early detection and prognosis ofovarian cancer using serum YKL-40. J ClinOncol, 2004; 22: 3330-3339.8. Cintin C, Johansen JS, ChristensenIJ., et al. Serum YKL-40 and colorectalcancer. Br J Cancer, 1999; 79: 1494-1499.9. Tanwar MK, Gilbert MR, HollandEC. Gene expression microarray analysisreveals YKL-40 to be a potential serummarker for malignant character in humanglioma. Cancer Res, 2002; 62: 4364-4368.10. Hormigo A, Gu B, Karimi S., et al.YKL-40 and matrix metalloproteinase-9 aspotential serum biomarkers for patients withhigh-grade gliomas. Clin Cancer Res, 2006;  / JofIMAB; Issue: vol. 16, book 3, 2010 /  7Address for correspondence:Maria Hristova KazakovaDepartment of Medical biology, Medical University of Plovdiv15A V. Aprilov Blvd., 4000 Plovdiv, BulgariaTel. +359/42/602 993e-mail: kazakova25@abv.bg12: 5698-5704.11. Schmidt H,  Johansen JS,  Gehl J.,et al. Elevated serum levels of YKL-40 isan independent prognostic factor for poorsurvival in patients with metastaticmelanoma. Cancer, 2006; 106: 1130-1139.12. Volck B, Johansen JS, StoltenbergM., et al. Studies on YKL-40 in knee jointsof patients with rheumatoid arthritis andosteoarthritis. Involvement of YKL-40 in thejoint pathology. Osteoarthritis Cartilage,2001; 9: 203-214.13. Johansen JS. Studies on serum YKL-40 as a bioarker in diseases withinflammation, tissue emodelling, fibroses andcancer. Dan Med Bull, 2006; 53:172-209.14. Kawazaki M, Hasegawa Y, KondoS., et al. Concentration and localization fYKL-40 in hip joint diseases. J Rheumatol,2001; 28: 341-345.15. Zivanovic. S, Rachov LP, VojvodicD, Yucetie D. Human cartilage glycoprotein39 – biomarker of joint damage in kneeosteoarthritis. International Orthopaedics,2009, 33: 1165-1170.16. Vind I, Johansen JS, Price PA,Munkholm P. Serum YKL-40, a potentialnew marker of disease activity in patientswith inflammatory bowel disease. Scand JGastroenterol, 2003; 38: 599-605.17. Johansen JS, Baslund B, GarbarschC., et al. YKL-40 in giant cells andmacrophages from patients with giant cellartheritis. Arthritis Rheum, 1999; 42: 2624-2630.18. Johansen JS, Milman N, HansenM., et al. Increased serum YKL-40 inpatients with pulmonary sarcoidosis. Aregulated kinase- and protein kinase B-mediated signalling pathways. Biochem J,2002; 365: 119-126.26. Mohanty AK, Singh G,Paramasivam M., et al. Crystal structure ofa novel regulatory 40-kDa mammary glandprotein (MGP-40) secreted duringinvolution. J Biol Chem, 2003; 278: 14451-14460.27. Roslind A, Johansen JS. YKL-40: anovel marker shared by chronicinflammation and oncogenic transformation.Methods Mol Biol, 2009; 511: 159-184.28. Cintin C, Johansen JS, ChristensenIJ., et al. Highserum YKL-40 level aftersurgery for colorectal carcinoma is relatedto short survival. Cancer,  2002; 95: 267-74.29. Johansen JS, Jensen BV, Roslind A,et al. Serum YKL-40, a new prognosticbiomarker in cancer patients? CancerEpidemiol Biomarkers Prev,  2006; 15: 194-202.30. Ringsholt M, Høgdall EVS,Johansen JS., et al. YKL-40 proteinexpression in normal adult human tissues –an immunohistochemical study. J Mol Hist,2007; 38: 33–43.31. Yamac D, Ozturk B, Coskun U., etal.  Serum YKL-40 levels as a prognosticfactor in patients with locally advancedbreast cancer. Advance in Therapy, 2008; 25:801-809.32. Shao R, Hamel K, Petersen L., et al.YKL-40, a secreted glycoprotein, promotestumor angiogenesis. Oncogene, 2009; 28:4456-4468.potential marker of disease activity? RespMed, 2005; 99: 396-402.19. Nrjgaard C, Johansen JS,Christensen E., et al. Serum levels of YKL-40 and PIIINP as prognostic markers inpatients with alcoholic liver disease. JHepatol, 2003, 39: 179-186.20. Johansen JS, Lottenburger T,Nielsen HJ., et al. Weekly and long-timevariation in serum concentrations of YKL-40 in healthy subjects. Cancer EpidemiolBiomarkers Prev, 2008;  5: 2603:2608.21. Johansen JS, Hryer PE, Larsen LA,et al. YKL-40 protein expression in the earlydeveloping human musculoskeletal system.J Histochem Cytochem, 2007; 55: 1213-1128.22. Ringsholt M, Hrgdall EVS, JohansenJS., et al. YKL-40 protein expression innormal adult human tissues - an immuno-histochemical study. J Mol Hist, 2007; 38:33–43.23. Bruunsgaard H, Pedersen M,Pedersen BK. Aging and proinflammatorycytokines. Curr Opin Hematol, 2001; 8:131-136.24. De Ceuninck F, Gaufillier S,Bonnaud A., et al. YKL-40 (Cartilage gp-39) induces proliferative events in culturedchondrocytes and synoviocytes andincreases glycosaminoglycan synthesis inchondrocytes. Biochem Biophys ResCommun, 2001; 285: 926-931.25. Recklies AD, White C, Ling H. Thechitinase 3-like protein human cartilageglycoprotein 39 (HC-gp39) stimulatesproliferation of human connective tissuecells and activates both extracellular signal-

Journal of IMAB - Annual Proceeding (Scientific Papers)

YKL-40 IN SERA OF BREAST TUMOR PATIENTS

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YKL-40 IN SERA OF BREAST TUMOR PATIENTS

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