The expression of Eps15 homology domain 1 is negatively correlated with disease-free survival and overall survival of osteosarcoma patients

To assess the prognostic capacity of EHD1 for osteosarcomas, formalin-fixed, paraffin-embedded (FFPE) SCLC tumor tissues were collected from 57 osteosarcoma patients who underwent surgery between January 2011 and September 2015. Inclusion criteria were patients recorded to have osteosarcoma by pathology reports and included cases from stage I to stage III. All cases, representing a spectrum of osteosarcomas, were retrieved from patients attending Harbin Medical University Cancer Hospital, Harbin, China. Exclusion criteria were having stage IV of disease, or a history of other cancers. For each patient, each tissue specimen type was resected during a single surgical procedure. Primary cancers were evaluated in accordance with the seventh edition of the American Joint Committee on Cancer (AJCC) staging system (TNM). All patients were followed up until their death or the study end date (January 21, 2014). The median follow-up time for survivors was 46.8 months (range 34.9–58.7 months). The study was approved by the Ethical Committee of Harbin Medical University. Informed consent was obtained from all patients. All investigators involved in the study, apart from the study statistician, were blinded to patient outcomes throughout all laboratory analyses.

The expression of EHD1 in FFPE sections was analyzed by immunohistochemistry. The tissue sections were first dried at 70 °C for 3 h. After deparaffinization and hydration, sections were washed in phosphate-buffered saline (PBS, 3 × 3 min). Endogenous peroxidase was quenched with 3% H₂O₂ for 15 min. After first being washed with distilled water, sections were washed in PBS (3 × 5 min). Antigen retrieval was performed in citrate buffer (pH 6.0). Each section was treated with 300–500 ml EHD1 rabbit polyclonal antibody solution (Abcam, Cambridge, UK, ab75886, diluted at 1:200) overnight at 4 °C. The sections were incubated with peroxidase-conjugated streptavidin for 30 min, and the reaction products were visualized with diaminobenzidine as a chromogen and counterstained with commercial hematoxylin. The percentage of positive cells was determined by counting 500 cells in five randomly selected fields per section. IHC staining was scored based on intensity as follows: 0 (no staining), 1 (weak staining = light yellow), 2 (moderate staining = yellow brown), and 3 (strong staining = brown). The percentage (0–100%) of the extent of reactivity was scored as follows: 0 (no positive tumor cells), 1 (fewer than 10% positive tumor cells), 2 (10–50% positive tumor cells), and 3 (greater than 50% positive tumor cells). Next, the cytoplasmic expression score was obtained by multiplying the intensity and reactivity rate values. Scores of < 4 were classified as low expression, and the remainders were classified as high expression. Two blinded, independent observers interpreted all slides.

All analyses were performed using statistical software (SPSS 19.0 for Windows; SPSS, Inc., Chicago, IL, USA). Differences were considered statistically significant when P < 0.05. Correlation of EHD1 expression levels with clinicopathological features of patients was assessed using the Pearson χ² test for categorical variables and the Student t test for continuous variables. Disease-free survival (DFS) was calculated from the date of surgery resection to the date of last follow-up or relapse. Cumulative DFS curves and overall survival (OS) curves were plotted using the Kaplan–Meier method, and the relationship between each of the variables and survival was assessed by a log-rank test using univariate analysis. Covariates with P ≤ 0.15 in the univariate analyses were included in multivariate analyses. The parameters were then tested using the multivariate Cox proportional hazards model, which was performed to identify independent variables for predicting survival. Risk ratios and their 95% confidence intervals (CIs) were recorded for each factor.

EHD1 protein expression was a positive expression in examined tumor tissues (Fig. 1). The cytoplasmic staining patterns observed for EHD1 were consistent with data from our previous studies.

Correlation of EHD1 expression with the clinicopathological features of patients, including age, gender, tumor histology, tumor size, and AJCC stage in SCLC patients was assessed (Table 1). High expression of EHD1 in tumor tissues was positively correlated only with the disease type (P = 0.025). No such significant correlations between EHD1 and other clinicopathological features were found in this study.

No significant correlations between clinicopathological features and DFS, based on univariate Cox regression models, were found in this study. Multivariate Cox proportional hazards model analysis of the same set of patient data showed that EHD1 expression (P = 0.020; HR, 5.582; 95% CI, 1.314–23.72) was independent of prognostic indicators for DFS in osteosarcoma patients (Table 2).

Univariate Cox regression model analysis showed that age (P = 0.015; HR, 0.945; 95% CI, 0.903–0.989), tumor size (P = 0.042; HR, 3.155; 95% CI, 1.045–9.525), and EHD1 expression (P = 0.026; HR, 3.202; 95% CI, 1.149–8.92) were independent prognostic indicators of OS in osteosarcoma patients (Table 3). Analysis of the same set of patient data using a multivariate Cox proportional hazards model showed that tumor size (P = 0.008; HR, 5.854; 95% CI, 1.587–21.597) and EHD1 expression (P = 0.007; HR, 6.372; 95% CI, 1.645–24.676) were independent prognostic indicators of OS in osteosarcoma patients (Table 3).

Kaplan–Meier survival curves stratified for EHD1 expression are shown in Fig. 2. The median DFS time in EHD1 high-expression patients was 46.8 months (95% CI, 34.9–58.7 months). The prognosis for patients who had low expression of EHD1 in osteosarcomas was significantly better (log-rank test, P = 0.045) compared with patients who had high expression of EHD1.

Kaplan–Meier survival curves stratified for EHD1 expression are shown in Fig. 3. The median OS time for patients with high expression of EHD1 was 46.8 months (95% CI, 29.8–63.8 months), and the median OS time for patients with low expression of EHD1 was 58.8 months (95% CI, 31.6–86.0 months). The prognosis for patients with low expression of EHD1 in osteosarcomas was significantly better (log-rank test, P = 0.019) compared with patients with high expression of EHD1.