Abstract
Background: Despite advanced diagnostics and multimodal treatments, the overall 5-year survival rate for patients with esophageal cancer remains low. In the past, several specific antibodies, including tyrosine kinase inhibitors, targeting different steps of carcinogenesis were investigated. We examined two receptor tyrosine kinases, platelet-derived growth factor receptor (PDGFR-α) and mast/stem cell growth factor receptor (CD117) in esophageal carcinomas. Materials and Methods: Tissue samples of 52 Norwegian patients who underwent esophagectomy were examined using immunohistochemistry. Results: PDGFR-α and CD117 expression was observed in cancer cells in all samples of both carcinoma types. A higher PDGFR-α immunoreactivity was detected in the squamous cell carcinoma group (p=0.032). Surprisingly, a higher number of PDGFR-α-positive cells in the analyzed samples for the entire population was associated with longer survival (p=0.05). Conclusion: The findings of our study need to be further validated as we examined a low number of patients. Both PDGFR-α and CD117 probably play an important role in the progression of esophageal carcinoma, and they may possibly be targets for biological anticancer therapy in the future.
Esophageal cancer is turning into one of the more common types of cancer in the world today. Its incidence shows great geographical variation, but the majority of cases are diagnosed in developing countries (1, 2). The two main histological types of the disease are: adenocarcinoma (AC) and squamous cell carcinoma (SCC). In Western countries, the number of ACs and SCCs is almost equal, while in developing countries, SCC is the most prevalent type (2). Despite advanced diagnostics and multimodal treatment, the overall 5-year survival rate for patients with esophageal cancer remains low and ranges between 10 and 26% (3-5). The rising incidence of this disease and poor prognosis associated with current treatment strategies demands a search for innovative therapies.
Platelet-derived growth factor receptor-alpha (PDGFR-α) is one of two receptors for platelet-derived growth factor (PDGF) which binds to all isoforms of PDGF. PDGFR-α is a cell surface receptor tyrosine kinase, which, upon activation by PDGF, dimerizes and undergoes autophosphorylation, leading to binding of co-factors and subsequent initiation of transduction signals (6-8). The pleiotropic effect of PDGF on various cell types, and the expression of PDGFR-α in a variety of tissues, suggests that the protein plays an important role in multiple processes, including embryonic development, cell proliferation and migration, angiogenesis and tumor progression (8-10). Expression of PDGFR-α has been reported in numerous malignancies and was associated with invasive behavior (11-14).
The other receptor tyrosine kinase analyzed in the study was the CD117 protein, known as a mast- and stem cell growth factor receptor. CD117 is a transmembrane protein belonging to the type III sub-family of the receptor tyrosine kinases, consisting of an extracellular, intramembranous and intracellular domain (15, 16). By binding to its ligand, CD117 plays an important part in cell differentiation, proliferation, cell adhesion and apoptosis (17). Expression of CD117 has been described in various normal cells, e.g. hematopoietic, germ and mast cells, as well as in malignancies such as mast cell leukemia, Ewing sarcoma, neuroblastoma, Gastrointestinal Stromal Tumor (GIST), ovarian and breast cancer, as well as small-cell lung cancer (18-23).
New multi-target tyrosine kinase inhibitors are being introduced as therapies of various tumors. In our study, we examined samples of both AC and SCC using tissue material obtained from Norwegian patients after esophagectomy. Using immunohistochemistry we examined differences in expression of PDGFR-α and CD117 in both carcinoma types.
Clinicopathological features of patients with esophageal carcinoma (n=52).
Materials and Methods
Patients and specimens. Fifty-two patients (24 with SCC and 28 with AC) undergoing esophagectomy at the Department of Surgery, The Norwegian Radium Hospital, between November 1988 and November 2002 were enrolled in this study. The clinical material has been described elsewhere (24). Esophagectomy was performed through laparotomy and a right-sided thoracotomy. Subsequent reconstitution was carried-out mostly by means of esophagogastrostomy using a gastric tube through the retrosternal route. The tissue samples were then fixed in neutral buffered formalin, embedded in paraffin and stained with hematoxylin and eosin.
