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Elevated Growth Differentiation Factor 15 Expression Predict Poor Prognosis in

Epithelial Ovarian Cancer Patients

Ying Zhang1,2*, Wei Hua3*, Li-chun Niu2, Shi-mei Li2, Ying-mei Wang3, Lei Shang4,

Cun Zhang1, Wei-na Li1, Rui Wang4, Bi-liang Chen5, Xiao-yan Xin5, Ying-qi Zhang1#,

Jian Wang5#

1 The State Key Laboratory of Cancer Biology, Biotechnology Center, School of

Pharmacy, the Fourth Military Medical University, Xi’an, Shaanxi 710032, China;2 Department of Gynecology and Obstetrics, the People's Liberation Army 323 Hospital,

Xi’an, Shaanxi 710045, China;

3 State Key Laboratory of Tumor Biology, Department of Pathology, Xijing Hospital,

The Fourth Military Medical University, Xi’an, Shaanxi 710033, China;4 Department of Health Service, School of Public Health, Fourth Military Medical

University, Xi’an, Shaanxi 710033, China;5 Department of Gynecology and Obstetrics, Xijing Hospital, Fourth Military Medical

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University, Xi’an, Shaanxi 710033, China;* Ying Zhang and Wei Hua contributed equally to this work.#Correspondence to: Dr. Jian Wang, Department of Gynecology and Obstetrics, Xijing

Hospital, Fourth Military Medical University, 169 Changle West Road, 710033 Xi’an,

Shaanxi, China;

Phone: +86-29-84775387, Email address: anran206@yeah.net

#Correspondence to: Ph.D. Ying-qi Zhang, Ph.D, The State Key Laboratory of Cancer

Biology, Biotechnology Center, School of Pharmacy, the Fourth Military Medical

University, 17 Changle West Road, 710032 Xi’an, Shaanxi, China

Phone: +86-29-84774773, Email: zhangyingqi710032@163.com

Running title: Elevated GDF15 Expression Predict Poor Prognosis in EOCs

Word count: Abstract: 172; Text: 4040.

ABSTRACT

Objective: The purpose of this study was to determine the expression of growth

differentiation factor 15(GDF15), and explore its clinical significance in epithelial

ovarian cancer (EOC) patients. Methods: The expression of GDF15 in EOC tissues and

serum samples was evaluated using immunohistochemistry and enzyme-linked

immunosorbent assay (ELISA) respectively. The association of GDF15 expression with

clinicopathologic parameters was analyzed. Survival time was assessed using the

Kaplan–Meier technique and Cox regression model. Results: Both in EOC tissues and

serum, high GDF15 levels were obviously related with advanced FIGO stage, lymph

node metastasis, ascites, and chemoresistance. Kaplan–Meier analysis indicated that

EOC patients with high GDF15 expression showed poorer progression-free survival

(PFS) and overall survival (OS). Multivariate analysis demonstrated that GDF15

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expression was an independent predictor of PFS in EOC patients. Conclusion: Our

study shows that elevated GDF15 expression was associated with poor prognosis in

EOC patients. We suggest that GDF15 is a novel biomarker for the early detection of

EOC, prediction of the response to chemotherapy, and screening for recurrence in EOC

patients.

Keywords: growth differentiation factor 15, epithelial ovarian cancer, biomarker,

chemotherapy, prognosis

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INTRODUCTION

Epithelial ovarian cancer (EOC) accounts for more than 80% of all malignant

cancers of the female reproductive system, and is the leading cause of death from

gynecological malignancies[1]. Owing to the lack of effective screening methods and

specific symptoms early in the disease, over 70% of patients are diagnosed at an

advanced stage, and thus, the prognosis of EOC patients remains poor, with a 5-year

overall survival (OS) rate of less than 25%[2, 3]. Serum cancer antigen 125 (CA125)

levels are widely used to distinguish malignant from benign pelvic masses, monitor the

response to cytoreduction and chemotherapy, and screen for disease recurrence in EOC

patients[4-7]. However, the sensitivity and specificity of this serum test are low[4, 5, 8].

Therefore, novel, clinically effective biomarkers that can sufficiently predict the

prognosis of EOC patients and identify platinum-resistant EOCs hold great promise to

improve the therapeutic effects in patients with ovarian cancer.

Growth differentiation factor 15 (GDF15) is a secreted protein of the transforming

growth factor-β (TGF-β) superfamily. GDF15 plays multiple roles in various

pathologies, including inflammation, cancer, cardiovascular diseases, and obesity[9-11].

GDF15 is weakly expressed in most tissues, but is highly overexpressed under

pathologic conditions such as injury, inflammation, and various cancers[10, 11]. Although

GDF15 has been reported to have both tumorigenic and anti-tumorigenic activities,

considerable evidence indicates that GDF15 plays an important role in carcinogenesis-

related activities, such as proliferation, migration, apoptosis, and angiogenesis[12-15].

