Association of Surgical Margins and Pathological Staging with Epidermal Growth Factor Receptor Expression in Oral Squamous Cell Carcinoma- A Prospective Cohort Study

Abstract

Oral squamous cell carcinoma (SCC) is one among the most frequent cancers across the globe. The postoperative overall survival (OS) has not improved over years much despite of advanced surgical techniques and various anticancer drugs. A better and good understanding of molecular mechanisms and identification of potential oncogenes in OSCC may provide new therapeutic decisions such as target therapy in management of these cancer patients. To find whether there is any association between surgical margins and pathological staging with epidermal growth factor receptor expression affecting the prognosis in oral squamous cell carcinomas. A prospective cohort study was performed in 25 patients with biopsy proven oral squamous cell carcinoma who presented to our hospital from July 2017 to June 2019. The data collected from their report were pTNM staging, surgical margins (anterior, posterior, superior and inferior) and scoring of EGFR expression. Surgical margins with EGFR expression was found to have p-value of 0.023 and pTNM staging with EGFR expression was found to have p-value of 0.051 which were statistically significant. This study concludes that there is strong association of EGFR expression with pTNM and surgical margins which may influence the prognosis of the patient. This study helps us to understand the need of adding EGFR targets like cetuximab along with radiation instead of conventional cisplatin therapy.

Introduction

OSCC are often challenging to manage because of their heterogeneity in the natural history and the growing demand for not only to have a better local tumour control but also to improvise functional and cosmetic results [1, 2]. The data of large randomized clinical trials(RCT) to address the optimization of radiation fractionation protocol collectively show that biologically based modifications in the schedule of fractionation have improved the control rate loco-regionally(LR) not only by 10–15% but have had tremendous impact on the OS rate [3–5]. The results of scores of various clinical trials testing combined modality treatment reveal that cytotoxic agents given before; induction or neoadjuvant chemotherapy or after;adjuvant chemotherapy. surgery or radiation does not improve the therapeutic outcome appreciably relative to LR treatment alone [5]. In contrast to the study, administering concurrent chemotherapy along with radiation has improved the overall survival (OS) rate by about 8% but at the cost of increased toxicity. However, inspite of decades of intensive clinical investigations, the outcome of patients presenting with stage III–IV OSCC is still very poor, with actuarial survival rates of 5 years fluctuating between 30 and 40% in most trials, depending on the patient eligibility criteria [6]. These findings underscore the need to develop novel strategies in the management of patients with advanced OSCC.

Recent advances in the understanding the molecular biology of OSCC have opened many new novel research directions [7]. An Increasing effort has been directed towards the developing molecular targeted therapies or a search for molecular markers that can be useful either in predicting outcome of treatment or in selection of patients for specific molecular targeted therapies based on particular tumour characteristics. There are numerous recently published review articles summarize the current status of the search for prognosis-predictive molecular biomarkers. Although none of the studies has identified any convincing data to warrant routine clinical application of any marker, encouraging leads have been generated for a number of molecules including EGFR and one of its ligands, p53, cyclin D and TGF [8].

This prospective study was done to find whether there is any association between clinic-pathological factors like surgical margins and pathological staging EGFR ffecting the prognosis in OSCC.

Materials & Methods

A prospective cohort study of 25 patients with biopsy proven oral squamous cell carcinoma who presented to our Out Patient Department (OPD) in Greater Kailash Hospital, Indore, Madhya Pradesh was performed. Patients of age group from 20 to 70 years with, squamous cell carcinomas of oral cavity were included in the study. All the tumours were classified according to the TNM of the International Union Against cancer (UICC-AJCC). Clinical examination of the primary tumour and neck was done. All patients underwent surgery with radical neck dissection. Postoperative histopathological reports were recorded in the study. The data collect from this report were pTNM staging, margins of tumour and scoring of EGFR. Audio- visual recording of the patients in their local language (Hindi) were taken to check epidermal growth factor expression status in previous operated wax blocks. Consents were signed from the patients and one of their attenders in both English and Hindi.