TNM classification was carried-out according to the Union for International Cancer Control (UICC) Global Cancer Control (25). The patients' age ranged from 38 to 87 years. The clinicopathological features of the patients are summarized in Table I.
Immunohistochemistry. PDGFR-α: The Dako EnVisionTMFLEX+ detection system (Code K8002; Dako, Carpinteria, CA, USA) was used for detection of immunostaining. Pre-treatment of the sections was performed using EnVision™ FLEX Target Retrieval Solution. After pre-treatment, the endogenous peroxidase activity was blocked for five minutes using the Peroxidase-Blocking Reagent. Sections were then incubated for 30 minutes at room temperature with primary antibody: anti-PDGFR-α (Abcam, Cambridge, UK). The sections were then rinsed twice in Dako wash buffer, incubated for 15 minutes with EnVision FLEX+Linker and incubated for 30 minutes with FLEX/Horseradish peroxidase. After being gently rinsed with Dako wash buffer, the sections were incubated for 10 minutes with 3’3-diaminobenzidine tetrahydrochloride (DAB). The sections were then counterstained with hematoxylin, dehydrated and mounted in Dako Toluene-Free Mounting Medium (Code CS705). Appropriate positive and negative controls were used and gave satisfactory results.
CD117: The Envision Plus detection system (Dako) was used for detection of immunostaining. The sections were de-paraffinized in xylene and then microwaved in 10 mm citrate buffer (pH 6.0) to unmask the epitopes. Endogenous peroxidase activity was blocked by incubation with 0.03% hydrogen peroxide in methanol for five minutes. Sections were incubated with a polyclonal rabbit antibody to human CD117 (code A4502, diluted 1:400; Dako, Glostrup, Denmark) for 30 minutes at room temperature. After being gently rinsed three times with washing buffer, the sections were incubated with peroxidase-labelled polymer conjugated to goat antirabbit IgG (Dako) for 30 minutes before being stained for five minutes with DAB, counterstained with hematoxylin, dehydrated and mounted in Diatex (A. P. Larson Co., NY, NY, USA). A known CD117-positive seminoma was used as positive control, and the same concentration of non-immune rabbit IgG was applied as a negative control. Both controls gave satisfactory results.
Semi-quantitative analysis. PDGFR-α and CD117 expression in tumor tissue was semiquantified using a visual grading system in which the staining intensity was categorized into four groups: 0+, 1+, 2+, 3+, where the 0 group is defined as having a complete absence of positive staining; groups 1-3 are defined as groups with presence of positive staining of increasing intensity compared to a positive control. The number of positive cells for PDGFR-α was categorized into four groups: 0=0% positive cells, 1=1-33%, 2=34-66%, 3 ≥66% positive cells, where group 0 is defined as having a total absence of positive cells, and groups 1-3 are defined as groups with a percentage of positive cells, as mentioned above. The number of positive cells for CD117 was categorized into two groups: 1: ≤1%, 2: >1% positive cells. This division was caused by the low number of positive cells found in tumor tissue. In order to guarantee quality control, the evaluation was performed independently by two experienced investigators who were unaware of the related clinical information before conducting statistical analyses.
Statistical analysis. Associations between PDGFR-α and CD117 expression and various clinicopathological features were assessed using the Pearson's Chi-square test and the Fischer's exact test as required. The Kaplan–Meier product-limit was applied to calculate the curves of survival. Differences between groups were analyzed using the log-rank test. A two-sided p-value of less than 0.05 was considered statistically significant. Statistical analyses were performed using the Statistical Package for the Social Science program, version 18.0 (SPSS, Chicago, IL, USA).
Results
Immunostaining of PDGFR-α. The entire patient population: There was a statistically significant correlation between the number of cells expressing PDGFR-α and the histological type of esophageal carcinoma and sex. We found more PDGFR-α-positive cells in the SCC (p=0.032) and the male group (p=0.035). There was no correlation between PDGFR-α expression, including both intensity and number of positive cells, and T, N, M, histological grade or local recurrence.
AC: We observed a correlation between the number of positive cells and patients' age. Older patients had more PDGFR-α-positive cells (p=0.006). No other correlations between PDGFR-α expression and clinicopathological features were observed in this group.
SCC: There was no correlation between PDGFR-α expression, including both intensity and number of positive cells and T, N, M, histological grade or local recurrence in the SCC group.
