Prognostic impact of epidermal growth factor receptor (EGFR) expression on loco-regional recurrence after preoperative radiotherapy in rectal cancer

In patients with rectal carcinoma, pelvic recurrence is a major source of morbidity and mortality. Despite improvements in surgical approaches, local recurrence may occur in up to 30% of patients treated with surgery including total mesorectal excision [1]. Since 2001, the Dutch Colorectal Cancer Group Trial [2] has confirmed that a short course of radiotherapy (RT) reduced the rate of pelvic recurrence at 3 years, from 10.1% to 3.4%. In addition, a meta-analysis of 19 randomized trials including preoperative RT tends to show that it provides a gain of three percent at 5 years in overall survival [3]. However despite these recent intensive clinical investigations, there is still a need to develop novel strategies in the management of patients with locally advanced rectal cancer.

Advances in the understanding of the molecular biology of rectal cancer have opened many new research directions. Increasing effort has been directed towards developing molecular targeted therapies or searching for molecular markers that are useful either in predicting treatment outcome or in selecting patients for specific molecular targeted therapies, based on particular tumor characteristics. None of the recent studies has identified convincing data to warrant routine clinical application of any marker such as p53 [4, 5], or apoptosis regulators [6].

To date, no data have become available that shed light on the impact of EGFR expression on local and distant relapse in patients treated with preoperative RT and extensive local surgery i.e. abdominoperineal excision or low anterior resection with total mesorectal excision. We present here the prognostic impact of EGFR expression on locoregional recurrence in 77 patients treated with preoperative RT at our institute.

The identification of parameters that reflect biological behavior of individual cancer tissues correlating with tumor aggressiveness is a key determinant of prognosis and a fundamental issue for the improvement of cancer therapy. Despite recent progress in defining the molecular mechanisms of cancer development and tumor progression, only a few individual biomarkers providing prognostic information have been identified. Among them, the EGFR pathways attracted the most attention of cancer investigators.

EGFR (HER1), a transmembrane glycoprotein, is a member of the large receptor tyrosine family encoded by a gene located in human chromosome 7p12. EGFR exists in inactive monomer form or in active dimer form. Dimerization can take place between different receptors in order to develop homologue (homodimers) or heterologue (heterodimers) dimers [9]. In either normal or malignant cells, the activation of EGF receptor cascades may have multiple consequences such as cell growth, differentiation, and proliferation. EGF receptor cascades may also promote malignant transformation, angiogenesis, and/or metastatic dissemination [10, 11].

In addition, the cell membrane has been known for some time to be a secondary target for ionizing radiation. This phenomenon may provoke the pathways of mitogen-activated protein kinase (MAPK), phosphatidyl inositol-3-phosphate kinase (PI3K), and MAPK8 activation [12], which can modulate cell proliferation or death. Preclinical and clinical studies associate EGFR expression with radioresistance [13–16]. Ionizing radiation produces several types of cellular response via activation of multiple transduction pathways resulting in cell death, differentiation, or proliferation. Following irradiation, the MAPK pathway was recently reported to be a cellular "SOS" signal initiator starting from EGF receptors [17]. MAPK pathway activation via EGFR receptors was reported in many malignant human cell lines [17–19]. This activation is similar to the one produced by physiological concentrations of EGF (0.1 nM), and seems to act as a radioprotector [16, 17, 19, 20]. Moreover, it has been recently shown that EGF-receptor and MAPK signal pathway activation following ionizing radiation depends on the proteolytic clivage of TGFβ precursor and functional activation of autocrine TGFβ [21]. STAT-3 signal pathway activation by phosphorylation via EGF receptors can be initiated by ionizing radiation, and it results with a radioprotective effect by apoptosis inhibition [22–24]. An inverse relation between the number of EGF receptors and tumor radiocurability is reported in several murine cell lines. In these models, radiation-induced apoptosis was decreased when important levels of EGF receptor were expressed on the cells [25, 26]. Clinical consequences of these findings would be tailoring treatment according to a simple predictive assay of radiosensitivity based on the EGF-receptor expression. Clinical data pertaining to the relationship between EGFR expression and the success of radiotherapy are sparse and equivocal. Nevertheless, with respect to squamous-cell cancer of the head and neck, EGFR is among the best-studied examples [27–33], and positive and negative correlations between EGFR levels and tumor recurrences were reported in laryngeal cancers after radiotherapy [34–36]. The relationship of EGFR levels to the prognosis in unresectable pharyngeal or nasopharyngeal cancer patients treated by chemo-radiotherapy was recently reported [37–39].

In colorectal cancer, EGFR expression was evaluated in resected tumors [40]. The authors found significantly higher EGFR levels in stage III cancers than in stages I and II. It was then concluded that high EGFR expression is associated with poor prognosis. Another group [41], found 72 cases of EGFR-positive expression in 82 resected colorectal adenocarcinomas (88%). The extent of EGFR expression (>50%) revealed significant differences in survival times. In our study, a significant correlation between the positive tumor cell percentage greater than 25% and the rate of locoregional recurrence was detected (P = 0.037).

