BMC Cancer BioMed Central

Research article Open Access

YKL-40 tissue expression and plasma levels in patients with ovarian
cancer
Estrid VS Høgdall*1,2, Merete Ringsholt3, Claus K Høgdall4, Ib
Jarle Christensen5, Julia S Johansen6, Susanne K Kjaer1,4, Jan Blaakaer7,
Lene Ostenfeld-Møller4, Paul A Price8 and Lise H Christensen9

Address: 1Department of Virus, Hormones and Cancer, Institute of Cancer Epidemiology, Danish Cancer Society, Copenhagen, Denmark,
2Department of Pathology, Herlev Hospital, University of Copenhagen, Copenhagen, Denmark, 3Faculty of Medical Laboratory Science, University

College Oeresund, Copenhagen, Denmark, 4The Gynaecologic Clinic, The Juliane Marie Centre, Rigshospitalet, University of Copenhagen,
Copenhagen, Denmark, 5Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter, University of Copenhagen, Copenhagen, Denmark,
6Department of Rheumatology, Herlev Hospital, University of Copenhagen, Denmark, 7Department of Gynecology and Obstetrics, Aarhus

University Hospital, Skejby, Aarhus, Denmark, 8Department of Biology, University of California, San Diego, La Jolla, CA, USA and 9Department
of Pathology, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark
Email: Estrid VS Høgdall* - [email protected]; Merete Ringsholt - [email protected]; Claus K Høgdall - [email protected]; Ib
Jarle Christensen - [email protected]; Julia S Johansen - [email protected]; Susanne K Kjaer - [email protected];
Jan Blaakaer - [email protected]; Lene Ostenfeld-Møller - [email protected]; Paul A Price - [email protected];
Lise H Christensen - [email protected]
* Corresponding author

Published: 9 January 2009 Received: 26 June 2008

Accepted: 9 January 2009
BMC Cancer 2009, 9:8 doi:10.1186/1471-2407-9-8
This article is available from: http://www.biomedcentral.com/1471-2407/9/8
© 2009 Høgdall et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: YKL-40 (chitinase-3-like-1) is a member of "mammalian chitinase-like proteins". The protein is
expressed in many types of cancer cells and the highest plasma YKL-40 levels have been found in patients with
metastatic disease, short recurrence/progression-free intervals, and short overall survival. The aim of the study
was to determine the expression of YKL-40 in tumor tissue and plasma in patients with borderline ovarian tumor
or epithelial ovarian cancer (OC), and investigate prognostic value of this marker.
Methods: YKL-40 protein expression was determined by immunohistochemistry in tissue arrays from 181
borderline tumors and 473 OC. Plasma YKL-40 was determined by ELISA in preoperative samples from 19
patients with borderline tumor and 76 OC patients.
Results: YKL-40 protein expression was found in cancer cells, tumor associated macrophages, neutrophils and
mast cells. The tumor cell expression was higher in OC than in borderline tumors (p = 0.001), and associated
with FIGO stage (p < 0.0001) and histological subtype (p = 0.0009). Positive YKL-40 expression (≥ 5% staining)
was not associated with reduced survival. Plasma YKL-40 was also higher in patients with OC than in patients
with borderline tumors (p < 0.0001), and it was positively correlated to serum CA-125 (p < 0.0001) and FIGO
stage (p = 0.0001). Univariate Cox analysis of plasma YKL-40 showed association with overall survival (p <
0.0001). Multivariate Cox analysis, including plasma YKL-40, serum CA125, FIGO stage, age and radicality after
primary surgery as variables, showed that elevated plasma YKL-40 was associated with a shorter survival (HR =
2.13, 95% CI: 1.40–3.25, p = 0.0004).
Conclusion: YKL-40 in OC tissue and plasma are related to stage and histology, but only plasma YKL-40 is a
prognostic biomarker in patients with OC.