Immunostaining of CD117. The entire patient population: We found a statistically significant correlation between the number of cells expressing CD117 and sex, demonstrating an increased number of positive cells in the female group (p=0.032). Otherwise, we did not find any correlation between CD117 expression and T, N, M, histological grade or local recurrence.
AC and SCC: There were no other correlations between CD117 expression and clinicopathological features in the carcinoma groups separately. PDGFR-α and CD117 expression in both types of esophageal carcinoma, including intensity and number of positive cells, is summarized in Table II.
Cellular pattern of immunostaining. All samples analyzed in this study were positive for PDGFR-α. A weak intensity was observed in 12 samples of AC and 11 samples of SCC. A middle intensity was observed in 14 and 12 of the AC and SCC samples respectively. Only two cases of AC and one case of SCC presented strong staining. As for the number of PDGFR-α-positive cells in the AC group, groups 1-3 were pictured as follows: 12, 11 and five samples respectively. In the SCC group, groups 1-3 presented the following number of positive cases: four, seven and 13 respectively. We found CD117-positive cells in all analyzed cases, but in general, the number of positive cells was low. In the group with low number of positive cells (≤1%), we found 11 cases of AC and 17 cases of SCC. The group with the number of positive cells >1% included 17 and seven cases of AC and SCC respectively (Table I). PDGFR-α expression was observed in the cytoplasm and the nuclear membrane of cancer cells. CD117 showed cytoplasmic and cancer cell membrane immunostaining (Figure 1). In addition, PDGFR-α and CD117 expression was also observed in some cases in both carcinoma types in tumor surrounding stromal cells and pericytes.
Correlation with the patient survival. Analysis of PDGFR-α expression in the whole population in relation to survival (log-rank test) revealed that survival was longer in the group with a high number of PDGFR-α-positive cells (>66%) than the group with a low number of positive cells (1-33%) (p=0.05) (Figure 2A). The median survival rates were 38 months and 22 months, respectively. The 5-year survival rate for the groups was 20% and 6.5%, respectively. No further correlation between the staining intensity for PDGFR-α or number of CD117-positive cells and survival of the patients was found in our study (Figure 2B and C).
Platelet-derived growth factor receptor-α and CD117 immunostaining in esophageal adenocarcinoma (n=28) and squamous cell carcinoma (n=24).
Discussion
The epidemiology of esophageal cancer has significantly changed, especially in Western countries. During the last decade, AC has almost replaced SCC as the predominant tumor entity in the esophagus (2). Despite advanced diagnostic methods and multimodal treatments suitable for both histological types, the prognosis of the disease still remains poor (1). Recently, however, the role of biological anticancer therapy has emerged, which seems to be a promising treatment option for various malignancies. However, data on such promising new therapies in esophageal carcinomas are rare. Biological anticancer therapy, for example, employs tyrosine kinase inhibitors, whose use considerably enriches the standard treatment.
With the present study, we aimed to investigate the expression of two receptor tyrosine kinases in esophageal carcinoma tissue using immunohistochemistry. Overexpression of both proteins has previously been described and related to tumor progression in various malignancies (11, 13, 14, 18-21). We examined tissue samples with both AC and SCC obtained from Norwegian patients after esophagectomy. Despite an annual increase in the number of diagnosed cases, the incidence of esophageal cancer in Norway is still low, as is the number of patients undergoing surgical treatment. Thus, we are aware of the limitations of the present study, since we examined a relatively small group of patients.
Immunoreactivity for platelet-derived growth factor receptor-α (A, B) and CD117 (C, D) in esophageal adenocarcinoma (A, C) and squamous cell carcinoma (B, D).
We observed a significant correlation between the number of cells expressing PDGFR-α and the histological type of carcinoma, showing a higher number of PDGFR-α-positive cells in the SCC group. This finding is consistent with Gockel et al.'s studies demonstrating a PDGFR-α expression rate of 91% and 100% for AC and SCC, respectively (26). This observation may lead to an assumption that on the molecular level, esophageal SCC is a more aggressive form of malignancy than AC, taking into consideration that PDGFR-α overexpression is related to tumor-associated angiogenesis and tumor progression. In fact, patients with AC are generally thought to have slightly better prognosis than patients with SCC. However, this conclusion is mostly based on other clinical data such as a possible early diagnosis, which is favorable for patients with AC, a higher perioperative risk and postoperative mortality for patients with SCC caused by extensive transthoracic surgery, and increased presence of secondary carcinoma in patients with SCC (27, 28).