Serum GDF15 levels are markedly increased in patients with pancreatic, prostate, and

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colorectal cancers[12, 13, 16, 17]. All the above evidence indicates that GDF15 may be a

useful biomarker for solid tumor detection. However, the role of GDF15 in the

development and progression of EOC is largely unknown. To determine whether

GDF15 can serve as a powerful diagnostic and prognostic factor in EOC, we evaluated

the expression of GDF15 in EOC tissues and analyzed its association with

clinicopathologic data. In addition, enzyme-linked immunosorbent assay (ELISA) was

used to determine serum GDF15 levels in EOC patients and healthy controls.

MATERIALS AND METHODS

Tumor tissues

Between January 2010 and December 2013, we enrolled 145 patients who had been

diagnosed with primary EOC at the Department of Obstetrics and Gynecology, Xijing

Hospital, Xi’an, China, and had available untreated tissue specimens. The eligibility

criteria for this study included the following: (1) histologically proven EOC; (2)

availability of clinical data and resected tissue; (3) postoperative treatment with

standard platinum-based adjuvant chemotherapy (cisplatin/paclitaxel or

cisplatin/cyclophosphamide/doxorubicin); and (4) follow-up from the time of surgical

intervention to 2014. The exclusion criteria for this study were as follows: (1)

histologic types other than EOC; (2) preoperative radiation or chemotherapy; and (3)

patients whose cause of death was unknown. The study was approved by the ethics

committee of Xijing Hospital, and the patients provided informed consent before their

inclusion into the study. Informed consent for the use of the tumor specimens was

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obtained either from the patients or from their next of kin. All patients underwent

follow-up gynecological examinations, transvaginal/abdominopelvic ultrasonography,

radiological investigations, and serum CA125 measurements. All patients were followed

up from the time of surgical intervention to 2014.

Tissue specimens were harvested intraoperatively, formalin-fixed, and paraffin-

embedded. Each EOC sample was cut into 4-μm sections. One section was stained with

hematoxylin–eosin (H&E) and used for morphological diagnosis, while the others were

used for immunohistochemical analysis. The EOC samples were histologically

classified according to the International Federation of Gynecology and Obstetrics

(FIGO) criteria[18]. The histologic type and grade were classified according to the World

Health Organization criteria[19]. All histologic specimens were analyzed by a single

operator.

Blood samples

Blood samples were obtained before surgery and any medical treatment from 120

EOC patients and 40 healthy controls (age, 46.3 ± 12.4 years) with no history of ovarian

pathology or other systemic disease. Blood samples from the patients and controls were

drawn into serum tubes and centrifuged at 1000 g for 10 min. All serum samples were

stored at -80°C until use.

Immunohistochemistry

A standard streptavidin–biotin complex method was used. Negative control slides

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for GDF15 antibody were prepared using non-specific mouse IgG, and breast cancer

specimens were used as positive controls. Tissue specimens were de-waxed with xylene

and gradually hydrated. After being blocked with endogenous peroxidase and 3% H2O2–

methanol for 10 min, the slides were incubated overnight at 4°C with the primary mouse

monoclonal anti-GDF15 antibodies (Novus Biologicals, Littleton, CO) at a dilution of

1:800. After being washed three times for 5 min each with phosphate-buffered saline,

the sections were incubated with a secondary antibody (Pierce, Rockford, IL) for 30 min

followed by incubation with the avidin–biotin complex for a further 30 min. 3-3ʹ-

Diaminobenzidine tetrahydochloride was used as a chromogen. All sections were

counterstained with Gill’s hematoxylin. A pathologist then reviewed the

immunohistochemical preparations in parallel with their corresponding H&E-stained

slides to confirm the diagnosis.

Evaluation of immune staining

The tumor cores were evaluated by specialist pathologists and oncologists blinded

to the clinicopathologic characteristics of the patients. The scale of staining was

semiquantitatively evaluated according to the percentage of stained cells and the

staining intensity as previously described[20]. A brown precipitate observed on tissue

sections indicated positive immunoreactivity with the primary GDF15 antibody. Whole-

field inspection of the core was included in the assessment, and the immune staining

was scored as follows: (1) the proportion of malignant cells positively stained with the

anti-GDF15 antibody was scored as 0 (0%–4%), 1 (5%–24%), 2 (25%–49%), 3 (50%–

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74%), or 4 (75%–100%); (2) the intensity of immunostaining was graded as 0

(negative), 1+ (weak), 2+ (moderate), or 3+ (strong); and (3) the two scores were

multiplied to obtain the final score. Final scores of 0–4 indicated low expression, scores

of 5–8 indicated moderate expression, and scores of 9–12 indicated high expression.