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Background [24], stage III [23], or recurrence of OC [25] and high

YKL-40 (chitinase-3-like-1) is a highly conserved protein plasma or serum levels of YKL-40 have shorter survival
[1] and a member of "mammalian chitinase-like proteins" compared to patients with normal levels of YKL-40. No
[1,2]. The protein is expressed in many types of cancer close association between serum YKL-40 and serum CA-
cells (dbest NCBI database), and elevated plasma levels are 125 and CA15-3 in patients with OC has been published
predictive of poor prognosis in patients with different [23-26]. In patients with FIGO stage III multivariate Cox
types of cancer [2-8]. The highest plasma YKL-40 levels analysis including preoperative plasma YKL-40, serum
have been found in patients with metastatic disease, short CA125, optimal vs. suboptimal primary surgery, age, and
recurrence/progression-free intervals, and short overall histological type of tumor, demonstrated that high
survival [2-8]. Furthermore, plasma YKL-40 has provided plasma YKL-40 was an independent biomarker of short
independent information on prognosis over clinical char- survival [23]. Furthermore, serum YKL-40 was a predictor
acteristics and biomarkers, such as serum CA-125, LDH, of chemoresistance in the second-line treatment of OC
PSA, CEA, and HER2 [2-8]. It has been suggested that YKL- patients [26].
40 is associated with cancer cell proliferation, differentia-
tion, metastatic potential, and extracellular tissue remod- The aim of this study was to determine YKL-40 tissue
elling, but in vivo proofs of these are yet to be obtained [2]. expression in borderline and epithelial OC tissues, its pos-
sible correlation to clinical-pathological parameters and
Immunohistochemical studies have demonstrated a plasma YKL-40 levels, and the prognostic value of both
strong cellular expression of YKL-40 in all germ layers of YKL-40 tissue expression and plasma levels.
human embryos and fetuses, including ecto-, meso- and
endoderm [9]. The expression is particular high in tissues Methods
characterized by rapid proliferation and marked differen- Patient Population
tiation, and in tissues undergoing morphogenetic changes The MALOVA study ("MALignant OVArian cancer study")
[9]. A similar pattern is seen in normal adult human tis- is a multidisciplinary Danish study covering epidemiol-
sue, where YKL-40 is highly expressed in cells with a high ogy, lifestyle factors, biochemistry and molecular biology
cellular activity [10]. with the purpose of identifying risk factors and prognostic
factors for OC. The design of the MALOVA study is
Neoplastic tissue has also been found to show a higher described in details elsewhere [27]. Briefly, preoperative
expression for YKL-40 than the normal counterpart [2]. In blood samples and tumor tissue samples were obtained
glioblastoma, YKL-40 protein expression has proven to be from the majority of the 681 OC patients and from the
a biomarker of histologic subtypes [11], with high gene- 235 women with borderline ovarian tumor were included
and protein expression being associated with poor radia- in the MALOVA study. FIGO stages were obtained from
tion response and early disease progression and death clinical records and reviewed by two gynecologists spe-
[11-13]. In addition, at recurrence YKL-40 is up-regulated cialized in OC. Patients were either classified as radically
in the tumor tissue [14] and elevated in serum [15]. operated with no macroscopic residual tumor or non-rad-
ically operated with macroscopic residual tumor after sur-
High YKL-40 protein expression has also been found in gery. On the retrospectively collected paraffin embedded
carcinoma cells from breast [16-18], colon [2], liver [19], tissues, histologic grading were performed individually by
cervix [2], and head/neck [20], and skin (melanoma) [2], two persons.
as well as within tumor-associated macrophages, mast
cells and leukocytes [10,18,20-22]. In a small study of Follow-up
patients with breast cancer high YKL-40 protein expres- In Denmark all inhabitants have a unique personal (10-
sion in the cancer cells was associated with short disease- digit) identification number (CPR number), which is used
free survival [16]. However, this could not be confirmed universally in the Danish society. These identification
in a recent large study of YKL-40 protein expression in numbers, which comprise information on date of birth
biopsies from 630 patients with primary breast cancer; in and sex, are registered in the computerized Danish
this study there was no association between YKL-40 pro- National Central Population Register. The register con-
tein expression in the breast cancer cells and disease free tains information on e.g. dates of death and emigration.
survival and overall survival [18]. All cases in this study were traced in the register and date
of death, emigration or August 23rd, 2007, whichever
There are no published studies on YKL-40 protein expres- came first, were registered. The relevant hospital files were
sion in OC tissue. Preoperative serum or plasma concen- collected and scrutinized and information on treatment
trations of YKL-40 are elevated in 65% of patients with (surgery and chemotherapy), was established. At the end
FIGO stage I and II, and in 74% to 91% of patients with of follow-up, a total of 490 OC patients had died (median
FIGO stage III and IV [23,24]. Patients with early-stage OC follow-up time: 23 months, range: 1–111), and 191 OC

