or.nsfc.gov.cnor.nsfc.gov.cn/bitstream/00001903-5/366806/1/1000014512558.docx · web viewelevated...
Post on 24-Mar-2018
217 Views
Preview:
TRANSCRIPT
1
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
2
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
3
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
4
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
5
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
6
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
7
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%–
8
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
9
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
10
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)
11
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).
12
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
13
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.
14
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.
15
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.
16
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
17
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.
18
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;
19
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
20
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).
21
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)
22
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)
23
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
24
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
25
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
26
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
27
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.
top related