ELISA

ELISA was used to measure serum GDF15 levels, according to the manufacturer’s

instructions (GDF15 ELISA Kit; USCNLIFE, Wukan, China). Serum CA125 levels

were determined at the Xijing hospital laboratory by using an immunoenzymometric

assay and an immunoelectrochemiluminescence detection technique with a CA125 II

ECLIA (electrochemiluminescence immunoassay) kit and Roche/Hitachi Modular

Analytics E170 (Roche Diagnostics GmbH, Mannheim, Germany).

Statistical analysis

Statistical analysis was performed using SPSS 17.0 for Windows (SPSS Inc.,

Chicago, IL). Associations between GDF15 expression in EOC tissues and

clinicopathologic variables were assessed using the chi-square test. Serum GDF15

levels were expressed as mean ± SD. The distributions of GDF15 values in serum were

asymmetric; therefore, nonparametric analyses (Mann–Whitney U test and Kruskal–

Wallis test) were used to compare median values between groups. The Fisher exact test

was used to compare the clinicopathologic characteristics according to the serum

GDF15 level. In addition, a receiver operating characteristic (ROC) curve was

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employed to obtain the area under the curve (AUC), sensitivity, and specificity. The

survival probabilities of patients according to the GDF15 expression level were

described using Kaplan–Meier curves and compared using the log-rank test. Factors that

showed significant prognostic value on univariate regression analysis were evaluated

with multivariate Cox regression analysis. A value of p < 0.05 was considered

statistically significant.

RESULTS

Characteristics of EOC patients

A total 145 patients diagnosed with primary EOC between January 2010 and

December 2013 were studied. The characteristics of all the patients are summarized in

Table 1. The age range of the patients was from 35 to 83 years (mean: 51.92 ± 15.95

years).

Table 1. Characteristics of patients with ovarian cancer

Characteristic Number of patients (%)

Age (years)

<60 119 (82.1)

≥60 26 (17.9)

Pathologic type

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Serous cystadenocarcinoma 111 (76.6)

Mucinous cystadenocarcinoma 14 (9.7)

Endometrioid adenocarcinoma 9 (6.2)

Undifferentiated carcinoma 11 (7.6)

Histologic differentiation

Well 20 (13.8)

Moderate 11 (7.6)

Poor 114 (78.6)

FIGO stage

I + II 41 (28.3)

III + IV 104 (71.7)

Ascites

Negative 23 (15.9)

Positive 122 (84.1)

Lymph node metastasis

Negative 72 (49.7)

Positive 73 (50.3)

Response to first-line chemotherapy

Sensitive 111 (76.6)

Resistant 34 (23.4)

Distant metastasis

Negative 117 (80.7)

Positive 28 (19.3)

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Recurrence

Negative 66 (45.5)

Positive 79 (54.5)

CA125 expression (U/ml)

<500 52 (35.9)

501–1000 43 (29.7)

≥1000 50 (34.5)

Increased GDF15 expression in EOC tissues

EOC tumor cells displayed cytoplasmic GDF15 staining (Fig. 1). The positive

expression rate of GDF15 was 82.39% in EOC tissues. Weak or negative staining was

observed in 93 samples (64.1%), moderate staining was observed in 24 samples

(16.6%), and high GDF15 protein expression was detected in 28 tumor samples

(19.3%). In contrast, GDF15 expression was never detected in normal ovarian tissues.

As shown in Table 2, the expression of GDF15 in EOC tissues was significantly higher

in patients with advanced FIGO stages (III + IV) than in patients with early-stage

tumors (I + II; p = 0.026). Further analysis showed that the expression of GDF15 in

EOC tissues was related with ascites (p = 0.017) and lymph node metastasis (p = 0.003).

EOC patients who were resistant to first-line chemotherapy more frequently showed

higher GDF15 expression than those who were sensitive to first-line chemotherapy (p =

0.030). However, GDF15 expression in EOC tissues did not differ with pathologic type

and histologic differentiation (p > 0.05).

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Table 2. GDF15 expression and clinicopathologic parameters

CharacteristicGDF15 expression

χ2 p valueLow Moderate High

Age (years)

7.012 0.031<60 82 18 19

≥60 11 6 9

Pathologic type

4.062 0.694

Serous 70 19 22

Mucinous 11 1 2

Endometrioid 4 3 2

Others 8 1 2

Histologic differentiation

Well 15 3 2

2.460 0.673Moderate 6 3 2

Poor 72 18 24

FIGO stage

I + II 33 5 37.296 0.026

III + IV 60 19 25

Ascites

Negative 19 4 08.165 0.017

Positive 74 20 28

Lymph node metastasis

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Negative 51 15 611.686 0.003

Positive 42 9 22

Response to first-line chemotherapy

Sensitive 72 22 176.611 0.030

Resistant 21 2 11

Figure 1. Immunohistochemical micrographs of GDF15 protein in different ovarian

tissues (400×).

(A) Negative, (B) low, (C) moderate, and (D) high GDF15 expression in EOC tissues.

