Breast cancer is one of the most frequently malignant tumors and a major cause of cancer-related death among women worldwide [
Secreted frizzled-related protein 2 (sFRP2), a member of the Wnt-binding protein family, approximately 30-35 kDa, with homology to the transmembrane Wnt-binding domain of frizzled receptors, is widely generated in many adult tissues, including the heart, lung, pancreas, prostate, kidney, and brain [
In this study, to explore the diagnostic and prognostic value of serum sFRP2 in breast cancer, we examined serum levels in breast cancer patients by enzyme-linked immunosorbent assay (ELISA) and assessed the association between serum sFRP2 and clinicopathological features.
Samples used in this study were collected at the First Affiliated Hospital of Sun Yat-sen University. Two hundred and seventy-four stage I-III primary breast cancer patients were investigated from January 2004 to January 2009. All breast cancer patients were diagnosed and confirmed independently by two pathologists, who performed pathological examination of specimens coming from biopsies or surgically resected tissues, according to the World Health Organization (WHO) criteria. All the enrolled subjects presented with tumors that were restricted to the breast, with no indication of distant metastasis or skin involvement at presentation. Patients who had previous malignancy or received neoadjuvant chemotherapy or preoperative radiation therapy prior to surgical operation were excluded from the study. All the patients received standard surgical treatment attached with endocrine therapy, adjuvant chemotherapy, and radiotherapy under the guidelines of National Comprehensive Cancer Network. All clinicopathological data, including age, histology, tumor size, lymph node status, and follow-up data, were collected from medical records. The tumor stage was determined according to the criteria for breast cancer of the American Joint Committee on Cancer. The control group contained 147 age-matched healthy volunteers who performed physical examination at the Department of Physical Health Examination of the First Affiliated Hospital of Sun Yat-sen University. All enrolled subjects were unrelated ethnic Han Chinese women. All subjects with breast cancer were followed up at intervals of three to 12 months (every three months for the first two years, then every six months for three years, and yearly thereafter) until June 2016. Progression-free survival (DFS) was defined as the date from diagnosis to the date the patient lives with the disease but it does not get worse.
The research protocol was reviewed and approved by the Human Research Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University in accordance with the principles of the Declaration of Helsinki. Each participant provided informed consent for participation in the study at the first visit.
All serum samples were collected directly from breast cancer patients at the time of first visit and stored immediately at -80°C. The serum concentration of human sFRP2 was measured using a commercially available sandwich ELISA kit (Elabscience Inc., Wuhan, China) with a detection range of 1.25-80 ng/mL. Measurements were performed completely blinded to the clinical information strictly following manufacturer’s instructions, and quality control was guaranteed. The optical density (OD value) was determined at 450 nm by SpectraMax M5 Microplate Reader (Molecular Devices, USA).
Statistical analyses were carried out using SPSS (Version 13.0, SPSS Inc., Chicago, IL, USA) and GraphPad Prism (Version 5.0, GraphPad Software Inc., San Diego, CA, USA). All variables with normal distribution were expressed as
The study recruited a total of 274 patients who were treated for breast cancer at the First Affiliated Hospital of Sun Yat-sen University and 147 normal healthy controls. The clinicopathological characteristics of the study subjects are displayed in Table
Clinicopathological characteristics of the study cohort.
Characteristics | No. of patients | (%) | No. of controls | (%) |
---|---|---|---|---|
Age (years) | ||||
≤45 | 131 | 47.8 | 80 | 54.4 |
>45 | 143 | 52.2 | 67 | 45.6 |
Menopausal status | ||||
Premenopausal | 166 | 60.6 | 96 | 65.3 |
Postmenopausal | 108 | 39.4 | 51 | 34.7 |
Tumor size (cm) | ||||
≤2 | 79 | 28.8 | ||
>2 | 173 | 63.2 | ||
Undetermined | 22 | 8.0 | ||
Histology | ||||
IDC | 213 | 77.8 | ||
DCIS | 59 | 21.5 | ||
Other | 2 | 0.7 | ||
TNM stage | ||||
I+II | 97 | 35.4 | ||
III | 155 | 56.6 | ||
Undetermined | 22 | 8.0 | ||
Lymph node metastases | ||||
Negative | 133 | 48.6 | ||
Positive | 136 | 49.6 | ||
Unknown | 5 | 1.8 | ||
ER | ||||
Negative | 97 | 35.4 | ||
Positive | 172 | 62.8 | ||
Unknown | 5 | 1.8 | ||
PR | ||||
Negative | 120 | 43.8 | ||
Positive | 149 | 54.4 | ||
Unknown | 5 | 1.8 | ||
HER2 | ||||
Negative | 198 | 72.3 | ||
Positive | 71 | 25.8 | ||
Unknown | 5 | 1.9 | ||
Ki67 | ||||
≤14% | 105 | 38.3 | ||
>14% | 164 | 59.8 | ||
Unknown | 5 | 1.9 |
Abbreviations: IDC: invasive ductal carcinoma; DCIS: ductal carcinoma in situ; ER: estrogen receptor; PR: progesterone receptor; HER2: human epidermal growth factor receptor 2.
We first measured serum sFRP2 levels in samples by ELISA. As displayed in Table
The association of serum sFRP2 levels with clinicopathological characteristics in breast cancer patients.
