The study on human subjects was approved by the Ethics Committee of Jinshan Hospital, Fudan University, Shanghai, China. Samples from patients who were primarily diagnosed with breast tumor at Jinshan Hospital from 2013 to 2014 were retrieved for the present study. None of the patients had received radiotherapy or chemotherapy before surgery. A total of 68 paraffin-embedded samples constituting 20 benign tumors and 48 malignant tumors were subjected to the histopathological examination and immunohistochemistry. The adjacent normal tissues were used as controls. The 10% formalin-fixed paraffin-embedded breast tissue specimens were prepared. Four micrometer thick sections of these specimens were stained by hematoxylin and eosin (H&E) to confirm the histological characteristics. The histological grades and clinical stages of tumor were classified by experienced surgeons and pathologists based on the WHO classification.

To evaluate the expression of NAMPT, VEGF and HER2 proteins in breast tumors, immunohistochemical staining was performed as described previously (19). Briefly, after blocking, the sections were incubated with a rabbit monoclonal anti-NAMPT, mouse monoclonal anti-VEGF or anti-HER2 antibody (all 1:250 dilution; Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with biotinylated secondary antibody (1:150 dilution; Maixin Bio, Fuzhou, China) at room temperature for 1 h. After washing, the signal was detected using a DAB kit (diaminobenzidine; Maixin Bio). Finally, the sections were counterstained with hematoxylin and photographed under a light microscope (BX43; Olympus, Tokyo, Japan).

Double blind scoring of NAMPT, VEGF and HER2 immunoreactive staining was performed independently by two examiners who had no prior knowledge of patient's clinical status. The sections were evaluated at original magnification, ×200. The proportion of cells exhibiting protein expression was scored by the extent of immunoreactive staining and was assigned to one of the following categories as described previously (19). Briefly, the percentage of positive cells was scored as follows: no reactivity as 0, ≤25% positive cells as 1, 26–50% positive cells as 2, 51–75% positive cells as 3 and >75% positive cells as 4. The intensity of staining was scored as follows: no staining as 0, weak staining as 1, moderate staining as 2, and strong staining as 3. The final staining index (SI) was developed based on the sum score of the positive staining and intensity. The SI score was then clustered into four groups: 0, ≤2 sum points; 1, 3–4 sum points; 2, 5–6 sum points; 3, 7 sum points. Finally, the cases were categorized based on the SI score 0–1 to be negative and 2–3 to be positive.

Blood samples were collected from 30 patients with a malignant tumor on a day before operation (M-BO) and the 7th day after operation (M-AO) as well as from 28 patients with benign tumor. For comparison, 30 blood samples from age- and body mass index (BMI)-matched healthy controls were obtained from the Health Check-Up Center of Jinshan Hospital in 2014. The median age of healthy controls and patients with malignant tumor was 50.00 and 52.00, respectively, and the median BMI of healthy controls and patients with malignant tumor were 23.05 and 23.73, respectively. All subjects (patients and healthy controls) who enrolled into the study signed a consent form prior to the collection of blood samples. Serum was prepared using a serum separator tube (SST) which allowed samples to clot for 30 min before centrifugation for 15 min at 1,000 × g, aliquoted, and stored at −80°C until use. None of the samples were previously thawed.

Serum levels of NAMPT, VEGF and HER2 were determined in patients and healthy individuals by enzyme-linked immunosorbent assay (ELISA). NAMPT and HER2 kits were purchased from Wuhan Xinqidi Biological Technology Co., Ltd. (Wuhan, Hubei, China), whereas VEGF kit was purchased from R&D Systems (Human VEGF Immunoassay, Quantikine^® ELISA; R&D Systems, Inc., Minneapolis, MN, USA). Briefly, after adding 100 µl assay diluent into each well, 100 µl standards and serum samples were, respectively, added into the wells and incubated at room temperature for 2 h. After washing away any unbound substances, 200 µl enzyme-linked polyclonal antibody specific to NAMPT, VEGF, or HER2 was added into each well and incubated at room temperature for 2 h. After washing 3 times, 200 µl substrate solution was added into each well and incubated at room temperature for 25 min. After adding 50 µl stop solution into each well, the optical density of each well was determined within 30 min using a microplate reader at 450 nm.

Statistical analyses were performed with SPSS Statistics 21.0 software (SPSS, Chicago, IL, USA). Based on the SI system, the categories on positivity and negativity were classified. Statistical evaluation was performed using χ² test to analyze the association between the expression of NAMPT/VEGF/HER2 and the clinicopathological characteristics and to compare the positivity between benign and malignant tumors. For comparison of each variable in breast malignant tumors, a McNemar test was performed. For multiple group comparison, an ANOVA was applied. Significant difference between 2 groups was analyzed by a Student's t-test. For testing correlation between different serum markers, a linear regression was applied. Data are presented as the mean ± standard deviation (SD) or standard error of mean (SEM) as indicated. P<0.05 was considered to indicate a statistically significant difference.

The expression of NAMPT, VEGF and HER2 in benign and malignant breast tumors was detected by immunohistochemical staining. The adjacent normal breast tissue was used as normal control. We found that the expression of NAMPT, VEGF and HER2 was undetectable or barely detectable in controls, whereas the aberrant expression of NAMPT, VEGF and HER2 was noted in human benign and malignant breast tumors (Fig. 1). By comparison of all breast tissues, the highest degree of the expression of NAMPT, VEGF and HER2 was observed in malignant tumors. After the assessment of the SI score as indicated above, we classified the expression level into positive and negative categories. Compared with benign tumors, the positive rate of NAMPT, VEGF and HER2 expression was significantly increased in malignant tumors (all P<0.01) (Table I). These data suggest that NAMPT, VEGF and HER2 may be the indicators or progression markers of breast cancer development.