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We next examined the relationship between GDF15 expression and prognostic

outcomes in EOC patients. All 145 EOC patients with optimally debulked tumors and

available outcome data were included in the survival analysis. The median follow-up

duration was 30.24 months (range, 40 to 79.3 months). The detailed clinical information

of the 145 EOC patients divided according to GDF15 expression level (low, moderate,

or high) was reviewed to determine the prognostic implications of GDF15 expression.

Analysis using the Kaplan–Meier method showed that patients with high GDF15

expression had significantly shorter OS than those with low or moderate GDF15

expression (19.13 months versus 58.62 months and 47.11 months, p = 0.000; Fig. 2).

Similarly, the median postoperative progression-free survival (PFS) time was lower in

patients with high GDF15 expression (11.13 months) than in those with low or

moderate GDF15 expression (33.62 months and 48.43 months, respectively, p = 0.000).

The Kaplan–Meier curves indicated that high GDF15 expression was significantly

associated with an increased risk of death.

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Figure 2. Kaplan–Meier curves of survival durations in EOC patients grouped

according to GDF15 expression. Both progression-free survival (PFS) and overall

survival (OS) were significantly shorter in patients with high GDF15 expression than in

those with low or moderate GDF15 expression.

To identify factors that affected OS, the five clinical factors listed in Table 3 and the

GDF15 levels were included in a multivariate Cox regression analysis. The analysis

revealed that tumor stage (p = 0.023), response to first-line chemotherapy (p = 0.004),

and GDF15 level (p = 0.014) were significantly associated with survival among the

EOC patients.

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Table 3. Multivariate analyses of factors associated with overall survival

Risk factor

Overall survival Progression-free survival

HR 95% CI p value HR 95% CI p value

Age (years)

(<60 vs. ≥60)1.54 0.51–2.82 0.495 1.52 0.75–2.78 0.323

Grade

(Poor vs. others)1.39 0.66–2.82 0.243 1.14 0.63–2.04 0.449

FIGO stage

(I + II vs. III + IV)0.41 0.16–0.43 0.023 0.41 0.32–0.87 0.019

Lymph node metastasis

(Positive vs. negative)2.27 1.45–10.51 0.016 2.25 1.07–2.94 0.040

Response to first-line chemotherapy

(Sensitive vs. resistant) 4.72 2.79–9.87 0.004 3.64 2.62–7.51 0.017

GDF15 level

(High vs. low)6.60 1.656–26.28 0.014 6.19 1.468–23.87 0.039

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Multivariate analysis and Cox proportional hazards regression model were used. Variables were adopted

because of their prognostic significance demonstrated on univariate analysis (p < 0.05).

HR, hazards ratio; CI, confidence interval; FIGO, International Federation of Gynecology and Obstetrics.

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Serum GDF15 level and its correlation with clinicopathologic characteristics

A total 120 blood samples taken from EOC patients before they underwent surgery

were compared with samples taken from 40 healthy controls. The mean serum GDF15

concentration was significantly higher in the patient group than in the healthy controls

(1302.12 ± 415.03 pg/ml vs. 418.17 ± 301.66 pg/ml, p = 0.047; Fig. 3).

Figure 3. Box plot of serum GDF15 levels in EOC patients and healthy controls. The

serum concentrations of GDF15 in the EOC patients ranged from 550.17 pg/ml to

2402.26 pg/ml (median, 1302.12 pg/ml), and were significantly higher than the GDF15

levels in the control cohort of 40 healthy female volunteers (72.6–1410.78 pg/ml;

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median, 618.17 pg/ml; p = 0.047, t-test).

As shown in Fig. 4, the area under the ROC (AUROC) was calculated based on the

serum GDF15 levels in 120 EOC patients and 40 healthy controls. The ROC analyses

revealed that the estimated AUROC of serum GDF15 was 0.894, which was not

superior to that of serum CA125 (0.924). The cutoff serum GDF15 level as determined

using the Youden index was 748 pg/ml. At this cutoff, GDF15 showed a similar

specificity (83.3%) to that of CA125 (88.1%), and a higher sensitivity (75.5%) than that

of CA125 (68.2%). The combined AUC value of GDF15 and CA125 was 0.944,

suggesting that the combination had a better performance than that of CA125 alone in

the detection of EOC.

Figure 4. ROC curves of serum CA125, GDF15 (A), and the combination of CA125

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with GDF15 (B) among EOC patients and healthy controls. (A) The estimated area

under the ROC curve of serum GDF15 was 0.894 (95% confidence interval [CI]: 0.791–

0.957, p < 0.001). (B) The AUC value of the combination of GDF15 with CA125 was

0.944 (95% CI: 0.856– 0.986, p < 0.001).

To determine the impact of elevated serum GDF15 levels in EOC patients, we

analyzed the relationship between serum GDF15 levels and clinicopathologic

characteristics (Table 4). Serum GDF15 levels were not correlated with pathologic type

and differentiation (p > 0.05), but were significantly correlated with FIGO stage.