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patients were still alive (median follow-up time: 91 order to demask antigens within the tissue, sections were
months, range: 66–121). pretreated in TEG buffer, pH 9.0 (Tris 10 mmol/l and
EGTA 0.5 mmol/l), followed by heating in a microwave
Tissue array oven to 98°C for 15 min. Sections were then left to cool
The paraffin embedded tissue from the tumors was used in the buffer at room temperature for an additional 15
for tissue array analyses. Forty-eight OC and 24 borderline min. To block endogen peroxidase activity the sections
ovarian tumors were excluded because of poor tissue were treated with 0.03% w/v hydrogen peroxide for 5
quality or lack of tumor tissue in the collected blocks, and min. To avoid background staining the sections were then
132 OC and 16 borderline ovarian tumors were excluded pre-incubated with 5% w/v purified bovine serum albu-
due to unreadable YKL-40 expression results, lack of min (BSA) (Dade Behring, Liederbach, Germany) diluted
tumor cells in the selected TA cylinders, loss of tissue dur- in TRIS buffer pH 7.6 for 10 min. The primary specific
ing the staining procedure or folding/tearing of sections mouse monoclonal antibody against human YKL-40
by the microtome. Finally, 42 non-epithelial tumors (28 (201.F9, 3.8 mg/ml, isotype IgG2b, κ, epitope GAWRGTT-
OC and 14 borderline ovarian tumors) were excluded GHHS, corresponding to the amino acids 210–220) was
because epithelial and non-epithelial ovarian tumors dif- diluted 1:100 (38 ng/ml) in 1% w/v purified BSA (Dade
fer in embryologic and pathologic characteristics. Behring) in TRIS buffer pH 7.6 and incubated for 60 min.
Together, these exclusions left 473 OC and 181 borderline The secondary antibody EnVisionTM+System-HRP (DAB)
ovarian tumors, which were suitable for the YKL-40 tissue (Dako, Glostrup, Denmark, Code K 400711-2, lot no.
expression and survival analyses. 104.225) was used for 30 min. The colour was developed
with DAB added with chromogen for 10 min. Sections
For the tissue array production, one to two representative were rinsed in water, counterstained in Mayers Hematox-
areas were marked on a freshly cut H&E stained section ylin for 3 min and finally dehydrated, mounted and cov-
from each selected block. Tissue cylinders with a diameter erslipped with Pertex. Washings with 5 mM Tris buffer,
of 2 mm were punched from corresponding areas in the pH 7.6 with NaCl 0.9% w/v and Tween 0.1% v/v (TBS)
donor tumor block and brought into 2–4 individual were used between all steps in the procedure. After heat-
recipient paraffin blocks (tissue array blocks), using a cus- ing all steps were performed at room temperature in a