To examine the association of the expression of NAMPT, VEGF, and HER2 with the clinicopathological characteristics of breast cancer, a Chi-square test was applied. All patient information was gathered by reviewing medical charts and the records of pathology. By comparison of 48 malignant tumors, we found that the expression of NAMPT, VEGF and HER2 was not significantly associated with age (≤50 vs. >50), tumor size (≤2 vs. >2 cm), lymph node metastasis (yes vs. no) and clinical stage (0–II vs. III–IV) in patients with breast cancer (all P>0.05) (Table II).

Next, we compared these triple variables with each other to see whether the overexpression of NMAPT, VEGF and HER2 in breast malignant tumor is correlated. Using a McNemar test, we found that there was no significant difference among the comparisons, such as NAMPT vs. VEGF, VEGF vs. HER2 and HER2 vs. NAMPT (all P>0.05) (Table III). These data in turn suggest that the expression of NAMPT, VEGF and HER2 may be correlated. Further analysis showed that the detection rate of NAMPT, VEGF and HER2 in combination was increased in breast cancer patients. The detection rate of NAMPT, VEGF and HER2 alone was 54.17, 64.58 and 60.42, respectively. The detection rate of NMAPT plus VEGF, VEGF plus HER2, and HER2 plus NAMPT was increased to 68.75, 87.50 and 81.25%, respectively. Finally, the detection rate of collective triplets reached 89.58%.

Since NAMPT, VEGF and HER2 are secretory proteins, next we examined their serum levels using ELISA and compared the difference of their concentrations in healthy controls and patients with benign and malignant breast tumors before and after tumor removal. Two factors, i.e., age and BMI, were considered when comparisons were performed, but no significant difference was found between healthy controls and breast cancer patients, indicating that their age and BMI were matched between the two groups. The basal levels of serum NAMPT, VEGF and HER2 in healthy controls (CTL; n=30) were 94.90±4.24 pg/ml, 87.02±2.41 pg/ml and 1.12±0.04 ng/ml, respectively. The concentrations of serum NAMPT, VEGF and HER2 in patients with benign tumor (BN; n=28) presented different trends. In patients with a benign tumor, serum NAMPT was slightly increased (P<0.05) (Fig. 2A), whereas serum VEGF was slightly decreased (P<0.01) (Fig. 2B), compared with healthy controls. However, no significant difference of serum HER2 was observed between healthy controls and patients with a benign tumor (P>0.05) (Fig. 2C). In patients with a malignant tumor before operation (M-BO, n=30), the serum levels of all three variables were significantly elevated (all P<0.001) and there were 6.64-, 1.76-, and 2.52-fold increases of the concentration of NAMPT, VEGF and HER2, respectively. Interestingly after the operation (M-AO), the elevated serum NAMPT, VEGF, and HER2 levels significantly declined 47, 41 and 23%, respectively (all P<0.05), and that of VEGF almost returned to the basal level of control (Fig. 2B).

We further examined whether serum levels of NAMPT, VEGF and HER2 are correlated with each other in patients with malignant tumors. Before operation, no correlation was observed between them (VEGF vs. NAMPT, NAMPT vs. HER2 and HER2 vs. VEGF; all P>0.05) (Fig. 3A–C); however after tumor removal, a significant correlation was found: VEGF vs. NAMPT (P=0.0336) (Fig. 3D), NAMPT vs. HER2 (P=0.0028) (Fig. 3E) and HER2 vs. VEGF (P=0.0025) (Fig. 3F).

Since the presence and absence of a tumor seemed to affect the serum levels of NAMPT, VEGF and HER2, next we examined the association between serum levels and tissue expression of these variables in patients with a malignant tumor before and after operation. We found that the serum levels of NAMPT and VEGF were higher in patients before operation than those after operation and that serum levels were not associated with the positivity (SI score ≥2) and negativity (SI score ≤1) of their expression in the tumor tissues (Fig. 4A and B). These data suggest that serum variables are more sensitive than the tissue counterparts. However, the change of the serum levels of HER2 was different between negative and positive groups. In patients with HER2-negative (SI score ≤1), the serum level of HER2 was significantly decreased after tumor removal (M-AO vs. M-BO; P<0.05), whereas in patients with HER2-positive (SI score ≥2), no change of serum HER2 was observed after tumor removal (P>0.05) (Fig. 4C).

Finally, the association of the serum levels of NAMPT, VEGF and HER2 with the clinicopathological features of breast cancer patients before and after tumor removal was examined. The serum levels of NAMPT as well as VEGF were significantly different in patients with breast cancer before and after operation and were found to be higher in patients before surgery (P<0.05) (Table IV), irrespective of the clinicopathological features. The serum level of HER2 was also higher in patients younger than 50, with tumor size ≤2 cm, without lymph node metastasis in clinical stage of 0–II before operation than those after operation (P<0.05). However in patients older than 50, with tumor size >2 cm and lymph node metastasis in clinical stage of III–IV, no significant difference of HER2 was observed before and after operation (P>0.05). By comparing with the clinicopathological features of patients, such as age (≤50 vs. >50), tumor size (≤2 vs. >2 cm), lymph node metastasis (yes vs. no), and clinical stage (0–II vs. III–IV), the serum levels of NAMPT, VEGF and HER2 were similar (all P>0.05).