Overexpression of IFITM3 predicts the high risk of lymphatic metastatic recurrence in pN0 esophageal squamous cell carcinoma after Ivor-Lewis esophagectomy

Patients

From January 2008 to January 2010, patients with midthoracic ESCC who underwent Ivor-Lewis esophagectomy with two-field lymphadenectomy in our department (Provincial Hospital Affiliated to Shandong University, China) were eligible for this study. In total, this study enrolled 104 patients, including 83 men and 21 women, with ages ranging from 40 to 75 years (Clinicopathological data are listed in Table 1).

All patients met the following inclusion criteria: (1) According to 2009 Union for International Cancer Control (UICC) standard for midthoracic ESCC, Ivor-Lewis esophagectomy with two-field lymph node dissection was conducted to achieve complete resection (R0), and the proximal and distal incisal margins as well as lateral margin were pathologically examined without residual foci (Arai et al., 2012). At the same time, the average number of lymph nodes dissected was 18 ± 5.8 (ranging from 12 to 25); (2) Patients enrolled in the study were restaged after surgery according to TNM staging for esophageal cancer; (3) Without history of previous malignancies or other severe diseases that may influence the outcome of our follow-up; (4) Patients were not eligible if preoperative neoadjuvant chemotherapy or postoperative adjuvant treatment was administered.

Surgical procedure of Ivor-Lewis esophagectomy

Four thoracic surgeons worked together to perform this type of surgery, and the thoracic operation was performed by two surgeons. The patient was placed in the 40°–45° left lateral decubitus position. After a right anterolateral thoracotomy, the chest was entered through the fourth intercostal space. The azygos vein arch was divided, and the esophagus was dissected from the esophagogastric junction to the apex of the chest. When the tumor invasion obviously extended outside the esophagus, the thoracic duct was routinely ligated above the diaphragm.

At the same time, an upper midline abdominal incision was made by another two surgeons, and the abdomen was explored. During mobilization of the stomach, care was taken to preserve the right gastroepiploic vessels and arcades. The left gastric artery and vein were isolated and doubly ligated at their origin. Pyloroplasty was not routinely performed. Then, the hiatus was enlarged and the stomach was pulled into the chest. An endto-side esophagogastric anastomosis was performed within the apex of the chest, and the stomach was secured in the mediastinum (Chen et al., 2009a; Chen et al., 2009b).

Specimens

The ESCC tissue and ANM (more than 5 cm from the margin of ESCC) were collected from surgical specimens of each selected patient. At the same time, the ANM did not exhibit tumor infiltration, deterioration or necrosis upon macroscopic and microscopic examination.

This study was approved by the Ethics Committee of Provincial Hospital Affiliated to Shandong University, and the approval number was 2008081. Written informed consent was obtained from all the participants.

Immunohistochemistry

The streptavidin-peroxidase immunohistochemical method was used to examine the expression of the IFITM3 protein. Formalin-fixed and paraffin-embedded surgical specimens were sequentially cut into 4-µm sections. Then, the sections were dewaxed, antigen retrieval and hydrogen peroxide incubation. Rabbit anti-IFITM3 monoclonal antibodies (GeneTex, Irvine, California, USA) were used at a dilution of 1:200 and incubated at 4 °C overnight. The monoclonal primary antibody was replaced by phosphate-buffered saline (PBS) as a negative control. Further experimental steps were followed according to the instructions of a secondary biotinylated antibody kit purchased from ZSGB Biotech (Beijing, China).

The expression of the IFITM3 protein was determined according to Sakakura’s criteria. Two pathologists blinded to the clinical data were invited to evaluate the IHC sections independently. The outcome was calculated by combining the proportion with the staining intensity. The proportion was scored as follows: 0 (0–10%), 1 (11–25%), 2 (26–50%), 3 (51–75%), and 4 (75–100%). The staining intensity was scored: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The final immunohistochemical score (IHS) was defined as the proportion score × staining intensity score. In this study, IHS ≥ 8 was considered to represent overexpression.