Characteristics | Number | sFRP2 ( |
|
---|---|---|---|
Patient group | 274 | ||
Control group | 147 | ||
Age (years) | |||
≤45 | 131 | 0.2415 | |
>45 | 143 | ||
Menopausal status | |||
Premenopausal | 166 | 0.1815 | |
Postmenopausal | 108 | ||
Tumor size (cm) | |||
≤2 | 79 | ||
>2 | 173 | ||
Histology | |||
IDC | 213 | 0.8597 | |
DCIS | 59 | ||
TNM stage | |||
I+II | 97 | ||
III | 155 | ||
Lymph node metastases | |||
Negative | 133 | ||
Positive | 136 | ||
ER | |||
Negative | 97 | 0.1572 | |
Positive | 172 | ||
PR | |||
Negative | 120 | 0.5532 | |
Positive | 149 | ||
HER2 | |||
Negative | 198 | 0.3460 | |
Positive | 71 | ||
Ki67 | |||
≤14% | 105 | ||
>14% | 164 |
Diagnostic performance of serum sFRP2 in breast cancer patients. (a) Serum sFRP2 levels in breast cancer patients and normal healthy controls. The mean level of serum sFRP2 for breast cancer patients was
To assess the performance of serum sFRP2 as a diagnostic biomarker in distinguishing breast cancer patients from normal healthy controls, receiver operating characteristic/area under the curve (ROC/AUC) was performed (Figure
Sensitivity for sFRP2, CEA, and CA15.3 among patients with breast cancer.
Marker | Controls vs. breast cancer patients: sensitivity (%) | ||
---|---|---|---|
90% specificity (%) | 95% specificity (%) | 98% specificity (%) | |
sFRP2 | 46 | 30 | 21 |
CEA | 32 | 15 | 10 |
CA15.3 | 41 | 23 | 16 |
To further explore the prognostic role of serum sFRP2 in patients with breast cancer, breast cancer patients’ outcomes were analyzed by Kaplan-Meier analysis. The breast cancer patients’ median concentration of serum sFRP2 (58 ng/mL) was categorized as a threshold to divide the 274 breast cancer patients into two groups: a high serum sFRP2 group (>58 ng/mL,
Kaplan-Meier survival curves of breast cancer patients. Progression-free survival rate of breast cancer patients with high (>58 ng/mL) and low (≤58 ng/mL) serum sFRP2 levels.
Univariate and multivariate Cox analysis of variables considered for progression-free survival rates of breast cancer patients.
Variables | Category | Univariate analysis | Multivariate analysis | ||||
---|---|---|---|---|---|---|---|
HR | 95% CI | HR | 95% CI | ||||
Age | ≤45 Y vs. >45 Y | 1.41 | 0.71-2.74 | 0.38 | 1.34 | 0.69-2.36 | 0.43 |
Menopausal status | Post vs. pre | 2.13 | 1.095-3.67 | 0.02 |
2.11 | 1.42-4.05 | 0.03 |
ER status | Negative vs. positive | 1.79 | 1.06-4.53 | 0.04 |
2.45 | 1.12-4.61 | 0.02 |
PR status | Negative vs. positive | 1.17 | 0.79-2.12 | 0.38 | 1.33 | 0.72-2.18 | 0.29 |
HER2 status | Positive vs. negative | 1.95 | 0.92-4.33 | 0.09 | 1.87 | 0.83-4.04 | 0.11 |
Size | >2 cm vs. ≤2 cm | 2.91 | 1.68-5.87 | 0.003 |
1.91 | 0.91-4.34 | 0.09 |
Lymph node status | Positive vs. negative | 4.15 | 1.85-7.03 | <0.001 |
2.72 | 1.51-5.95 | 0.002 |
TNM stage | III vs. I+II | 2.27 | 1.17-3.79 | 0.03 |
2.67 | 1.27-5.72 | 0.004 |
Serum sFRP2 | >58.0 vs. ≤58.0 ng/mL | 4.14 | 2.07-8.78 | <0.001 |
3.89 | 1.95-7.68 | 0.001 |
Abbreviations: HR: hazard ratio; CI: confidence interval; Y: years; ER status: estrogen receptor status; PR status: progesterone receptor status; HER2 status: human epidermal growth factor receptor 2 status.
sFRP2, approximately 300 amino acids in length, belongs to a large family of sFRPs, which are circulating soluble proteins with a highly homologous cysteine-rich domain for cell surface frizzled receptors [
In this study, we found that serum sFRP2 concentrations were increased in breast cancer patients compared with normal healthy controls. We also found that serum sFRP2 concentrations were associated with breast cancer tumor size, TNM stage, and lymph node metastasis status. Kaplan-Meier and Cox regression analysis also found that elevated sFRP2 level was associated with a poor prognosis and can be an independent prognostic factor for breast cancer patients. Furthermore, ROC analysis showed that serum sFRP2 had the potential to distinguish breast cancer patients from normal healthy controls with high sensitivity. Because serum sFRP2 can be conveniently measured by the application of a commercial ELISA kit, our initial results demonstrate the potential value of sFRP2 as a diagnostic biomarker for breast cancer.
Metastasis is the main cause of cancer-associated deaths and is involved in cancer cell processes from the primary tumor to translocation and colonization at the secondary site [
Our group aimed to identify a novel serum biomarker for cancer diagnosis [
In conclusion, serum sFRP2 can act as a noninvasive biomarker with high sensitivity for diagnosis and prognosis of breast cancer.
The data used to support the findings of this study are available from the corresponding author upon request.
The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors indicated no potential conflicts of interest.
LSL and XDX designed the experiment, interpreted the data, and prepared the manuscript. CMH, ZJY, CMH, JXW, JBL, ML, XDX, and LSL conducted the experiment, collected the data, and helped to prepare the manuscript. All authors read and approved the final manuscript. Chumei Huang and Zhuangjian Ye contributed equally to this work.
This research was supported by grants from the Natural Science Foundation of Guangdong Province of China (Grant no. 2015A030313035 to LSL).