EGFR Grading System

Staining Procedure: Immunohistochemistry

Sections of about 3 μm thick, were mounted on positive charged microscope slides. After dewaxing in xylene, sections were dehydrated in ethanol, rinsed in distilled water, placed in 3% H2O2 for 10 min and rinsed in distilled water for 15 min for antigen retrieval procedure. Slides were placed in citrate buffer solution, PH = 6, in microwave at 92 °C for 10 min. After cooling at room temperature for 20 min, slides were exposed to primary antibodies (BioGenex polyclonal rabbit anti-human EGFR antibody AR335-5RE; Clone EP38Y IgG1, BioGenex monoclonal mouse anti-human HER-2/neu Receptor antibody; Clone CB11; IgG1)were used (Diluted in PBS, pH 7.6, containing 1% BSA and 0.09% sodium azide) in dilution of 1:10–20.Sections were washed again and incubated with secondary antibody for 30 min followed by the streptavidin biotin- peroxidase (strept ABC complex, HRP duet kit, Dako) for 30 min at room temperature. Reactions were developed with a solution containing 0.6 mg/mL of 3,3’diaminobenzidinedihydrochloride (DAB, sigma) and 0.01% H2O2 and counter stained with Mayer’s hematoxylin for about 2 min. The validation of the reaction was achieved by using negative external controls, with primary antibody omission and below mentioned positive controls. There are many laboratories in India who are doing EGFR by IHC like CORE diagnostics, Lal path labs, Roche diagnostics charging around 1200–1600INR per wax block. The EGFR IHC for all the patients in our study were done in medical college where the clinical trial was carried out at cost of 200 INR per block. So it was 800 INR for 4 blocks (margins) for each patient. The cost was economical and was given by the patient.

EGFR expression was evaluated according to a previously defined four point scale based on the Immuno-labelling of tumour cell membranes proposed by Diniz-Freitas et al. as follows [9]

1. No labelling or labelling in < 10% of tumour cells.

2. Weak labelling, homogenous or patchy in > 10% of the tumour cells

3. Moderate labelling, homogenous or patchy in > 10%- 50% of the tumour cells.

4. Intense labelling, homogenous or patchy in > 50% of the tumour cells.

Results

Table 1 predicts that there is strong association of surgical margins with EGFR expression which is statistically significant with p-value of 0.023. Out of 125 margins, 79 margins showed > 50% of EGFR expression.

Table 1.

Association of margins with EGFR expression

Margins	0 (negative)	1 (< 10%)	2 (10–50%)	3 (> 50%)	Total
Anterior	1	1	3	19	25
Posterior	1	0	5	19	25
Inferior	2	0	3	18	25
Node	20	0	1	4	25
Superior	1	1	4	19	25
Chi square	11.716
p-value	0.023*

^*p-value < 0.05 is significant

Table 2 predicts that there is strong association of surgical margins with EGFR expression which is statistically significant with p-value of 0.051. 38 margins of stage IVA patients showed more > 50% EGFR expression which predicts as the stage increases along with high EGFR expression, the prognosis of the patient will be poor.

Table 2.

Association with stage and expression of EGFR

Stage	0 (negative)	1 (< 10%)	2 (10–50%)	3 (> 50%)	Total
I	9	2	7	10	31
II	2	0	0	7	8
III	1	0	2	4	7
IVA	5	0	4	38	47
Chi square	21.661
p-value	0.051*