tom-made manual Tissue Array Instrument (Beecher humidity chamber to avoid air-drying of the sections. The
Instruments, Silver Spring, MD, USA). Once the cores staining process was done manually.
were laid into the recipient block, the paraffin was slightly
melted in order to bind the cores into the block. This treat- YKL-40 scoring of tissue expression
ment secured the cores during sectioning. Melting was Two observers, both experienced in evaluating immuno-
achieved by placing the blocks in an oven at 37°C for 10 histochemical stained tissues, simultaneously assessed the
min. patterns of YKL-40 protein expression of each tumor sam-
ple. The observers had no knowledge of clinical parame-
A total of 2 to 4 tissue arrays consisting of corresponding ters and study endpoint. Standardization of scoring was
viable and representative tissues from each ovarian tumor achieved by comparison of the scores, and any discrepan-
were constructed in order to minimize the risk of intra- cies were resolved by consensus. Scoring for YKL-40 pro-
tumor variability. The tissue arrays were sorted with tein expression was based on the proportion of cells in a
respect to histological subtype and FIGO stage. Four cores given tumor specimen exhibiting distinct cytoplasmic
of control tissue (2 from kidney, 2 from liver) were placed immunopositivity as well as the intensity of staining (per-
strategically in each tissue array to ensure unique orienta- centage scale: 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and
tion of every tissue array together with a maximum of 31 100). Secondly, the YKL-40 scoring results were trans-
different patient tumor samples. formed into two scales: a two-tiered scale (1: negative; 2:
≥ 5% positive tumor cells) and a four-tiered scale (1: neg-
Tissue preparation and immunostaining of YKL-40 ative; 2: 5% positive tumor cells; 3: 10% positive tumor
Two μm sections (Section Transfer System, STS, Ergostar cells; 4: ≥ 20% positive tumor cells). The four tissue con-
HM200, MICROM International GmbH, Walldorf, Ger- trol cores of kidney and liver, respectively, showed con-
many) from the tissue array blocks were transferred to sistent staining results. For the general description and the
glass slides (DAKO Chem-Mate Capillary Cap Microscope prognostic evaluation both scales were used.
Slides, 57 mm, DAKO A/S, Glostrup, Denmark). Slides
were stored at 4°C for a maximum of 8 days until staining Plasma YKL-40 analysis
for YKL-40. Blood samples were obtained no earlier than two weeks
prior to surgery from the 95 patients operated at Herlev
Prior to staining, the sections were deparaffinized in Hospital and Rigshospitalet, University of Copenhagen.
xylene and rehydrated in graded dilutions of alcohol. In EDTA-blood samples were left on the blood cells at room