Median serum GDF15 levels were obviously higher in patients with advanced stages of

EOC (III + IV) than in patients with early-stage EOC (I + II; 563.49 pg/ml vs. 987.23

pg/ml, p = 0.004). Moreover, the median serum GDF15 levels were higher in patients

with ascites than in patients without ascites (586.3 pg/ml vs. 898.9 pg/ml, p = 0.026).

Similarly, GDF15 levels were higher in patients with lymph node metastasis than in

those without lymph node metastasis (663.8 pg/ml vs. 1044.8 pg/ml, p = 0.039).

Additionally, the serum level of GDF15 was significantly higher in EOC patients who

were resistant to first-line chemotherapy than those who were sensitive to first-line

chemotherapy (692.0 pg/ml vs. 1096.6 pg/ml, p = 0.030).

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Table 4. Serum GDF15 levels by clinicopathologic characteristics of the patients

Characteristic GDF15 (pg/ml)

Median (range)

p value χ2 value CA125 (U/ml)

Median (range)

p value χ2 value

Pathologic type

Serous 748.8 (37.9–2704.0)0.128 5.682

764.3 (75.0–2897.0)0.120 2.419

Non-serous 868.8 (287.9-1475.2) 481.8(35.6-1329.3)

Differentiation

Well 541.9 (37.9–1266.8)0.221 3.019

566.90 (37.0–1808.0)0.011 6.443

Moderate 782.8 (308.7–1214.0) 1038.27 (22.6–2492.8)

Poor 1006.8 (256.0–2704.0) 1285.79 (97.5–2897.0)

FIGO stage

I + II 563.49 (37.9–1266.8)0.004 13.158

488.92 (22.6–2492.8)0.039 4.263

III + IV 987.23 (289.9–2704.0) 1318.84 (575.0–2897.0)

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Ascites

Negative 586.3 (37.9–1652.2)0.026 4.937

700.9 (75.0–2518.0)0.022 9.609

Positive 998.9 (69.2–2704.0) 1134.0 (118.1–2897.0)

Lymph node metastasis

Negative 663.8 (37.9–1402.2)0.039 4.263

818.6 (75.0–2518.0)0.120 2.419

Positive 1044.8 (69.2–2704.0) 1317.9 (127.7–2897.0)

Response to first-line chemotherapy

Sensitive 692.0 (37.9–1173.2)0.037 2.245

795.1 (75.0–2225.0)0.009 2.876

Resistant 1096.6 (256.7–2704.0) 1546.9 (171.0–2897.0)

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DISCUSSION

EOC is the leading cause of death among gynecologic malignancies. In spite of

recent advances, over three-quarters of EOC patients are diagnosed at an advanced stage

because of the asymptomatic nature of the early stage of the disease and the rapid

progression of chemoresistant disease. It is worth noting that ovarian cancer represents a

very diverse group of tumors. The epithelial category, which accounts for 90% of all

ovarian cancers, is classified into the following subtypes: (1) serous (50%); (2)

endometrioid (10%–25%); (3) mucinous (5%–10%); (4) clear cell (4%–5%); (5)

undifferentiated (5%); and (6) transitional cell cancer (rare)[21]. Over the last three

decades, CA125 has been used for distinguishing malignant from benign pelvic masses,

detecting recurrent disease, monitoring response to treatment, and for early detection[4, 6,

8]. However, serum CA125 is not an ideal biomarker for EOC screening because of its

low sensitivity and specificity. Høgdall et al.[22] found that the test for CA125 is positive

in 85%–90% of serous tumors, 40%–65% of clear cell and endometrioid tumors, and

only 6%–12% of mucinous tumors. Furthermore, serum CA125 levels may be in the

normal range in 50% of symptomatic stage I patients and in about 10%–20% of

advanced-stage patients[23-26]. The identification of valuable diagnostic and prognostic

biomarkers to improve the outcomes of EOC patients remains a challenge.

Under normal physiological conditions, GDF15, a member of the TGF-β

superfamily, is largely expressed in the placenta, and is expressed at low levels in the

liver, lungs, kidneys, and neuroepithelium[11, 27]. GDF15 has been found to play a role in

cell cycle regulation and cell proliferation, differentiation, and apoptosis[10, 11, 27]. Studies

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have demonstrated that GDF15 is markedly increased in many cancer lines and tissues,

including breast cancer, gastric adenomas, oral squamous cell carcinoma (OSCC),

glioblastoma, prostate cancer, and colon cancer[28-35]. In OSCC, low GDF15 expression

predicts better survival, especially overall and distant metastasis–free survival[l,[33].

Wallin et al.[34] reported that colorectal cancer patients with moderate-to-high levels of

GDF15 had higher recurrence rates than did patients with no or low GDF15 expression.

