Abstract
Introduction
Eukaryotic initiation factor 4 E (eIF4E) is known to play an important role in protein synthesis. 1 It is also overexpressed in a number of cancers, including breast,2,3 colon 4 and bladder carcinoma,5,6 suggesting that it is the product of a proto-oncogene and has potential as a tumour marker.2–5 In the present study, expression of eIF4E in hypopharyngeal carcinoma was assessed qualitatively and quantitatively using immunohistochemistry and Western blotting, and the relationships between eIF4E expression and tumour–node–metastasis stage, histological differentiation and the initial or recurrent status of the tumour were investigated.
Patients and methods
Patients and samples
Specimens from hypopharyngeal carcinomas and benign hypopharyngeal lesions were obtained retrospectively from consecutive patients who underwent surgery at the Department of Otolaryngology, Affiliated Hospital of Qingdao University, Qingdao, China, between June 2008 and October 2012. There were no further inclusion or exclusion criteria. The study protocol was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University and all study participants gave verbal informed consent.
Each specimen was divided in two, with one half being were frozen at −80℃ within 15 min of removal (for use in Western blotting) and the other half being preserved in 10% formaldehyde before being embedded in paraffin wax and cut into4-µm thick sections) for use in immunohistochemical staining.
Tumours were classified using the tumour–node–metastasis (TNM) staging system. 7 The tumour specimens were examined pathologically to confirm the diagnosis of hypopharyngeal carcinoma and assess the degree of histological differentiation. Specimens were classified as highly, moderately or poorly differentiated on the basis of a comprehensive analysis of the tissue structure and cell atypia under light microscopy. Expression of eIF4E in samples from all the study participants was analysed using immunohistochemical staining and Western blotting, described below.
Immunohistochemical staining
Paraffin wax-embedded sections were deparaffinizedwith 100% xylene and ethyl alcohol; endogenous enzyme was inactivated by treatment with 3% hydrogen peroxide for 5–10 min. After heat-induced antigen retrieval by heating to boiling point twice in 0.01 M citrate buffer solution (pH 6.0), sections were blocked using 5% rabbit serum for 20 min at room temperature. Immunohistochemical staining was performed using a streptavidin–biotin complex immunohistochemistry kit (Boshide Biotechnology, Wuhan, China). Briefly, sections were incubated overnight at 4℃ with 1 : 50 diluted goat antihuman eIF4E polyclonal antibody (clone N-19, SC-6967; Santa Cruz Biotechnology, Santa Cruz, CA, USA), then washed three times in phosphate-buffered saline (PBS; pH 7.4) Sections were then treated with biotinylated rabbit antigoat immunoglobulin (Boshide Biotechnology) for 20 min at room temperature and washed three times in PBS, followed by incubation with streptavidin–biotin–peroxidase complex for 20 min at room temperature. Immunostaining was visualized using a DAB kit (Boshide Biotechnology). Sections were incubated with fresh 3,3′-diaminobenzidine (DAB) for 5–15 min at room temperature, counterstained with haematoxylin and mounted in neutral gum.
Specimens were viewed using a light microscope; positive expression of eIF4E was seen as pale-brown staining in the cytoplasm and around the nucleus. The level of expression was assessed using the semiquantitative method of Pavelic et al, 8 in which the staining strength is multiplied by the percentage of positive cells, to give an eIF4E score. Staining strength was classified as colourless (scoring 0), weak (scoring 100), moderate (scoring 200) or strong (scoring 300). To calculate the percentage of positive cells, the most uniformly stained area with the most positive cells visible at low magnification was chosen. Within this area, five high-power fields were chosen and 100 cells counted within each field. The mean number of positive cells was calculated to give the overall percentage of positive cells.
Positive (known to express eIF4E) and negative (PBS used instead of eIF4E antibody) control sections were also prepared and examined for comparison.