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temperature for less than 8 hours. Following the samples analyses the YKL-40 expression scale in tumor tissue was
were separated by centrifugation at 2000 g for 10 min. at dichotomized as < 5% vs. ≥ 5% and the plasma YKL-40
room temperature and plasma EDTA samples stored in levels by the actual value on the log scale (natural) (log
aliquots at -80°C until analysis. EDTA plasma samples transformed). The multivariate Cox regression multivari-
collected from patients included in the MALOVA study, ate analysis was based on FIGO stage, residual tumor after
who had been treated at other hospitals were not used for primary surgery, serum CA-125, age and plasma YKL-40
plasma YKL-40 measurements, since the plasma obtained levels by the actual value on the log scale (natural) (log
from these centers had not been separated from the blood transformed). Confidence intervals (95% CI) were based
cells within 8 hours after sampling. It is known that EDTA on Wald's test statistic for the corresponding parameters
plasma YKL-40 is only stable when samples are processed in the Cox regression model, i.e. on the log-scale for the
within 8 hours [28]. hazard ratios (HR). Model assessment was done using
graphical methods, Schoenfeld and martingale residuals.
Plasma levels of YKL-40 were determined in duplicates by P-values less than 5% were considered significant. All cal-
a commercial two-site, sandwich-type enzyme-linked culations were performed using SAS (version 9.1, SAS
immunosorbent assay (Quidel, Santa Clara, CA, USA) Institute, Cary, NC, USA).
using streptavidin coated microplate wells, a biotinylated-
Fab monoclonal capture antibody, and an alkaline phos- Results
phatase-labeled polyclonal detection antibody. The detec- YKL-40 protein expression in borderline ovarian tumors
tion limit was 20 μg/l. The intra-assay coefficient of and in ovarian cancer tissue
variation (CV) was ≤ 5.0% and inter-assay CVs ≤ 10.2%. Tissue arrays for YKL-40 immunohistochemical analysis
The samples were analyzed blinded to clinical parameters were available from 181 patients with borderline ovarian
and study endpoint. tumors (median age 54 years, range 33 – 80) and from
473 patients with OC (median age 59 years, range 26 –
Healthy subjects 80).
The reference interval for plasma YKL-40 was determined
in 144 healthy women (median age 51 years, range 18–79 The distribution of the YKL-40 score in the borderline
years) characterized by not being on medication and hav- ovarian tumors was: level 1 (negative): 44 (24%); level 2:
ing no signs of pre-existing disorders such as joint, liver, 74 (41%); level 3: 35 (19%) and level 4: 28 (16%). The
metabolic or endocrine disease or malignancy. distribution of the YKL-40 score in the OC samples was:
level 1 (negative): 112 (24%); level 2: 101 (21%); level 3:
Ethics 138 (29%) and level 4: 122 (26%). Figure 1 illustrates rep-
Written informed consent was obtained from all patients. resentative examples of the different levels of YKL-40
The study has been approved by the scientific ethical com- staining in borderline ovarian tumors and OC. The cancer
mittee in the study area (KF01-384/95). cells showed a diffuse granular staining of the cytoplasm.
Staining of membranes and nuclei were not observed.
Statistical analyses Borderline ovarian tumors appeared with the same stain-
Statistical comparisons between groups were carried out ing pattern as the carcinomas, but with reduced staining
using Chi-square or rank sum tests. Association between intensity (Figure 1, b–d). YKL-40 protein expression was
YKL-40 tissue expression levels and plasma YKL-40 levels found in all the different histological types of OC (Figure
was assessed using the Spearman correlation. The clinical 1). The YKL-40 protein was also expressed in inflamma-
endpoint in the OC patients was survival determined as tory cells such as macrophages, mast cells and neu-
the time from baseline blood sample before operation to trophils. These results have been confirmed by double-
time of death updated at August 23rd, 2007. Patients who labeling methods verifying co-expression of YKL-40 and
died from non-related OC were censored in the survival tryptase (mast cells) and CD68 (macrophages), respec-
analyses at the date of death. Cases in which patients were tively (data not shown).
alive by this date were censored. Survival probabilities
were estimated by the Kaplan-Meier method and tests for The association between clinico-pathological variables
differences between strata were done using the log-rank and YKL-40 score is shown in Table 1 and Table 2. The
statistic. Graphical presentation using Kaplan-Meier esti- YKL-40 protein expression (staining percentage scale) in
mates of survival were shown grouping the patients by OC was associated with FIGO stage (p < 0.0001) and his-
YKL-40 expression in the tumor tissue dichotomized at tological type of tumor (p < 0.0001), and the YKL-40
5%. Graphical presentation using Kaplan-Meier estimates expression (staining percentage scale) was higher in the
of survival were shown grouping the patients by plasma OC than in the borderline tumors (p = 0.0019). Also a
YKL-40 levels dichotomized as normal or elevated com- tendency towards a correlation between reduced YKL-40
pared to age-matched controls. In the univariate survival