In addition, colorectal cancer patients with high plasma GDF15 levels had statistically

shorter time to recurrence (p = 0.041) and reduced overall survival (p = 0.002)[34].

The clinical utility of GDF15 as a biomarker for EOC has not fully been explored.

Staff et al. reported that high GDF15 concentration was detected on ELISA in both

plasma and ascitic fluid samples obtained from patients with advanced ovarian cancer[36,

37]. As the plasma GDF15 concentration correlated inversely with survival time, GDF15

was proposed to be a potentially useful prognostic biomarker in ovarian cancer[36].

However, the study by Staff et al.[36] was restricted to serous ovarian cancer and limited

to patients with advanced tumors. Considering the complexity of the histologic subtypes

of EOCs and the diverse tumor properties, the value of GDF15 as a tumor biomarker for

early detection, surveillance treatment response, and prognosis prediction warranted

further investigation.

Therefore, in our study, we analyzed the expression of GDF15 in EOC tissues and

serum samples, and found that the serum levels of GDF15 were elevated, which was

consistent with the results observed in the EOC tissue samples. Importantly, we also

assessed the value of GDF15 as a diagnostic indicator in different stages of EOC, and

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investigated the potential of serum GDF15 for predicting tumor progression and

chemoresistance.

First, we found that cytoplasmic GDF15 expression was observed in 79.3% of EOC

tumor tissues on immunohistochemical analysis. We further analyzed the correlation

between GDF15 expression and clinicopathologic characteristics in EOC patients. The

data showed that increased GDF15 expression was associated with advanced FIGO

stage, lymph node metastasis, ascites, and chemoresistance. However, GDF15

expression in the tumor tissues was not associated with histologic grade or type.

Survival analysis revealed that EOC patients with high GDF15 expression exhibited

significantly poorer PFS and OS than did EOC patients with low GDF15 expression.

Multivariate analysis demonstrated that GDF15 expression was an independent

predictor of PFS in EOC patients. Consistent with data from previous studies[36], our

clinical data suggest that GDF15 expression may be an independent prognostic

predictor in EOC patients.

GDF15 concentration in effusion fluid has been correlated positively with the

GDF15 expression in EOC cells[36]. As a secreted protein, high plasma GDF15

concentrations most likely reflect the tumor burden of EOC. GDF15 has been suggested

to be a serological marker for the early diagnosis of and progression of EOC. In our

study, serum GDF15 levels were significantly higher in EOC patients than in healthy

controls. Furthermore, serum GDF15 levels in EOC patients were significantly

correlated with FIGO stage, ascites, and lymph node metastasis. Our results are in

agreement with the findings of Staff et al.[36], who found that GDF15 levels significantly

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differed with FIGO stage and survival duration.

Therefore, to further evaluate the clinical value of GDF15, we compared the

diagnostic usefulness of GDF15 and CA125, which is often used as an EOC marker in

clinical practice. ROC analyses revealed that GDF15 had higher sensitivity than CA125

(75.5% vs. 68.2%) for the detection of EOC, although the specificity of both markers

was similar (83.3% vs. 88.1%). Notably, the AUC value of the combination of GDF15

with CA125 was 0.944, compared with 0.924 for CA125 alone, suggesting that the

combination of serum GDF15 and CA125 had a better performance than CA125 alone

in the detection of EOC. These data suggested that GDF15 could serve as a sensitive

marker for the detection of EOC, and the combination of GDF15 with CA125 may yield

a superior diagnostic performance than that of CA125 alone. Our study only compared

serum GDF15 levels between EOC patients and healthy controls; we did not include

patients with benign ovarian tumors and borderline ovarian tumors. Further studies are

needed to conclude whether GDF15 can serve as a more valuable tumor biomarker than

CA125 in the detection of EOC.

Another interesting finding of our study is that GDF15 levels, both in the EOC

tissues and serum samples, were significantly higher in EOC patients who were resistant

to first-line chemotherapy than in those who were sensitive to first-line chemotherapy.

Therefore, we detected the expression of GDF15 protein in two pairs of platinum-

resistant cell lines and their parental platinum-sensitive cell lines by using western blot

analysis (data not shown). We found that the GDF15 levels were obviously higher in the

platinum-resistant cell lines than in the parental platinum-sensitive cell lines, and that

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platinum sensitivity significantly declined after increasing GDF15 expression by

GDF15 adenovirus (unpublished data). We have enough reason to presume that GDF15

plays a potential role in predicting the response to first-line chemotherapy in EOC

patients. In 2015, Meier et al. [38]used whole genome microarrays and linear model

analysis to identify potential resistance-related genes by comparing the expression

profiles of the parental human ovarian cancer model A2780 and its cisplatin-resistant

variant A2780cis, before and after carboplatin treatment in vivo. It was found that

GDF15 levels to be notably increased during carboplatin treatment in the A2780 but not

in A2780cis in vivo. In accordance with microarray and qRT-PCR data, serum GDF15

levels were obviously increased during carboplatin short-treatment in A2780 tumor-

bearing mice compared to vehicle treatment, but only slightly in A2780cis tumor-

bearing mice. Additionally, basal GDF15 plasma levels were higher in A2780cis-

bearing mice than in mice with A2780 tumors. Furthermore, knockdown of GDF15 in

A2780cis in vivo resulted in enhanced subcutaneous tumor growth in mice but

increased sensitivity to carboplatin treatment. In summary, there are enough reasons to

believe that GDF15 levels is correlated with platinum-resistance in ovarian cancer cells,

and GDF15 might serve as a novel treatment target in women with platinumresistant

ovarian cancer.

