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Cancer Chemother Pharmacol (2014) 73:631–637DOI 10.1007/s00280-014-2396-x

ORIGINAL ARTICLE

Increased serum level of epidermal growth factor receptor (EGFR) is associated with poor progression‑free survival in patients with epithelial ovarian cancer

Faruk Tas · Senem Karabulut · Murat Serilmez · Rumeysa Ciftci · Derya Duranyildiz

Received: 24 November 2013 / Accepted: 18 January 2014 / Published online: 30 January 2014 © Springer-Verlag Berlin Heidelberg 2014

Keywords Serum · EGFR · Ovarian cancer · Prognostic factor

Introduction

Epidermal growth factor receptor (EGFR) is the expression product of oncogene c-erbB1, and it belongs to the human epidermal receptor (HER) family, a structure composed of an extracellular ligand-binding domain, a transmembrane lipophilic glycoprotein, and an intracellular tyrosine kinase domain [1–5]. EGFR is widely expressed in the surface of mammalian epithelial cells and other many cell types [1, 2]. Many studies have demonstrated that EGFR has a physiologically favorable role during embryonic and post-natal development [1, 2]. Moreover, EGFR plays an impor-tant role in the pathogenesis of multiple malignancies and its expression strongly also affects the outcomes of cancer patients [1, 2]. It has been found to act as a strong prognos-tic indicator in many cancer types, with increased expres-sion being associated with poor progression-free or overall survival rates.

EGFR overexpression is detected in 30–98 % of epithe-lial ovarian cancers (EOCs) [1]. Accumulating evidences suggest that dysregulation of EGFR may contribute to ovarian cancer similar to other tumors via promotion of cell proliferation, migration and invasion, and angiogenesis, as well as resistance to cell apoptosis [1, 2]. Although some investigators have found EGFR expression to exhibit no relationship with known prognostic factors or outcomes of EOC, others have observed associations between EGFR expression and various demographical, clinical, and patho-logical prognostic indicators and survivals [2].

Given the ability to quantify circulating soluble ErbB isoforms (sErbBs) using immunoassay methods, serum

Abstract Purpose Epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of multiple malignan-cies, and its expression also strongly affects the outcomes of cancer patients. The objective of this study was to deter-mine the clinical significance of the serum levels of EGFR in epithelial ovarian cancer (EOC) patients.Materials and methods A total of 50 patients with a pathologically confirmed diagnosis of EOC were enrolled into this study. Serum EGFR levels were determined by the solid-phase sandwich ELISA method. Age and sex matched 20 healthy controls were included in the analysis.Results Median age of patients was 56.5 years old, range 22–83 years. Majority of the patients had advanced dis-ease (FIGO stage III-IV) (90 %). No significant difference in baseline serum EGFR levels between EOC patients and controls (65.9 vs. 65.4 ng/mL, p = 0.86). Patients with nor-mal CA 125 had higher serum EGFR level compared with the higher CA 125 level (p = 0.02). No other clinical vari-ables including histology, stage of disease, and response to chemotherapy were found to be correlated with serum EGFR assay (p > 0.05). The patients with increased serum EGFR levels had poor progression-free survival than those with lower levels (median survival 4 vs. 12 months, respec-tively, p = 0.01). However, serum EGFR level was found no prognostic role for overall survival (p = 0.15).Conclusion Increased serum level of EGFR is associated with poor progression-free survival in EOC patients.

F. Tas (*) · S. Karabulut · M. Serilmez · R. Ciftci · D. Duranyildiz Institute of Oncology, Istanbul University, Capa, 34390 Istanbul, Turkeye-mail: faruktas2002@yahoo.com

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sErbB isoforms are being investigated as potential bio-markers for a variety of cancers [2]. To date, although several of these assays have been used to study sErbBs in the serum of ovarian cancer patients, only one immunoas-say has been developed to quantify sEGFR/sErbB1 [2–5]. In particular, serum concentrations of sEGFR have been evaluated in ovarian cancer patients using ALISA immuno-assay [3–5]. Several reports have demonstrated that serum sEGFR concentrations are lower in ovarian cancer patients compared with healthy women, to women with benign ovarian tumors, or to women with other gynecological con-ditions [3–5]; hence, sEGFR may be a useful biomarker for risk assessment, early detection, and/or diagnosis of this disease [3, 4].

The present study was designed to assess the clinical significance of serum EGFR using the solid-phase sand-wich ELISA method in EOC patients.

