Epidermal growth factor receptor expression and adjuvant chemoradiotherapy in rectal cancer

Abstract

AimEpidermal Growth Factor Receptor (EGFR) is a trans-membrane protein with tyrosine kinase activity and is expressed in 25-80% of colon cancer cases. EFGR expression is prognostic in patients with metastatic colorectal cancer, and anti-EGR- based therapies are routinely used in the treatment of patients with metastatic colorectal cancer. To the best of our knowledge, the relationship between EGFR expression and prognosis in directly operated patients who did not receive neoadjuvant treatment and subsequently received chemo-radiotherapy is unknown. Therefore, we retrospectively evaluated patients with stage 3 rectal cancer who underwent surgery without any preoperative treatment in our center and aimed to investigate the relationship between EGFR expression and prognosis in patients who received adjuvant chemoradiotherapy.MethodsThe data of patients who underwent surgery for rectal cancer and received chemoradiotherapy between 2010 and 2016 at Manisa State Hospital were retrospectively analyzed.ResultsAccording to EGFR expression, it was 127.01 (95% CI, 85.43-168.59) months in the group with 10% less staining and 47.44 (95% CI, 26.77-68.12) months in the group with 10% or more staining. Lymphovascular invasion (p=0.032), perineural invasion (p=0.023), histologic grade (p=0.004) and EGFR expression percentage (p=0.005) were significantly associated with survival in multivariate analysesConclusionThe presence of 10% or more EGFR expression, LVI, PNI, and histological grade are significantly associated with survival in stage 3 rectal cancer patients who have undergone surgery and received postoperative chemotherapy. These markers can be used as prognostic biomarkers in the follow-up and treatment of these patients.

Keywords

Introduction

According to the Global Cancer Observatory (GLOBACAN), colorectal cancer is the 4th most common cancer worldwide and ranks 3rd in terms of mortality. Mortality rates are gradually decreasing in parallel with successful developments in screening programs and treatment.¹ Similar to these developments, oncological statistics for 2017 also defined a decrease in incidence and mortality rates.²
Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein with tyrosine kinase activity and is expressed in 25-80% of colon cancer cases.³ EGFR is known to be more highly expressed in left colon tumors, and its expression has been reported to be associated with cancer cell proliferation, apoptosis, angiogenesis, tumor invasion, and distant metastasis.⁴ Therapies targeting EGFR in metastatic patients are currently used in daily practice in lung, colon, and head and neck cancers.
Currently, neoadjuvant radiochemotherapy is recommended as the standard treatment for patients with clinical > T3 or lymph node-positive rectal cancer.⁵ However, in addition to prejudice against radiotherapy, fear and anxiety about disease progression during the neoadjuvant treatment period, surgery may sometimes be preferred as the first treatment method in rectal cancer cases due to patient preference, the presence of clinical symptoms, or urgent conditions such as ileus or bleeding, or due to differences between radiological stage and actual pathological stage.
To the best of our knowledge, the relationship between EGFR expression and prognosis in directly operated patients who did not receive neoadjuvant treatment and subsequently received chemoradiotherapy is unknown. Therefore, we retrospectively evaluated patients with stage 3 rectal cancer who underwent surgery without any preoperative treatment in our center and aimed to investigate the relationship between EGFR expression and prognosis in patients who received adjuvant radiotherapy/radiochemotherapy.

Materials and Methods

Study PopulationBetween 2010 and 2016, n = 89 patients diagnosed with “Adenocarcinoma of the Rectum” at Manisa State Hospital were retrospectively reevaluated. Among these patients, 50 who received neoadjuvant treatment were excluded, and 39 patients with stage 3 who received postoperative adjuvant treatment were included in the study.Data CollectionMorphological and clinical prognostic parameters (age, gender, tumor location, tumor size, histological grade of tumor, postoperative TNM stage, perineural invasion [PNI], lymphovascular invasion [LVI], EGFR staining status and severity) were evaluated. The relationship between defined prognostic findings and survival (disease-free survival, overall survival, recurrence, metastasis development, serum CEA, CA19-9 levels at the time of diagnosis) values and EGFR expressions were retrospectively analyzed.
EGFR expression was investigated by immunohistochemistry in rectal adenocarcinoma patients who received adjuvant treatment and evaluated in comparison with morphologic, clinical prognostic parameters, and survival time. Overall survival was defined as the time from the date of diagnosis until death. Disease-free survival was defined as postoperative disease progression or death due to any cause.
The hematoxylin-eosin stained slides of the cases were removed from the archive and re-evaluated, and blank sections were prepared for immunohistochemical examination from the blocks of the selected appropriate slides (sufficient tumor in the biopsy and/or surgical material). Immunohistochemistry was performed automatically on 3–5 micron thick sections prepared from formalin-fixed paraffin blocks of the cases using EGFR antibody on a Leica BondMax immunohistochemistry device. EGFR (clone: EGFR.113, Leica-Novocastra, 1:20 dilution) primary antibodies were investigated by immunohistochemistry. The severity and intensity of positive staining were evaluated. Membranous and/or cytoplasmic staining was considered positive. EGFR staining intensity was interpreted semi-quantitatively (staining intensity +1, +2, +3 positive; staining intensity “negative”, “less than 10% positive”, “10%-49% positive”, “50% or more positive”). EGFR staining and evaluation were performed on the resection material.Ethical ApprovalThe study was conducted in accordance with the principles of the Declaration of Helsinki and reviewed and approved by the Health Sciences Ethics Committee of Ege University (reference number 70198063-0500.06.04, identification number 16-12.1/1, Date: 27.01.2017).Ethical ApprovalEthics Committee approval for the study was obtained.Statistical AnalysisDescriptive statistics were presented as mean, standard deviation, median, minimum–maximum values for numerical variables and as numbers and percentages for categorical variables. Survival analyses were performed using the Kaplan-Meier method. Factors affecting survival were examined using Cox regression. p<0.05 was considered significant in all statistical analyses.