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Figure 1
Immunohistochemical
(×40) analysis of YKL-40 protein expression in paraffin-sections of different types of ovarian tumor tissue
Immunohistochemical analysis of YKL-40 protein expression in paraffin-sections of different types of ovarian
tumor tissue (×40). A positive immunostaining appears as a cytoplasmic, granular brown-colored staining. a: Normal ovarium
surface epithelium, no staining; b: borderline serous tumor, no staining; c: borderline mucinous tumor, no staining, d: border-
line mucinous tumor, 30% positivity; e: well-differentiated serous adenocarcinoma, 50% positivity; f: well-differentiated muci-
nous adenocarcinom, no staining; g: moderately differentiated serous adenocarcinoma, 30% positivity; h: moderately
differentiated mucinous adenocarcinoma, 40% positivity; i: moderately differentiated clear cell carcinoma, 40% positivity; k:
moderately differentiated endometroid adenocarcinoma, 70% positivity; l: moderately differentiated serous adenocarcinoma,
70% positivity; and m: moderately differentiated endometroid adenocarcinoma, 80% positivity.

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Table 1: Clinical characteristics and YKL-40 expression in tumor tissue and plasma YKL-40 levels from patients diagnosed with
borderline ovarian tumor#

Characteristic YKL-40 protein expression (N = 181) Plasma YKL-40 (μg/l) (N = 19)

N (%) Median (range) N (%) Median (range)

FIGO stage:
I 160 (88) 5 (0–90) 19 (100) 42 (20–180)
II 5 (3) 5 (0–30)
III 16 (9) 7.5 (0–30)

Histological type of tumor:

Cystadenoma (NOS), borderline 14 (8) 7.5 (5–20) 2 (10) 39 (38–40)
Serous cystadenoma, borderline 95 (52) 5 (0–80) 6 (32) 42 (31–142)
Mucinous cystadenoma, borderline 69 (38) 5 (0–90) 11 (58) 37 (20–180)
Endometroid cystadenoma, borderline 3 (2) 5 (5–40)

# Scoring of YKL-40 expression was based on the proportion of cells in a given tumor specimen exhibiting distinct cytoplasmic immunopositivity as
well as intensity of staining percentage scale: 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100. The YKL-40 scoring results was transformed into a
four-tiered scale: 1 = negative; 2 = 5% positive tumor cells; 3 = 10% positive tumor cells and 4 = 20% or more positive tumor cells.

protein expression and radicality of surgery was observed Preoperative plasma YKL-40 in patients with borderline
(p = 0.05). ovarian tumors and in OC
Plasma samples for YKL-40 analysis were available from
The highest frequency of any level of positivity of YKL-40 19 patients with borderline ovarian tumors (median age
protein expression was observed in serous adenocarcino- 45 years, range 38 – 76 years) and from 76 patients with
mas, where 123 out of 249 tissues were scored positive OC (median age 63 years, range 37 – 79 years). The
(49%). median preoperative plasma level of YKL-40 in the OC
patients was 125 μg/l (range 20 – 2650 μg/l) and signifi-
cantly higher than the level in 144 healthy women

Table 2: Clinical characteristics and YKL-40 expression in tumor tissue and plasma YKL-40 levels from patients diagnosed with ovarian
cancer#

Characteristic YKL-40 protein expression (N = 473) Plasma YKL-40 (μg/l) (N = 76)

N (%) Median (range) N (%) Median (range)

FIGO stage:
I 157 (33) 10 (0–90) 17 (22) 59 (20–340)
II 48 (10) 5 (0–30) 4 (5) 53 (39–259)
III 245 (52) 10 (0–70) 47 (62) 168 (32–1808)
IV 23 (5) 20 (5–40) 8 (11) 320 (86–2650)

Histological type of tumor:

Undifferentiated carcinoma 9 (2) 20 (0–40) 3 (4) 576 (156–1808)
Adenocarcinoma (NOS) 23 (5) 10 (0–30) 5 (6) 452 (237–2650)
Serous adenocarcinoma 249 (53) 5 (0–70) 50 (66) 112 (20–548)
Mucinous adenocarcinoma 53 (11) 10 (0–80) 7 (9) 102 (20–856)
Endometroid adenocarcinoma 69 (15) 10 (0–90) 8 (11) 93 (29–193)
Clear-cell neoplasm 46 (10) 10 (0–40) 2 (3) 220 (59–380)
No information - - 1 (1) 340

Histological grade of tumor:

Grade 1 124 (26) 10 (0–60) 17 (22) 61 (20–356)
Grade 2 165 (35) 10 (0–80) 27 (36) 162 (32–856)
Grade 3 183 (39) 10 (0–90) 26 (34) 144 (20–2650)
No information - - 6 (8) 360 (29–1808)

# Scoring for YKL-40 expression was based on the proportion of cells in a given tumor specimen exhibiting distinct cytoplasmic immunopositivity
as well as intensity of staining percentage scale: 0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100. The YKL-40 scoring results was transformed into a
four-tiered scale: 1 = negative; 2 = 5% positive tumor cells; 3 = 10% positive tumor cells and 4 = 20% or more positive tumor cells.