Materials and methods

Patients

A total of 50 EOC patients with histologically proven diag-nosis treated at Istanbul University, Institute of Oncology were enrolled into the study. The staging was established in accordance with International Federation of Gynecologists and Obstetricians (FIGO) classification. Patients stage III with bulky disease or stage IV disease were initially treated with neoadjuvant chemotherapy and operated afterward. Patients with operable stage III disease who had undergone primary surgery consist of total abdominal hysterectomy, bilateral salpingoophorectomy, appendectomy, omentec-tomy, and pelvic and/or paraaortic lymphadenectomy were treated with adjuvant chemotherapy. All patients received standard paclitaxel–carboplatin containing chemotherapy regimen.

For comparison of serum EGFR levels, age and sex matched 20 healthy controls were included in the analy-sis. Informed consent was obtained from all patients, and the study was reviewed and approved by a local ethical committee.

Measurement of serum EGFR levels

Serum samples were obtained on first admission before any adjuvant and metastatic treatment was given or follow-up patients. Blood samples were obtained from patients and healthy controls by venipuncture and clotted at room tem-perature. The sera were collected following centrifugation and frozen immediately at −20 °C until analysis.

Serum EGFR (R&D Systems, USA) levels were deter-mined by the solid-phase sandwich ELISA method. The

EGFR ELISA (R&D Systems, USA) uses a double-anti-body sandwich enzyme-linked immunosorbent assay to determine the level of human epidermal growth factor receptor (EGFR) in samples. Serum samples and stand-ards were added to the wells which were pre-coated with human EGFR monoclonal antibody and allowed to incu-bate for 2 h. Unbound material was washed away EGFR combined with Streptavidin–HRP were added to form immune complex and then allowed to incubate for 2 h. Unbound material was washed away. Chromogen solu-tion was added and incubate for 30 min (protect from light) for the conversion of the colorless solution to a blue solution, the intensity of which was proportional

Table 1 Characteristics of the patient and disease

Variables n

No. of patients 50

Age of patients (year)

<55 24

≥55 26

Histology

Serous papillary 21

Endometrioid 5

Mixed 4

Clear cell 2

Mucinous 1

Undifferentiated 17

Histological grade

I 2

II 6

III 13

Stage of disease

I–II 5

III 33

IV 12

Debulking surgery (optimal)

Yes 22

No 28

Serum LDH level (480 U/L)

Normal 31

High 13

Ca 125 level (35 IU/ml)

Normal 10

High 40

Response to chemotherapy

Yes 25

No 12

Platinum sensitivity in relapse

Sensitive 8

Resistant 15

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to the amount of EGFR in the sample. As the effect of the acidic stop solution, the color became yellow. The colored reaction product was measured using an auto-mated ELISA reader (Rayto, RT-1904C Chemistry Ana-lyzer, Atlanta GA, USA) at 450 nm. The results were expressed as ng/mL.

Statistical analysis

Continuous variables were categorized using median val-ues as cutoff point. Assessment of relationships and com-parisons between various clinical/laboratory parameters were accomplished using Mann–Whitney U test. After the tests, Bonferroni correction was applied. Overall survival was calculated from the date of first admission to the clin-ics to disease-related death or date of last contact with the patient or any family member. Progression-free survival was calculated from the date of admission to the date of first radiologic progression with/without increased serum tumor marker. Kaplan–Meier method was used for esti-mation of survival distribution, and differences in survival

were assessed by the log-rank statistics. A p value <0.05 was considered significant. Statistical analysis was carried out using SPSS 16.0 software.

Results

A total of 50 patients with a pathologically confirmed diag-nosis of EOC were enrolled into this study. Baseline his-topathological characteristics and demographic features of patients are listed in Table 1. Median age of patients was 56.5 years old, range 22–83 years. Majority of the patients had advanced disease (FIGO stage III–IV) (90 %).

No significant difference in baseline serum EGFR levels between EOC patients and the controls (65.9 vs. 65.4 ng/mL, p = 0.86) (Table 2; Fig. 1). Table 3 shows the cor-relations between the serum EGFR levels and the known clinicopathological variables. Patients with normal CA 125 had higher serum EGFR levels compared with the higher CA 125 level (p = 0.02). However, no other clinical vari-ables including histology, stage of disease, and response to chemotherapy were found to be correlated with serum EGFR assay (p > 0.05).