RNA extraction and RT-PCR

Total RNA was extracted from fresh frozen tissue using Trizol (Invitrogen) according to the manufacturer’s protocols. The purity of RNA was measured by UV spectrophotometer (NanoDrop 2000) and the OD 260/280 value ranging from 1.8 to 2.0 was used to reverse transcription. The detailed RT-PCR procedure was followed by a CWBio two-step RT-PCR kit (JiangSu, China). The primer sequences of the IFITM3 gene were 5′-CAAGGAGGAGCACGAGG-3′ (forward primer) and 5′-TTGAACAGGGACCAGACG-3′ (reverse primer). β-actin was used as internal control, and the primer sequences of β-actin were 5′-AGAGCCTCGCCTTTGCCGATCC-3′ (forward primer) and 5′-ATACACCCGCTGCTCCGGGTC-3′ (reverse primer). The PCR products of IFITM3 were further separated on 1% agarose gel electrophoresis. Azure C2000 (Azure Biosystems, Dublin, California, USA) was used for electrophoresis gelatin image formation analysis.

Follow-up

According to our plan, patients were examined every 3–6 months after surgery, and the checklist was described in our previous study (Akhtar et al., 2014). We compared the imaging data preoperatively and postoperatively in detail to differentiate whether recurrence occurred or not. If the lymph nodes were swollen or the minor axis was more than 1 cm, the clinical diagnosis of lymphatic recurrence was made. Fine-needle aspiration biospy and PET-CT were also assisted to make the diagnosis of recurrence. The follow-up was ended in July 2013, and the complete 3-year follow-up data was reviewed.

Statistical analysis

The χ² test was used to analyze the relationship between IFITM3 expression and clinicopathological variables. The recurrence curves and survival curves were calculated by the Kaplan–Meier method. Univariate log-rank test and Cox regression analysis were respectively performed to evaluate the recurrence risk and prognostic factors. A satatistically significant difference was represented by a two-tailed p value less than 0.05. All statistical analyses were performed using SPSS version 17.0 (Chicago, Illinois, USA).

IFITM3 expression analysis in ESCC tissue and ANM

The immunohistochemistry assay was used to detect the expression level of the IFITM3 protein. Overexpression was presented as yellow or brownish yellow staining in the cytoplasm of the tumor cell. As is shown in Fig. 1C, the significant immunoreaction of positive expression can be readily differentiated. However, there was low or undetected staining in ANM (Fig. 1A). Furthermore, according to the criteria of IHS, we divided all the specimens into two groups: 59 cases (56.7%) were categorized as the overexpression group (Fig. 1C) and 45 cases (43.3%) were in the low expression group (Fig. 1B).

To verify this aberrant upregulation of IFITM3, we examined the mRNA expression level by RT-PCR with 20 pairs of specimens randomly selected from the overexpression group and 20 pairs of tissues that originated from the low expression group. The results showed that the mRNA expression level was consistent with protein expression as demonstrated by IHC (Fig. 2).

IFITM3 expression and clinicopathological characteristics

According to the inclusion criteria mentioned above, a total of 104 ESCC patients were enrolled in this study with different ages, genders, tumor sizes, degrees of differentiation, T status and IFITM3 expression levels (Table 1). χ² analysis demonstrated that the expression level of IFITM3 had a close relationship with T status of tumor (p = 0.015). In contrast, there were no significant differences between expression level and age, gender, tumor size and degree of differentiation (p > 0.05).

IFITM3 expression and lymphatic metastatic recurrence

Through thorough follow-up, a total of 42 cases (40.4%) were confirmed to have first lymph node metastatic recurrence within 3 years, in which IFITM3 overexpression was detected in 30 patients (71.4%). In the low IFITM3 expression group, the 3-year lymphatic recurrence rate was only 26.7%. Conversely, in the overexpression group, this rate reached up to 50.8% (Table 1). As is shown in Fig. 3, Kaplan–Meier analysis showed that the recurrence rate was significantly increased in patients with IFITM3 overexpression, and the log-rank test calculated that these two curves were significantly different (p = 0.010).

In addition to the expression level of IFITM3, T status of tumor (p = 0.008) was also elucidated to be associated with the lymphatic recurrence in pN0 ESCC (Table 1). Multivariate Cox regression analysis revealed that these two variables were independent recurrence risk factors (p < 0.05, Table 2). Patients with early T status and low expression of IFITM3 may have a lower recurrence risk of lymphatic metastasis (Fig. 3).

IFITM3 expression and overall survival

As is shown in our data, the 3-year OS rate of patients with IFITM3 protein overexpression and low expression were respectively 64.4% and 88.9%. Figure 4 demonstrated that the long survival of patients was associated with moderate-high differentiation, early T status of tumor and low IFITM3 expression. Univariate analysis and multivariate analysis revealed that these are independent and significant prognostic factors (p < 0.05, Table 3).