Conclusions

In summary, our results demonstrated that GDF15 may be involved in the

progression of EOC, and high levels of GDF15, both in the serum and EOC tissue, may

28

be related with advanced FIGO stage, lymph node metastasis, ascites, and

chemoresistance. GDF15 expression was significantly associated with poor survival,

and was an independent predictor of PFS in EOC patients. GDF15 has the potential to

expedite the clinical diagnosis of EOC and aid in predicting patient outcomes and the

response to chemotherapy.

29

REFERENCES

1 R. Siegel, J. Ma, Z. Zou, et al. Cancer statistics, 2014[J]. CA: a cancer journal for clinicians, 2014,64(1):9-29.2 A. Jemal, F. Bray, M. M. Center, et al. Global cancer statistics[J]. CA: a cancer journal for clinicians, 2011,61(2):69-90.3 B. S. Gloss and G. Samimi. Epigenetic biomarkers in epithelial ovarian cancer[J]. Cancer letters, 2014,342(2):257-263.4 M. Felder, A. Kapur, J. Gonzalez-Bosquet, et al. MUC16 (CA125): tumor biomarker to cancer therapy, a work in progress[J]. Molecular cancer, 2014,13:129.5 J. Menczer, E. Ben-Shem, A. Golan, et al. The Significance of Normal Pretreatment Levels of CA125 (<35 U/mL) in Epithelial Ovarian Carcinoma[J]. Rambam Maimonides medical journal, 2015,6(1):e0005.6 J. G. Cohen, M. White, A. Cruz, et al. In 2014, can we do better than CA125 in the early detection of ovarian cancer?[J]. World journal of biological chemistry, 2014,5(3):286-300.7 A. K. Karam and B. Y. Karlan. Ovarian cancer: the duplicity of CA125 measurement[J]. Nature reviews. Clinical oncology, 2010,7(6):335-339.8 M. J. Duffy, J. M. Bonfrer, J. Kulpa, et al. CA125 in ovarian cancer: European Group on Tumor Markers guidelines for clinical use[J]. International journal of gynecological cancer : official journal of the International Gynecological Cancer Society, 2005,15(5):679-691.9 T. Ago and J. Sadoshima. GDF15, a cardioprotective TGF-beta superfamily protein[J]. Circulation research, 2006,98(3):294-297.10 T. E. Eling, S. J. Baek, M. Shim, et al. NSAID activated gene (NAG-1), a modulator of tumorigenesis[J]. Journal of biochemistry and molecular biology, 2006,39(6):649-655.11 S. N. Breit, H. Johnen, A. D. Cook, et al. The TGF-beta superfamily cytokine, MIC-1/GDF15: a pleotrophic cytokine with roles in inflammation, cancer and metabolism[J]. Growth factors, 2011,29(5):187-195.12 D. A. Brown, R. L. Ward, P. Buckhaults, et al. MIC-1 serum level and genotype: associations with progress and prognosis of colorectal carcinoma[J]. Clinical cancer research : an official journal of the American Association for Cancer Research, 2003,9(7):2642-2650.13 G. Yang, Q. Tan, Y. Xie, et al. Variations in NAG-1 expression of human gastric carcinoma and normal gastric tissues[J]. Experimental and therapeutic medicine, 2014,7(1):241-245.14 S. Kaur, S. Chakraborty, M. J. Baine, et al. Potentials of plasma NGAL and MIC-1 as biomarker(s) in the diagnosis of lethal pancreatic cancer[J]. PloS one, 2013,8(2):e55171.15 R. S. Mehta, M. Song, N. Bezawada, et al. A prospective study of macrophage inhibitory cytokine-1 (MIC-1/GDF15) and risk of colorectal cancer[J]. Journal of the National Cancer Institute, 2014,106(4):dju016.