We did not assess the predictive value of EGFR on tumor response after preoperative treatment. This question was recently tackled by Giralt et al [42]. The authors analyzed EGFR expression of 45 locally advanced-rectal-cancer patients treated with preoperative radiotherapy and total mesorectal resection. Immunochemistry for EGFR was determined at the preradiation diagnostic biopsy and in the resected specimens. EGFR positivity was observed in 29 of 45 tumors (64%) and was associated with neither clinical tumor stage nor clinical nodal stage. The overall response rate was 34% in EGFR positive patients vs. 62% in those who did not express EGFR (P = 0.07). Only one of the seven pathologic complete remission patients was EGFR positive (P = 0.003). The link between the positive EGFR expression and the microscopic response on surgical specimen seems to be logical, but we fail to assess it in our series. Such a relationship should be based on a large tumor sampling and needs a very strict procedure at the macroscopic level, to ensure that the whole tumor is analyzed after a neoadjuvant treatment. An exhaustive tissue material should allow a precise analysis of the entire spectrum of tumor regression, i.e., complete, partial or none, as it has been proposed by Dvorack et al [43]. It should then be of interest to correlate these well documented histopathological data with biological parameters such as EGFR.

In our study EGFR expression was not found to be an independent prognostic factor for overall survival in patients with rectal cancer. Other studies, described above [40, 41], have reported variable results making it difficult to draw firm conclusions about a possible relationship between EGFR expression and overall survival. Probably, the variation in results is due to (i) the use of different laboratory tests, (ii) varying extent of follow-up, and (iii) heterogeneity in the population of colon- and rectum-cancer patients. (i) EGFR assessment in previous studies was obtained by using different antibodies, different methods of antigen retrieval, and different cut-off values. In this study, we used uniform reagents provided by a kit allowing minimized variations in results and a reproducible method. Therefore, our results detected a low percentage of EGFR immunopositivity (56%) as compared with other colorectal cancer trials [44] probably due to the numerous IHC techniques. (ii) In this study we did not have a sufficiently long follow-up to give definitive conclusions on the prognostic impact on EGFR expression and overall survival. In fact, our analysis was only based on a group of patients with rectal carcinoma, a disease with a natural history different from that of colon carcinoma especially with respect to the tendency to recur locally. (iii) Reasons for recurrence after curative resection for colorectal carcinoma are not completely clear. Several theories have been put forward including, amongst others, microscopic deposits in the lymphatics, inadequate distant and lateral resection margins, exfoliated tumor cells at time of surgery, presence of malignant cells in the anastomosis and, finally, tumor aggressiveness related to biological behaviour. It is known that reported recurrence after resection for rectal carcinoma is commonly higher than after colon carcinoma [45, 46], and differences in prognosis have also been reported between high and low rectal carcinomas [47]. Risk factors that have previously been associated with increased recurrence rates include amongst others patient age, gender, tumor stage, site of lesion (colon vs rectum), infiltration of adjacent organs, histopathological criteria, tumor size, lymph-node involvement and radial resection margins [45, 46, 48–50]. In rectal cancer, in particular, the impact of surgery and adequate lymph-node dissection related to the risk of local recurrence have been highlighted [51]. Several studies have evaluated the prognostic significance of EGFR on survival in colorectal cancer but, to our knowledge, not specifically focusing on rectal cancer recurrence.

In our study, multivariate statistical model identified EGFR expression as a significant independent predictor of recurrence following preoperative and curative surgery for rectal cancer. Two possible explanations for this relationship are to be considered. Firstly, EGFR overexpressing tumors exhibit a more aggressive behavior leading to more pelvic recurrences and in a lesser extent to more distant metastases. A second possible explanation is that EGFR overexpressing tumors present decreased intrinsic radiosensitivity as explained at the first part of this discussion and lead to more pelvic recurrences. This explanation is supported by the fact that the majority of the observed recurrences in our study appeared in the irradiated areas.

Therapeutic approaches targeting EGFR signaling pathways either alone or in combination with radiation or cytotoxic agents are being intensively investigated [52]. Strategies that are in various stages of development include blockade of the extracellular receptor domain [44] and inhibition of the intracellular tyrosine kinase activity [53]. Data presented in Figure 2 suggest that tumor radiation-sensitization through the inhibition of EGFR signaling could yield a therapeutic gain by increasing the locoregional control rate in patients with EGFR-overexpressing rectal cancer.

Knowledge of expression of EGFR in rectal cancer can contribute to the identification of patients with an increased risk of recurrences. Our results have to be related to several confounding factors such as the small number of events and the retrospective approach. Nevertheless, the potential of introducing routine EGFR immunohistochemistry as a diagnostic tool into the clinical practice of rectal cancer management still has to be undertaken, and may allow clinicians to deliver targeted therapies even in patients with a poorer prognosis.