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(median 32 μg/l, range 20 – 143 μg/l; p < 0.001). 67% sion (negative or ≥ 5% positive tumor cells) in a
(51/76) of the OC patients had a plasma YKL-40 level multivariate Cox regression analysis with FIGO stage,
above the age-adjusted 95th percentile of the healthy patient age, radicality of surgery and serum CA-125 as
women. Patients with borderline ovarian tumors did not other parameters, showed no prognostic impact of YKL-
have elevated plasma YKL-40 (median 42 μg/l, range 20 – 40 tissue expression (HR = 0.97; 95% CI: 0.99 – 1.01)
180 μg/l) compared to healthy women. 21% (4/19) of the (data not shown).
patients with borderline tumors had a plasma YKL-40
level above the age-adjusted 95th percentile of the healthy Plasma YKL-40 and prognosis
women. Table 1 and Table 2 gives the relationship During follow-up 55 (72%) of the 76 OC patients died.
between preoperative plasma YKL-40 and standard clini- Univariate Cox analysis of plasma YKL-40 (log trans-
cal-pathological variables. Plasma YKL-40 increased with formed and treated as a continuous covariate) showed sig-
increasing FIGO stage among OC patients (p = 0.0001). nificant association with overall survival (HR = 2.85, 95%
Furthermore, plasma YKL-40 was correlated with serum CI: 2.04 – 3.97, p < 0.0001). Univariate Cox analysis of
CA-125 (Spearmans rho = 0.47, p < 0.0001) and age (rho plasma YKL-40 (dichotomized as normal vs. high age-cor-
= 0.59, p < 0.0001). No correlation was found between rected plasma YKL-40 level) showed also significant asso-
plasma YKL-40 and the YKL-40 tissue expression percent- ciation with overall survival (HR = 3.51, 95% CI: 1.82 –
age score (rho = 0.032, p = 0.081). 6.75, p < 0.0001). Figure 3 illustrates the Kaplan-Meier
estimates of survival stratified by baseline plasma YKL-40
YKL-40 expression in OC and prognosis dichotomized as normal vs. high age-corrected plasma
Univariate survival analysis demonstrated that YKL-40 tis- YKL-40 level.
sue expression (negative or ≥ 5% positive tumor cells) was
not associated with survival (HR = 1.2; 95% CI: 0.95 – Performing a multivariate Cox analysis including plasma
1.53) (Figure 2). Similarly, using the four-tiered scale YKL- YKL-40 (log transformed and treated as a continuous cov-
40 tissue expression was confirmed of no evidence of sep- ariate), serum CA-125 (log transformed and treated as a
aration (data not shown). Including YKL-40 tissue expres- continuous covariate), FIGO stage (I-IV), residual tumor

Figure 2
Kaplan-Meier survival curves showing the association between YKL-40 protein expression and overall survival
Kaplan-Meier survival curves showing the association between YKL-40 protein expression and overall survival.
YKL-40 is dichotomized in cellular percentage score in two groups (< 5% vs. ≥ 5% positivity og the cancer cells).

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Figure 3
Kaplan-Meier survival curves showing the association between preoperative plasma YKL-40 and overall survival
Kaplan-Meier survival curves showing the association between preoperative plasma YKL-40 and overall sur-
vival. Plasma YKL-40 is dichotomized in normal vs. elevated plasma YKL-40 levels. The cut-off point is the age-adjusted plasma
YKL-40 value corresponding to the 95th percentile in healthy controls. The P-value refers to the log-rank test for equality of
strata.