Western blot
Different concentrations of standard bovine serum albumin (0, 0.125, 0.25, 0.5, 0.75 and 1 mg/ml) were diluted to 40 µl using double-distilled water. A bicinchoninic acid (BCA) protein testing kit (Pierce Biotechnology, Rockford, IL, USA) and ultraviolet light spectrophotometer absorbance at 562 nm were used to create a standard curve for the determination of protein concentration. A 20-mg sample from each frozen tissue specimen was homogenized in 1 ml Tris-acetate storage buffer (pH 8.0; Boshide Biotechnology), centrifuged at 9000 r.p.m. (4℃) for 20 min (SIGMA® 1-15 K microfuge, Sigma–Aldrich, St Louis, MO, USA) and the supernatant was deposited in an Eppendorf tube and stored at −80℃. Then, 3 µl of each supernatant sample was diluted to 40 µl in double-distilled water. The protein concentration in each sample was then measured using the BCA kit and compared with the standard curve; the mean of two measurements was calculated for each sample.
Western blotting was performed using a kit provided by Boshide Biotechnology and the proteins were separated by gel electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA) using a 12% separation gel and a 5% stacking gel. Specimen samples were diluted to a concentration of 5 µg/ml using protein loading buffer (2 × Tris-acetate loading buffer, pH 6.8; Boshide Biotechnology) and heated in a water bath at 100℃ for 5 min to denature the protein. Standard protein markers (20.1 kDa and 31.0 kDa; Xibas Biotechnology, Shanghai, China) were treated in the same way. The specimen samples, standard protein markers and eIF4E cell culture protein extract (Bio-Rad Laboratories) as an internal reference were loaded into wells and a constant current of 20 mA was applied for 60 min. Separated proteins were then transferred to nitrocellulose membranes using a voltage of 100 mV applied for 100 min. Mmembranes were incubated with Ponceau S (Boshide Biotechnology) for 5–10 min to locate the standard protein band. Immunoreactions and DAB staining (Boshide Biotechnology) were performed on the nitrocellulose membranes to show the specimen bands. Briefly, membranes were incubated overnight at 4℃ with 1 : 300 diluted goat antihuman eIF4E polyclonal antibody (clone N-19, SC-6967; Santa Cruz Biotechnology). Membranes were then washed three times with PBS-Tween (PBST; pH 7.2), treated with 1 : 400 diluted biotinylated rabbit antigoat immunoglobulin (Boshide Biotechnology) for 1 h at 37℃, then washed four times with PBST. Immunostaining was visualized using a DAB kit (Boshide Biotechnology). Membranes were incubated with fresh DAB for 5 min at room temperature. The integrated optical density (IOD) of each band was measured using ImageTool software, version 3.0 (UT Health Science Center, San Antonio, TX, USA).
Statistical analyses
Results were expressed as mean ± SD. One-way analysis of variance was used to compare eIF4E scores and IOD values in different patient groups. A
Results
Relationships between eukaryotic initiation factor 4 E (eIF4E) score or eIF4E integrated optical density (IOD) and various clinicopathological parameters in patients with hypopharyngeal carcinoma (
Data presented as mean ± SD. *
Expression of eIF4E on immunohistochemical staining
Pale-brown staining was widely seen in the cytoplasm of hypopharyngeal carcinoma cells (Figure 1a]. Under the light microscope, three to five layers of eIF4E-positive cells were seen, spread as a band in the stratum basale and acanthocyte layers. Staining was also seen around the keratin pearl and more rarely in horn pearls and central parakeratotic cells. In the malignant tumours with higher TNM classifications, heavily stained cells were spread more widely, compared with other tumours. The most strongly stained cells were seen at the border of carcinomatous and normal tissue. In benign lesions, staining was absent or minimal (Figure 1b).
Immunohistochemical staining of eukaryotic initiation factor 4 E in (a) hypopharyngeal carcinoma tissue and (b) benign hypopharyngeal tissue samples. The colour version of this figure is available at: http://imr.sagepub.com.
In the carcinoma specimens, eIF4E scores ranged between 28.7 and 291.60, with a mean ± SD of 141.26 ± 18.73. However, in the benign lesions, eIF4E scores ranged between 0 and 13.2, with a mean ± SD of 3.96 ± 1.28. The difference in eIF4E score between these two groups was statistically significant (
Expression of eIF4E on Western blotting
Hypopharyngeal carcinoma specimens showed an electrophoretic band stained yellow/brown at the 25.0 kDa location (Figure 2). IOD values ranged between 4.21 and 16.09 kg/m3. Specimens from benign lesions showed no or weaker staining at the 25.0 kDa location, and had IOD values ranging between 0.63 and 4.15 kg/m3. The difference in IOD values between these two groups was statistically significant ( Eukaryotic initiation factor 4 E (eIF4E) expression demonstrated using Western blotting with 3,3′-diaminobenzidine staining, showing eIF4E protein as internal reference (A), benign hypopharyngeal tissue (B), hypopharyngeal carcinoma tissue (C1–C6) and standard protein marker (M). The colour version of this figure is available at: http://imr.sagepub.com.