The median follow-up time was 12 months (range 1–78 months). Median progression-free survival for all patients was 8 ± 2.8 months (95 % CI 3–14 months). One-year progression-free survival rate was 27.3 % (95 % CI 8.7–45.9). Median overall survival for all patients was 52 ± 7.5 months (95 % CI 37–67 months). One-, two-, and three-year overall survival rates were 84.9 % (95 % CI

Table 2 The values of serum marker levels in patients with EOC and in healthy controls

Assay Patients (n = 50) Controls (n = 20) p

Median Range Median Range

Serum EGFR level (ng/mL)

65.9 44.7–89.5 65.4 52.9–76.8 0.86

Fig. 1 The values of serum EGFR assays in EOC patients and healthy controls (p = 0.86)

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73.5–96.2), 77 % (95 % CI 62.3–91.7), and 55 % (95 % CI 27–83), respectively. Patients with no chemotherapy responsive and with platinum-resistant in relapsed disease had worse outcome in both of progression-free survival and overall survival analyses (Table 4). The patients with increased serum EGFR levels had poor progression-free survival than the ones with lower levels (median survival 4 vs. 12 months, respectively, p = 0.01) (Table 4, Fig. 2). However, serum EGFR level was found no prognostic role for overall survival (median survival 23.4 vs. 59.2 months, respectively, p = 0.15) (Table 4; Fig. 3).

Discussion

ErbB receptors and their ligands play an important physi-ologic role in development, proliferation, differentiation, metabolism, motility, and survival of normal cells, as well as

a critical role in cell growth, adhesion and motility, stromal proliferation, angiogenesis, and apoptosis of tumor cells [1–5]. Thus, it is not surprising that aberrations of ErbB receptor signaling pathways, involving gene amplification, mutation, and/or receptor overexpression, as well as ligand overexpres-sion, may result in increased cellular growth and cellular transformation of epithelial cells [1–5]. Indeed, ERBB gene amplification and/or ErbB receptor overexpression has been observed in a variety of human carcinomas, including ovar-ian cancer [1, 2]. ERBB oncogene amplification and ErbB receptor overexpression in ovarian tumors have been associ-ated with patient prognosis (paradoxically, as both positive and negative predictive factors), as well as with responsive-ness to therapy [1, 2]. Paradoxically, these prognostic studies report that EGFR overexpression may provide a favorable, neutral, or adverse effect on patient survival. The basis for this discrepancy is likely related to the failure of most EGFR expression assays to distinguish between various transmem-brane, soluble, and truncated EGFR isoforms [2].

Several studies have analyzed EGFR protein expression by immunohistochemistry (IHC) in normal human adult ovary [1, 2]. In general, EGFR is expressed by the ovar-ian surface epithelium (OSE) of most adult ovaries, with reduced or no EGFR immunostaining of stromal fibroblasts or endothelial cells [1, 2]. Overall, IHC demonstrates that normal adult ovary expresses low-to-moderate levels of EGFR in the OSE, with lower levels of expression in stro-mal fibroblasts and endothelial cells. ErbB receptors and their ligands appear to be important for certain aspects of normal ovarian cellular function and physiology. There-fore, it is not surprising that ErbB receptor and/or ligand overexpression is associated with ovarian surface epithelial cell transformation and oncogenesis, where receptor and/or ligand overexpression (or mutation) results in increased tyrosine kinase activity and improper activation of certain signal transduction pathways [2].

EGFR expression by IHC in ovarian cancers appears to be highly variable. The average frequency of EGFR expression is 48 % (between 4 and 100 %) [2]. Mostly, some investigators have found EGFR expression to exhibit no relationship with known prognostic factors or surviv-als of EOC. For example, no association has been found between EGFR expression and patient age, disease stage, tumor grade, histological subtype, presence of ascites, lymph node metastases, tumor size, or residual disease [2]. Likewise, no difference in EGFR staining intensity has been observed among benign, borderline, and malig-nant ovarian tumors, or between primary and recurrent metastatic tumors [2]. Moreover, no association has been found between EGFR expression and responsiveness to treatment, progression-free survival, or overall survival [2]. In contrast, other investigators have observed associations between EGFR expression and various demographical,

Table 3 Comparisons between serum EGFR levels and various clini-cal/laboratory variables

Variables EGFR(ng/mL)Median (range)

Age, years (p) 0.07

Young (<55) 66.8 (50.0–89.5)

Older (≥55) 61.9 (44.7–85.7)

Histology (p) 0.49

Serous papillary 65.8 (44.7–83.2)

Others 66.0 (50.0–89.5)

Grade (p) 0.55

I − II 68.3 (57.8–75.0)

III 65.8 (44.7–84.3)

Stage (p) 0.87

Non-metastatic 65.2 (44.7–89.5)

Metastatic 67.6 (50.0–85.7)

Debulking surgery (p) 0.15

Yes 67.7 (53.8–83.2)

No 63.4 (44.7–89.5)