^*p-value < 0.05 is significant

Discussion

EGFR are the family members of EGF growth factor receptor tyrosine kinase and are usually found on the surface of the cell. The receptor for the epidermal growth factor is 170–180 kD transmembrane glycoprotein tyrosine kinase receptor which is identified in various types of cancer which includes pulmonary cancer, prostate cancer, breast cancer, bladder cancer among with head and neck cancers [9]. Studies confirm that there is overexpression of EGFR in over three quarters of the OSCC analyzed cases. EGFR expression is involved in all the epithelial layers in specimens of oral SCC while in normal oral epithelium it was just localized to the basal cell layer which is documented in other studies too [10]. The extent and intensity scores of EGFR expression revealed by other studies which suggest that carcinomas expressing EGFR display certain pathological features of more aggression which may be attributable for the activation of different pathways of signalling process that can control diverse biological processes [11]. In the current study done by us, we found a statistically significant correlation between the EGFR expression, tumour surgical margins and stage of the tumour while there was no correlation with other clinical parameters in cases of OSCC. Various series of reports have been shown with a connection between expression of EGFR and the resistance to radiation therapy [12, 13]. In vivo, EGFR inhibition by a monoclonal antibody called cetuximab which increases the response of OSCC to irradiation. On the contrary, research studies done by Yamada et al. and Bernardes et al. [14] found the carcinomas expressing EGFR to be well differentiated in many of the cases. These clinical studies documented those lesions having positive staining for EGFR often presented as low-grade tumours, revealing no association with outcome of the patient. Smith et al. [15] in their study emphasized on the fact that overexpression of EGFR had a protective role against loco-regional recurrence and is always related to increased radio sensitivity. Hence the EGFR antigen represents an attractive target for specific therapies which use monoclonal antibodies or tyrosine kinase inhibitors to these patients.

Bonner et al. in 2000 [16] studied and put-forward a pannel of OSCC cell lines that the combination of cetuximab (5 μg/l) delivered concurrently with radiotherapy (3 Gy) resulted in a greater decrement in cellular proliferation than either radiotherapy treatment alone, regardless of the inherent EGFR expression of the cell line. These promising results served as preclinical background for clinical investigations involving C225/radiation in OSCC.

The same author later conducted a multinational phase III study of 424 patients having advanced locoregionally OSCC treated with curative intent. Patients were randomized in two groups- between definitive RT, and the same RT regimen concurrently with weekly administration of cetuximab. Median follow-up of the patient was 54 months. Radiation therapy plus weekly cetuximab significantly prolonged progression-freesurvival (hazard ratio for disease progression or death: 0.70 (0.54–0.90); P = 0.006). The median duration of overall survival in the definitive RT arm was 29.3 months, against 49.0 months in the arm combining radiation therapy plus cetuximab with P value of 0.03.

The survival rate of 3 years was 45% for those patients who were receiving RT alone, and 55% for those patients who were receiving cetuximab with RT. There were grade 3–5 acneiform rash more common in the study arm with cetuximab which was around 17% than in the RT alone arm which was around 1% only. This difference was statistically highly significant with a P value of < 0.001. Most Important, the use of cetuximab did.

not appear to exacerbate or increase the radiation-induced mucositis (grades 3–5: P = 0.44; all grades: P = 0.84) nor any other toxicities. This was the first clinical study to ever demonstrate a good survival benefit related to the use of an EGFR Inhibitor mainly cetuximab when combined to RT in head and neck cancer mainly in OSCC, thereby confirming the promising results provided by previous phase II clinical studies. Studies has to be still confined to give more promising results, as the control arm in this study was unfortunately RT alone but not concomitant chemo-radiation [17, 18]. This trial was designed and executed in a period when RT alone was still considered as an acceptable and standard method in the management of advanced head and neck cancer patients. During this time, concurrent chemo-radiation has assumed an important role for these patients. This clinical trial enabled atleast an unencumbered assessment regarding the capacity of cetuximab to augment radiation response and outcome without the confounding variable of chemotherapy [19, 20]. The promising results from this study in this phase III clinical trial will further require a cross-validation from additional clinical trials to confirm the outcome advantage for the combination of cetuximab with concurrent chemo-radiation therapy. A broad series of new clinical trials are currently on its way [21, 22].

Despite of decades of development in intensive clinical investigations, the patient’s outcome reporting with stage III–IV HNSCC is still very poor, with actuarial survival rates of 5 years fluctuating between 30 to 40% in most of the clinical trials. These findings underline the urgent need to study and develop novel strategies in the management of advanced HNSCC patients [6]. Accelerated radiotherapy schemes lead to an enhanced 5-year local control from 60–70% which in turn is associated with an improved disease-free survival (DFS), but with almost nil benefit regarding overall survival (OS). Most of the clinical trials testing for combined or multi-modality therapy demonstrate that cytotoxic drugs given before like induction or neoadjuvant chemotherapy or after like adjuvant chemotherapy, surgery or radiation do not significantly improve control of the disease both locally and distantly.