Cumulative survival analyses by the Kaplan–Meier method according to platelet-derived growth factor receptor-α (PDGFR-α) positivity (p=0.05; A), PDGFR-α intensity (p>0.05; B) and CD117 positivity (p>0.05; C).
While analyzing CD117 expression in esophageal carcinomas, we did not find any spectacular results except for a correlation between the number of cells expressing the protein and sex, showing an increased number of CD117-positive cells in the female group. However, other studies, including a previous study from our Center, demonstrated that overexpression of CD117 in carcinomas is associated with impeded apoptosis, facilitating tumor invasion and metastasis and thus resulting in poor prognosis (29, 30). We are aware that this lack of results in this field of our study may be caused by the low number of included patients, which is a considerable limitation as mentioned before.
All samples analyzed in this study were positive for both PDGFR-α and CD117, demonstrating the proteins' expression in cancer cells, but also in tumor stroma and pericytes in certain cases. We observed cytoplasmic immunoreactivity for both proteins as well as nuclear and cell membranous immunostaining for PDGFR-α and CD117 respectively. This finding confirms the well-known cellular localizations of these proteins, which are crucial for their function (6, 29). Tumor progression is considered to be a consequence of a complex, dysregulated molecular process. Receptor tyrosine kinases containing extracellular ligand-binding domains as well as intracellular catalytic domains contribute to this process through their signaling pathways, including e.g. the threonine-specific protein kinase and mitogen-activated protein kinase cascades (13, 31). Constitutive activation of the receptor tyrosine kinases, either by gene alteration or defective down-regulation, may enhance tumor progression. The presence of proteins in all analyzed samples confirms their important role in the progression of both carcinoma types at all stages of their cell differentiation.
Numerous previous studies have demonstrated that tumors with PDGFR-α overexpression detected by immunochemistry had an unfavorable outcome (11, 12). Surprisingly, in our study, the analysis of PDGFR-α expression and patient survival in the entire population group revealed the unexpected clear tendency (p=0.05) that the group with the higher number of PDGFR-α-positive cells was associated with longer survival. Similar findings have been described in only a few studies: studies investigating PDGFR-α expression in astrocytomas and Kern et al.'s study investigating the effect of mutations and expression of tyrosine-protein kinase Kit (KIT) and PDGFR-α in GISTs (32-35). It is not really clear why the well-known tumor progression-promoting protein may be a favorable factor in these carcinoma types, but a possible explanation may be an intratumoral heterogeneity in astrocytomas and site-dependent clinical behavior of GISTs. In our study, we analyzed two histologically different tumor types, and the obtained result may be associated with the essential histological difference. However, this explanation should be interpreted with caution. We did not observe any association between CD117 expression in cancer cells and survival.
To summarize, several specific antibodies, including tyrosine kinase inhibitors, targeting various crucial steps of carcinogenesis have recently been investigated. We examined two receptor tyrosine kinases, PDGFR-α and CD117, in esophageal carcinomas. Both proteins were expressed in cancer cells of all carcinoma samples; however, a higher PDGFR-α immunoreactivity was detected in the SCC group. Surprisingly, we observed a clear tendency: a higher number of PDGFR-α-positive cells in the analyzed samples was associated with longer survival. These findings need to be validated in future studies. Furthermore, further research of PDGFR-α and CD117 is warranted to explore their potential use in prognostic prediction and targeted therapy for esophageal carcinoma.
Acknowledgements
The Authors would like to thank the technicians working with immunohistochemistry at the Division of Pathology, The Norwegian Radium Hospital. The Authors thank Alexandra Goscinski-Bloesch for the linguistic revision of the text. This work was supported by funding from the Norwegian Cancer Society. The study was approved by the Regional Ethical Committee.
- Received March 5, 2015.
- Revision received April 5, 2015.
- Accepted April 5, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