after surgery (yes or no), and age showed that only plasma arrays of breast cancer [18]. Our present results of OC are
YKL-40 (HR = 2.13, 95% CI: 1.40 – 3.25, p = 0.0004) was in accordance with this study of breast cancer, where no
found of independent prognostic value of overall survival. association was found between YKL-40 score and progno-
Age at surgery (HR = 1.02, 95% CI: 0.99 – 1.06), radicality sis [18]. However, this is in contrast to tumor samples
after primary surgery (HR = 1.20, 95% CI: 0.44 – 3.33), from glioblastomas, where YKL-40 acted as a biomarker of
serum CA-125 (HR = 1.08, 95% CI: 0.94 – 1.23) and genetic and histological subtypes, radiation therapeutic
FIGO stage (p = 0.63, FIGO 1 vs 4: HR = 0.61, 95% CI: response and prognosis [11,12,14,29,30]. Pair-wise com-
0.11 – 3.34, FIGO 2 vs 4: HR = 0.25, 95% CI: 0.03–2.37, binations of markers have identified epidermal growth
FIGO 3 vs 4: HR = 0.66, 95% CI: 0.28–1.58) were found factor receptor variant III (EGFRvIII) and YKL-40 as prog-
of no independent prognostic value in OC patients. nostically important in patients with glioblastoma, pro-
viding patients with EGFRvIII-negative/YKL-40-negative
Discussion tumours with the best prognosis [29].
In the present study we found YKL-40 protein expression
in OC cells with a higher expression score in OC than in There are no published studies on YKL-40 protein expres-
borderline tumors, and a clear association with FIGO sion in OC tissue, and to our knowledge no publications
stage and type of histology, but not with survival. We used exist on a close association between serum YKL-40, serum
tissue micro array with a tissue core diameter of 2.0 mm, CA-125 and CA15-3 in patients with OC [23-26]. It has
but although this is comparatively large we do not know previously been shown that preoperative serum or plasma
whether the degree of heterogeneity of YKL-40 protein concentrations of YKL-40 are elevated in 65% of patients
expression in OC tissue could have influenced our results. with FIGO stage I and II, and in 74% to 91% of patients
This was, however, not the case in an immunohistochem- with FIGO stage III and IV [23,24]. In addition, patients
ical analysis of YKL-40 protein expression in tissue micro with high serum or plasma levels of YKL-40 with early-

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stage OC [24], stage III [23], or recurrent of OC [25] had LHC contributed to the conception of immunohisto-
shorter survival than OC patients with normal levels of chemical method and interpretation of the results. IJC
YKL-40. contributed to the understanding of statistical analysis
and interpretation of results. PAP contributed to the con-
In the present study plasma was available from a small ception of method and the YKL-40 AAb.
subgroup of patients with borderline ovarian tumors and
OCs, and in accordance with the studies mentioned above All authors read and approved the final paper.
[23-26] we also found that elevated preoperative plasma
concentrations of YKL-40 significantly predicted short sur- Acknowledgements
vival in patients with OC. However, we did not find any We thank all nurses and doctors on the gynecological and pathological
correlation between plasma and tissue score YKL-40 in departments for their tremendous work. The authors are grateful to Heidi
corresponding tissue and YKL-40 level in plasma samples Marie Paulsen (Rigshopitalet), Vibeke Reese (Danish State Serum Institute),
Tonni Løve Hansen, and Debbie Nadelmann (Herlev Hospital) for technical
in this subgroup of OC patients. Two persons simultane-
assistance. This work was supported by grants from Mermaid1, The Danish
ously assessed the patterns of YKL-40 protein expression Cancer Society and National Cancer Institute, Bethesda, USA (RO1 CA
of each tumor sample and discussed each sample if result 61107), "Direktør Jens Aage Sørensen og Hustru Edith Ingeborg Sørensens
were not concordant. It is often the case that there is no Mindefond" and "Vera og Carl Johan Michaelsens legat". Quidel provided
correlation between protein expression in cancer tissue the study with YKL-40 ELISA kits.
and circulating levels of the protein. The reason for this
observation is possibly due to a considerable contribution References
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