30

16 A. B. Marjono, D. A. Brown, K. E. Horton, et al. Macrophage inhibitory cytokine-1 in gestational tissues and maternal serum in normal and pre-eclamptic pregnancy[J]. Placenta, 2003,24(1):100-106.17 J. Koopmann, P. Buckhaults, D. A. Brown, et al. Serum macrophage inhibitory cytokine 1 as a marker of pancreatic and other periampullary cancers[J]. Clinical cancer research : an official journal of the American Association for Cancer Research, 2004,10(7):2386-2392.18 D. G. Mutch and J. Prat. 2014 FIGO staging for ovarian, fallopian tube and peritoneal cancer[J]. Gynecologic oncology, 2014,133(3):401-404.19 T. Kaku, S. Watanabe and Y. Ohishi. [Pathology of ovarian cancer][J]. Nihon rinsho. Japanese journal of clinical medicine, 2012,70 Suppl 4:512-516.20 R. Simon, M. Mirlacher and G. Sauter. Immunohistochemical analysis of tissue microarrays[J]. Methods in molecular biology, 2010,664:113-126.21 S. Vaughan, J. I. Coward, R. C. Bast, Jr., et al. Rethinking ovarian cancer: recommendations for improving outcomes[J]. Nature reviews. Cancer, 2011,11(10):719-725.22 E. V. Hogdall, L. Christensen, S. K. Kjaer, et al. CA125 expression pattern, prognosis and correlation with serum CA125 in ovarian tumor patients. From The Danish "MALOVA" Ovarian Cancer Study[J]. Gynecologic oncology, 2007,104(3):508-515.23 V. Nossov, M. Amneus, F. Su, et al. The early detection of ovarian cancer: from traditional methods to proteomics. Can we really do better than serum CA-125?[J]. American journal of obstetrics and gynecology, 2008,199(3):215-223.24 J. R. van Nagell, Jr. and E. J. Pavlik. Ovarian cancer screening[J]. Clinical obstetrics and gynecology, 2012,55(1):43-51.25 O. Dorigo and J. S. Berek. Personalizing CA125 levels for ovarian cancer screening[J]. Cancer prevention research, 2011,4(9):1356-1359.26 W. D. Kang, H. S. Choi and S. M. Kim. Value of serum CA125 levels in patients with high-risk, early stage epithelial ovarian cancer[J]. Gynecologic oncology, 2010,116(1):57-60.27 X. Wang, S. J. Baek and T. E. Eling. The diverse roles of nonsteroidal anti-inflammatory drug activated gene (NAG-1/GDF15) in cancer[J]. Biochemical pharmacology, 2013,85(5):597-606.28 J. Xu, T. R. Kimball, J. N. Lorenz, et al. GDF15/MIC-1 functions as a protective and antihypertrophic factor released from the myocardium in association with SMAD protein activation[J]. Circulation research, 2006,98(3):342-350.29 P. Buckhaults, C. Rago, B. St Croix, et al. Secreted and cell surface genes expressed in benign and malignant colorectal tumors[J]. Cancer research, 2001,61(19):6996-7001.30 J. Y. Park, K. H. Park, S. Bang, et al. Expression of nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) inversely correlates with tumor progression in gastric adenomas and carcinomas[J]. Journal of cancer research and clinical oncology, 2008,134(9):1029-1035.31 M. Blanco-Calvo, N. Tarrio, M. Reboredo, et al. Circulating levels of GDF15,

31

MMP7 and miR-200c as a poor prognostic signature in gastric cancer[J]. Future oncology, 2014,10(7):1187-1202.32 E. Schiegnitz, P. W. Kammerer, F. P. Koch, et al. GDF 15 as an anti-apoptotic, diagnostic and prognostic marker in oral squamous cell carcinoma[J]. Oral oncology, 2012,48(7):608-614.33 C. Z. Yang, J. Ma, D. W. Zhu, et al. GDF15 is a potential predictive biomarker for TPF induction chemotherapy and promotes tumorigenesis and progression in oral squamous cell carcinoma[J]. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO, 2014,25(6):1215-1222.34 U. Wallin, B. Glimelius, K. Jirstrom, et al. Growth differentiation factor 15: a prognostic marker for recurrence in colorectal cancer[J]. British journal of cancer, 2011,104(10):1619-1627.35 S. Shnaper, I. Desbaillets, D. A. Brown, et al. Elevated levels of MIC-1/GDF15 in the cerebrospinal fluid of patients are associated with glioblastoma and worse outcome[J]. International journal of cancer. Journal international du cancer, 2009,125(11):2624-2630.36 A. C. Staff, A. J. Bock, C. Becker, et al. Growth differentiation factor-15 as a prognostic biomarker in ovarian cancer[J]. Gynecologic oncology, 2010,118(3):237-243.37 A. J. Bock, H. T. Stavnes, T. Kempf, et al. Expression and clinical role of growth differentiation factor-15 in ovarian carcinoma effusions[J]. International journal of gynecological cancer : official journal of the International Gynecological Cancer Society, 2010,20(9):1448-1455.38 J. C. Meier, B. Haendler, H. Seidel, et al. Knockdown of platinum-induced growth differentiation factor 15 abrogates p27-mediated tumor growth delay in the chemoresistant ovarian cancer model A2780cis[J]. Cancer medicine, 2015,4(2):253-267.