Results

Thirty-nine patients were included in this study. The pathology blocks of 11 patients were excluded due to deterioration from poor storage conditions, and the remaining n = 28 patients were analyzed. Of these, 13 (46.4%) were female and 15 (53.6%) were male. The mean age of the patients was 60.93 ± 9.86 years (Table 1).
All patients received oral capecitabine chemotherapy concurrently with adjuvant radiotherapy, followed by 6 months of capecitabine–oxaliplatin. At the time of evaluation, the median follow-up period was 60 (18.06-168.59) months. During follow-up, 2 (7.14%) patients developed local recurrence, 5 (17.86%) developed liver metastasis, 4 (14.29%) developed lung metastasis, 2 (7.14%) developed lymph node metastasis, and 1 (3.57%) developed brain metastasis. EGFR expression was < 10% in 11 (29.95%) patients and ≥ 10% in 17 (71.05%) patients.
Overall survival was defined as 59.63 (95% CI, 18.08-108.38) months in the group with ≥ 10% staining. According to EGFR expression, survival was 127.01 (95% CI, 85.43-168.59) months in the group with < 10% staining and 47.44 (95% CI, 26.77-68.12) months in the group with ≥ 10% staining.
In univariate analyses, survival was significantly associated with histologic grade (p=0.006), N1 stage (p=0.036), N2 stage (p=0.002), LVI (p=0.012), PNI (p=0.011), and EGFR expression percentage (p=0.009). In multivariate analyses, LVI (p=0.032), PNI (p=0.023), histologic grade (p=0.004), and EGFR expression percentage (p=0.005) were significantly associated with survival (Table 2, Figure 1).

Discussion

When we look at studies conducted on patients receiving radiotherapy in rectal cancer, since preoperative radiochemotherapy or radiotherapy is the standard treatment in locally advanced disease, most studies include patients receiving preoperative treatment. However, in daily practice, patients may sometimes be operated directly for various reasons and then receive adjuvant radiotherapy. We conducted this study to examine the relationship between EGFR expression and prognosis in this group. In our study, we found a relationship between EGFR expression and the prognosis of patients with stage 3 rectal cancer who received adjuvant chemoradiotherapy.
Studies began with the identification of epidermal growth factor (EGF) as a protein in baby mice by Staley Cohen in 1962, followed by the identification of the EGFR receptor in 1975, and the determination of increased phosphorylation and tyrosine kinase activity when squamous cell carcinoma cells bind to EGF in the 1980s. EGFR is a transmembrane protein with tyrosine kinase activity and has been investigated in many cancers. Its overexpression has been reported to be associated with proliferation, apoptosis, angiogenesis, tumor invasion, and distant metastasis in many tumors.^4,6 Anti-EGFR-based therapies are widely used in metastatic colon, lung, and head and neck cancers.
Studies have reported that patients with EGFR overexpression have more advanced-stage disease, worse histologic grade, and lymphovascular invasion.⁷ Longer survival times are observed with treatments including anti-EGFR therapy compared to those without.^8,9 While EGFR expression was defined between 25-80% in colon cancers in different studies, this rate was reported to be around 50-60% in rectal cancers.¹⁰ Despite observed EGFR expression, some studies found EGFR expression associated with prognosis in colon cancer independent of tumor stage, while others found no association.^10-11-12 Differences in results have been attributed to staining evaluation difficulties, kit differences, center differences, and retrospective pathology sample issues.¹⁰ In our study, staining was not detected in 28% of patients due to storage conditions of old preparations and tissue loss during sectioning.
Huang et al. studied non-metastatic patients who were operated and received adjuvant chemotherapy, and found a correlation between EGFR expression and prognosis in stage 3 colorectal cancers. They reported EGFR expression as a negative predictive factor for disease recurrence.¹¹
Previous studies have reported that EGFR expression activates intracellular communication pathways, causing malignant transformation and tumor progression through increased cell proliferation, long-term survival, angiogenesis, antiapoptosis, invasion, and metastasis. In radiotherapy studies, EGFR overexpression was associated with resistant tumors, more local recurrence, lower stage, and worse prognosis.^{3,7-8-9-10-11-12-13-14-15}
Although the standard treatment for stage III rectal cancer is neoadjuvant radiochemotherapy/radiotherapy, stage III patients are rarely referred for postoperative adjuvant treatment in some cases. In our study, patients with EGFR expression ≥ 10% had shorter survival times, and this group had a poor prognosis similar to patients who received preoperative radiochemotherapy/radiotherapy.
We also confirmed the poor relationship between LVI and PNI and prognosis in accordance with the literature.^{16-17-18-19-20-21} However, the lack of a relationship with T, N stage, and histologic grade, which are normally associated with prognosis in colorectal cancers, may be due to the small number of cases and the small number of patients in each group (for example, only 3 patients with grade 1).
The retrospective nature of our study and the small number of patients constitute its weaknesses. However, despite these limitations, we believe it contributes to the deficiency in the literature. Our study is important because it is the first to correlate EGFR expression with prognosis in rectal cancer patients who received postoperative radiotherapy, filling a gap in the literature.

Conclusion

According to our study, the presence of EGFR expression of 10% or more, LVI, PNI and histologic grade are significantly associated with survival in operated stage 3 rectal cancer patients. These markers can be used as prognostic biomarkers in the follow-up and treatment of these patients.

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