Serum LDH level (p) 0.67

Normal 64.7 (44.7–82.3)

High 66.2 (56.8–85.7)

Serum CA 125 level (p) 0.02

Normal 68.7 (64.5–83.2)

High 63.9 (44.7–89.5)

Response to chemotherapy (p) 0.67

Yes 64.7 (44.7–89.5)

No 66.3 (52.4–84.3)

Platinum sensitivity in relapse (p) 0.24

Sensitive 59.3 (54.8–83.2)

Resistant 64.0 (49.6–85.7)

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clinical, and pathological prognostic variables and out-comes [2]. In these studies, EGFR expression has been shown to be significantly lower in LMP tumors compared with ovarian carcinomas, and increased EGFR expression has been associated with older patients, advanced stage dis-ease, poorly differentiated or high grade tumors, metastatic spread, and residual disease following surgery [2]. Simi-larly, EGFR expression also has been reported to be higher in serous compared with clear cell ovarian carcinomas, and in metastases compared with primary tumors [2]. Addition-ally, EGFR expression has been associated with disease progression as measured by progression-free survival and tumor recurrence rates, as well as with decreased overall survival [2].

In order to quantify circulating sErbBs using immu-noassay methods, serum sErbB isoforms are being inves-tigated as potential biomarkers for a variety of cancers [2]. Similar to IHC studies of ErbB receptors, serum sErbB immunoassay studies may yield different results

because of their specific characteristics (e.g., analytical standards, antibody reagents, assay format, and measure-ment units). To date, several of assays have been used to study sErbBs in the serum of ovarian cancer patients [2]. The major circulating sEGFR/sErbB1 isoform arises from an alternately spliced EGFR transcript [3–5]. Although several sEGFR/sErbB1 immunoassays have been devel-oped, only some of these assays have been well charac-terized and have become available commercially. The two sEGFR/sErbB1 immunoassays commercially available in the United States are both sandwich type ELISAs [2]. In addition to these two commercially available sEGFR ELI-SAs, Baron et al. [3–5] have developed a well character-ized, sensitive acridinium-linked immunosorbent assay (ALISA) for determining sEGFR/sErbB1 concentrations in ovarian cancer patients (Table 5). These reports have demonstrated that sErbB1 concentrations may be altered by normal physiological processes such as pregnancy, age, and menopause.

Table 4 Univariate analyses of progression-free and overall survivals

NR means not reached

Variables Survival (month)

Event/Total Progression-free survivalMedian (±SE)

Event/Total Overall survivalMedian (±SE)

Age, years (p) 0.60 0.12

Young (<55) 9/24 29.3 (9.9) 3/24 61.2 (8.9)

Older (≥55) 13/26 12.6 (2.6) 7/26 24.1 (2.3)

Histology (p) 0.85 0.74

Serous papillary 9/21 10.4 (2.2) 4/21 26.8 (2.6)

Others 13/29 27.3 (7.1) 6/29 52.6 (9.5)

Grade (p) 0.78 0.17

I − II 1/8 10.0 (0.0) 0/8 NR

III 5/13 9.4 (3.1) 3/13 NR

Stage (p) 0.76 0.34

Non-metastatic 14/38 25.0 (7.0) 7/38 55.4 (8.3)

Metastatic 8/12 14.5 (3.6) 3/12 24.2 (4.3)

Debulking surgery (p) 0.47 0.09

Yes 4/22 26.1 (6.3) 2/22 45.4 (9.0)

No 18/28 9.8 (3.7) 8/28 30.3 (1.7)

Serum CA 125 level (p) 0.06 0.11

Normal 1/10 52.0 (0.0) 0/10 NR

High 21/40 8.3 (1.4) 10/39 NR

Response to chemotherapy (p) <0.001 <0.001

Yes 12/25 34.2 (7.0) 4/25 60.2 (7.9)

No 9/12 3.3 (0.5) 6/12 14.5 (4.5)

Platin-sensitivity in relapse (p) 0.001 0.003

Sensitive 8/8 46.2 (5.3) 1/8 66.0 (10.4)

Resistant 14/15 6.9 (1.5) 9/15 15.1 (3.2)

Serum EGFR level (p) 0.01 0.15

High (>median) 9/23 4.0 (1.5) 6/23 23.4 (2.8)

Low (<median) 12/25 12.0 (0.9) 4/25 59.2 (8.7)