Relationships between eIF4E expression and clinicopathological characteristics
Expression of eIF4E was compared in patients with hypopharyngeal carcinoma according to various clinicopathological criteria. Using the TNM classification, patients were divided into a T1 + T2 group and a T3 + T4 group, and also into a N0 group and an N1 + N2 + N3 group. In addition, they were divided into a poorly differentiated group, a moderately differentiated group and a well-differentiated group on the basis of pathological appearance (Table 1).
The eIF4E score and IOD value were both significantly associated with tumour stage, lymph node metastasis and degree of differentiation (
Discussion
Tumour development is a multistage, multistep process; the development of malignant tumours can be considered as a mutation of cells within an abnormal environment.9,10 The effect of the eIF4E gene as a proto-oncogene in head and neck neoplasm has not been extensively studied. Nathan et al. 11 reported the overexpression of eIF4E in head and neck squamous cell carcinoma, and proposed that the probability of recurrence was associated with the expression of eIF4E at the edge of the incised specimen. eIF4E may have a role as the product of a proto-oncogene and as a tumour marker, and expression of eIF4E may be helpful in predicting prognosis and choosing appropriate therapeutic schedules.12,13
Since considerable protein synthesis occurs during the development of a solid tumour, expression of eIF4E is likely to be of importance. Overexpression of eIF4E can promote the expression of growth factors and cell growth-regulating factors, which are known to be closely related to tumour formation. 14 In the present study, increased expression of eIF4E in hypopharyngeal carcinoma, with a significantly higher eIF4E score and IOD value in tumours compared with benign lesions, suggests that eIF4E plays an important role in hypopharyngeal carcinoma. This is consistent with the conclusions of Menezes et al. 15 On the basis that change at the molecular level occurs before change at the cellular level, 16 eIF4E could be considered as a sensitive molecular marker. For example, the detection of overexpression of eIF4E in intraoperative specimens could be used to indicate the need for an extended radical resection and rigorous follow-up. In addition, staining for eIF4E may enable a diagnosis to be made, even if the tissue sample is very small or extruded.
Both the TNMstage and the degree of differentiation reflect the severity of the malignancy and are important in choosing appropriate therapy. In some cases it is difficult to determine both the TMN stage and the degree of differentiation on the basis of clinical diagnosis and pathological microscopic examination alone.17,18 In the present study, the eIF4E score and IOD were significantly associated with tumour stage and degree of differentiation in hypopharyngeal carcinoma, and therefore we consider that eIF4E expression could be used as a sensitive marker for tumour severity.
There were some limitations to the present study. For example, the number of cases studied was relatively small; analysis of a larger number of cases may have provided clearer results. In addition, the present study was undertaken retrospectively. In future it would be better to conduct a prospective study, in order to define more accurately the prognostic implications of eIF4E scores in hypopharyngeal carcinoma.
Lymph node metastasis is a gold standard in judging the prognosis of various tumours.19–21 In the present study, eIF4E scores and IOD were significantly higher in patients with hypopharyngeal carcinoma who had lymph node metastasis than in those with no lymphatic metastasis, suggesting that those with a high eIF4E score or IOD have a higher risk of tumour recurrence. Indicators of eIF4E expression could therefore be used to help predict prognosis and inform therapeutic choices, thereby improving patient survival and health-related quality of life. Although immunohistochemistry can give a semiquantitative indication of eIF4E expression, Western blotting provides a more objective quantitative assessment of the level of eIF4E protein expression.
In conclusion, eIF4E may play an important role in the development and metastasis of hypopharyngeal carcinoma. eIF4E expression, ideally assessed via Western blotting, may be helpful in establishing the diagnosis, stage and prognosis of this tumour type.