In contrast for administering the concurrent chemotherapy with RT has improved the 5-year OS rate by around 8%, but has cost and increased local toxicity [23, 24]. The advantage and magnitude of the survival benefit in respect to chemoradiation is almost similar regardless the use of mono-chemotherapy or poly-chemotherapy. Chemotherapy agents like cisplatin and 5-FU appear to be more effective than mitomycin C or carboplatin with nil RCT data available for newer cytostatic drugs like taxanes which are being used since decades and shown to be effective in OSCC. Presently chemotherapy and radiation therapy have reached their limits,

which might give some minor improvements in the coming years. There are various research projects which are currently developing new management strategies by integrating novel targeted therapies in oncology practice [25–27]. In OSCC, EGFR is not only an independent prognostic factor of outcome in multivariate analysis but it is also a first option for therapeutic target. There was a demonstration affecting a survival benefit when combining external RT with cetuximab which is a major breakthrough in the treatment of OSCC, establishing a new treatment modality for locally advanced OSCC. This clinical trial also put forwarded an important proof of principle that targeting a specific pathway of signalling can enhance the response of radiation to the tumors.

When considering the loco-regional improvement rate, it has been within the range in previous studied achieved with concurrent radiotherapy and chemotherapy and about more than half of patients receiving cetuximab along with radiotherapy still experienced local–regional relapse [28]. Hence there is a strict need for further prospective studies to improve the outcome of the disease. There are numerous devoted ongoing clinical trials just to address a better clinical outcome after addition of cetuximab to concurrent chemo-radiation which is given by RTOG study 0522 [29]. At this particular point, it must also be taken in consideration that cancer cells rely on sometimes several redundant activation pathways and EGFR is only one among them. The risk of failure of treatment is real, if we target only one receptor and hence there is huge and keen interest in combining broader range tyrosine kinase inhibitors such as CI-1033, which targets all the four Erb family members (pan ErbB) [30].

Finally when considering ionizing radiation, it stimulates the nitric oxide (NO) pathway as well as the production of vascular endothelial growth factor (VEGF). Angiogenesis inhibitors bear a tremendous potential to reinforce the cytotoxic action of RT.A small molecule tyrosine kinase inhibitor of EGFR and VEGF called as ZD6474 looks to give a very promising for the future [30].

In recent decades, molecular target EGFR has been considered most promising target for monoclonal antibody therapy. It is of much importance to execute studies which can help to determine the series of mutations in the EGFR 2 gene in order to have a better look into the mechanisms responsible for over expression of this frequently activated biomarker in human OSCC [31]. Oral cancers with EGFR overexpression exhibit a very high proportion of complete responses to chemotherapy than tumors with low-level expression of EGF. Overexpression of EGFR presumably may be due to higher intrinsic proliferative activity which could result in higher sensitivity to drug therapy cytotoxic to cells undergoing mitogenesis. Various in-vivo trials have neem done which shows their anti-tumour activity against tumour cell lines by a wide range of mechanisms that include an antiproliferative effect or direct cytotoxicity or the potentiation of the cytotoxic effects of radiotherapy or chemotherapy. Although there has been a tremendous development in the oncology field in India as well but it still needs more clinical research and validation in a large scale population.

Conclusion

This study concludes that there is strong association of EGFR expression with pTNM and surgical margins which may influence the prognosis of the patient. This study helps us to understand the need of adding EGFR targets like cetuximab along with radiation instead of conventional cisplatin therapy.

Declarations

Conflict of interest

The author declares that they have no competing of interest.

Ethical Approval

Institutional Human Ethical Committee approval taken with Ref no- SDC/PHD/04 on 14/07/2017. Trial registered in Clinical trial registry of India (CTRI) with no – CTRI/2018/05/014222.