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Previous studies have demonstrated that circulating sErbB concentrations may be altered by pathological con-ditions including ovarian cancer [2, 3]. In particular, serum concentrations of sEGFR have been evaluated in ovarian cancer patients using ALISA immunoassay [3–5] (Table 5). ALISA specifically measures a 110-kDa sErbB1 protein in serum samples of healthy women, women with benign gynecologic condition and ovarian cancer patients. These studies showed that serum sEGFR concentrations are lower

in ovarian cancer patients compared to healthy women, to women with benign ovarian tumors, or to women with other gynecological benign conditions [3–5]; hence, sEGFR may be a useful biomarker for risk assessment, early detection, and/or diagnosis of this disease [2–4]. In a study comparing serum CA 125 levels and sEGFR concentrations in women with EOC to women with benign gynecologic conditions of ovarian and non-ovarian origin, while serum sEGFR con-centrations are lower in EOC patients than in women with

Fig. 2 Progression-free sur-vival curves in EOC patients according to serum EGFR levels (p = 0.01)

Fig. 3 Overall survival curves in EOC patients according to serum EGFR levels (p = 0.15)

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benign gynecologic conditions, serum CA 125 levels were found to be higher in EOC patients compared with women benign gynecologic conditions [5]. Therefore, the com-bined use of serologic sEGFR and CA125 may improve the potential of these biomarkers in screening and diagnosing EOC by increasing their ability to detect and differentiate benign from malignant ovarian tumors [2, 5]. Moreover, serum sEGFR concentrations were not associated with dis-ease stage and tumor grade [3]. Serum sEGFR levels are significantly higher in healthy premenopausal than post-menopausal women. This means that sEGFR concentra-tions decrease with age in healthy women; however, similar finding was not found in ovarian cancer patients [3].

Similar to the literature [3–5], we also found that no associations between serum EGFR levels and known clin-icopathological variables including age of patient, histol-ogy, tumor grade, stage of disease, and response to chemo-therapy. However, only a correlation was determined in the current study. The patients with normal CA 125 had higher serum EGFR level. We also showed that no significant difference in baseline serum EGFR levels between EOC patients and controls in spite of the differences in the lit-erature [3–5]. A novel and interesting finding of our study was to determine a difference in progression-free survival between patients with higher and lower serum EGFR levels. Moreover, although similar data were provided for overall survival, it was not reached to statistical significance.

In conclusion, although little is known, evidence to date suggests that the EGFR family may be involved in the eti-ology and progression of EOC. However, there are much conflicting evidences in the literature regarding the patterns

of expression of these gene products; therefore, the pre-cise functional relevance of these alterations is not yet well understood. Similar comments were true for patterns of quantifying of the circulating serum EGFR concentrations. The present study contributes to the literature, because we performed it preliminary in literature. A standardized method remains to be established and validated in larger series of patients in prospective studies.

Conflict of interest None.

References

1. Gui T, Shen K (2012) The epidermal growth factor receptor as a therapeutic target in epithelial ovarian cancer. Cancer Epidemiol 36:490–496

2. Lafky JM, Wilken JA, Baron AT, Maihle NJ (2008) Clinical implications of the ErbB/epidermal growth factor (EGF) receptor family and its ligands in ovarian cancer. Biochim Biophys Acta 1785:232–265

3. Baron AT, Cora EM, Lafky JM, Boardman CH, Buenafe MC, Rademaker A et al (2003) Soluble epidermal growth factor recep-tor (sEGFR/sErbB1) as a potential risk, screening, and diagnostic serum biomarker of epithelial ovarian cancer. Cancer Epidemiol Biomarkers Prev 12:103–113

4. Baron AT, Lafky JM, Boardman CH, Balasubramaniam S, Suman VJ, Podratz KC et al (1999) Serum sErbB1 and epidermal growth factor levels as tumor biomarkers in women with stage III or IV epithelial ovarian cancer. Cancer Epidemiol Biomarkers Prev 8:129–137

5. Baron AT, Boardman CH, Lafky JM, Rademaker A, Liu D, Fish-man DA et al (2005) Soluble epidermal growth factor receptor (sEGFR) and cancer antigen 125 (CA125) as screening and diag-nostic tests for epithelial ovarian cancer. Cancer Epidemiol Bio-markers Prev 14:306–318

Table 5 Serum EGFR trials in EOC patients [2]

Analyte measured Assay used Cohort References

sEGFR ALISA Healthy women (n = 94)Ovarian cancer, preoperative (n = 21) postoperative (n = 73)

[4]

sEGFR ALISA Healthy women (n = 144)Ovarian cancer, preoperative (n = 225)

[3]

sEGFR ALISA Benign ovarian tumor, preoperative (n = 246)Benign gynecologic, preoperative (n = 253)Ovarian cancer, preoperative (n = 225)

[5]