HER 2/neu Overexpression in Oral Squamous Cell Carcinoma and Its Clinico-Pathological Association at a Tertiary Care Center in Eastern India

Abstract

Human epidermal growth factor receptor 2/neu (HER2/neu) is known to serve as a prognostic and predictive biomarker in several cancers such as breast, gastric and ovarian cancers. In head and neck squamous cell carcinoma, HER2/neu expression is seen but in a fluctuated manner. Hence, its role as a prognostic factor in oral squamous cell carcinoma (OSCC) needs evaluation. To determine the HER 2/neu overexpression in OSCC patients and its association with clinical and pathological parameters. 74 patients of OSCC treated between 2016 and 2018 were included in the study. Immunohistochemistry was done on tissue samples from these patients and HER2/neu expression was measured. Both biopsy and resected specimens were considered for the study. Out of 74 patients, 47.3% (35) were operated and 52.7% (39) were not operated due to loss to follow-up. No significant association was found (p = 0.636, OR = 0.68, CI = 0.14–3.34) between lymphovascular invasion (LVI) and HER2/neu expression. Similar results were seen for perineural invasion (PNI) (p = 0.490, OR = 0.53, CI = 0.88–3.24), depth of invasion (p = 0.21), grade of tumor (p = 0.214), clinical-stage (p = 0.511) and pathological stage (p = 0.091). No significant association existed between HER2/neu expression and LVI, PNI, clinical-stage, the grade of tumor and the pathological stage of oral squamous cell carcinoma.

Introduction

Cancer is a major public health problem and is the second leading cause of death worldwide. The greatest number of deaths are from cancers of the lung, prostate and colorectal region in men and the lung, breast, and colorectal in women [1]. Head and neck cancers represent the sixth most common cancer worldwide with approximately 630,000 new patients diagnosed annually resulting in more than 350,000 deaths every year. Head and neck squamous cell carcinoma (HNSCC) accounts for 5–10% of all new cancer cases and there is a wide geographical variation in the incidence and anatomic distribution of HNSCC worldwide. Oral squamous cell carcinomas (OSCC) or oral cavity cancers are a subset of HNSCC referring to cancers of the soft and hard palates, between the vermilion border of lips and the posterior third of the tongue. The habits of tobacco use and alcohol consumption contribute to the development of almost 80% of all HNSCC diagnosed globally. In India, oral squamous cell carcinoma is the most common cancer found in men and the third most common cancer in women [2]. Ninety percent of all these HNCs are squamous cell carcinomas (SCC), which are derived from mucosal linings of the upper aero-digestive tract. In India, 60–80% of the patients present in an advanced stage of the disease (stages III and IV) at the time of diagnosis. Therefore, the 5-year survival rate of these patients is around 40–60% with an elevated rate of recurrences [3]. HNSCC constitutes about one-third of all cancers in India, in contrast to 4–5% in the developed world. A hospital-based 5-year retrospective study at a regional cancer center in the state of Odisha reported that 66.49% of the patients included in the study were OSCC patients. This was the maximum occurring cancer among others [4].

Squamous cell carcinoma (SCC) of the head and neck is considered as a malignant epithelial disease arising from the mucosa of the upper aero digestive tract including the oral cavity, larynx, oropharynx, and hypo-pharynx [5]. Recent treatment options for HNSCC include combination therapies such as surgery, radiation, chemotherapy. New targeted tumour therapy options are widely employed in other cancer treatments, whereas limited treatment options exist for recurrent or metastatic disease in HNSCC [5, 6].

Human epidermal growth factor receptor 2 (HER2)/neu proto-oncogene(neu) is a proven molecular prognostic marker in the breast, gastric and ovarian cancers [7]. It has recently become an integral part of the treatment protocols of many malignancies such as breast cancer and is also included in clinical studies of HNSCC [8].

In our previous study, we found that HER2/neu is significantly expressed in the head and neck squamous cell carcinoma but its overexpression does not have any correlation with its epidemiological parameters [9]. HER2/neu overexpression or amplification has prognostic or clinic-pathological associations. This study aimed to determine the association of HER2 with clinico-pathological parameters like Lymphovascular invasion (LVI), Perineural invasion (PNI), depth of invasion, the grade of differentiation, clinical and pathological stage of the tumour. The prognostic association of Her-2/neu in HNSCC might provide new therapeutic designs and management of the disease for better patient outcome.

Methodology

In an observational study, all cases of HNSCC reported over 2 years from 2016 to 2018 were evaluated. Non-SCC, oropharynx, hypopharynx, CUP (Cancer unknown primary) patients; those who had not completed radiation therapy; those who did not have metastatic recurrent cancer; and those who did not consent to participate were excluded from the study. For participants who were operated, data were obtained on HER2/neu expression, the grade of differentiation, depth of invasion, LVI, PNI, clinical and pathological stage. For those who were not operated, data were analysed for HER2/neu expression, the grade of differentiation and clinical stage only.

Ethical clearance was obtained from the Institutional Ethics Committee. All participants were informed about the study and informed written consent was obtained.

The specimens were routinely fixed in 10% neutral buffered formalin (NBF). Dimensions of the biopsy specimen were noted; the lesion was identified and relevant tissue was submitted for processing in labeled capsules. The tissue was embedded in paraffin wax and 4 µm thick sections were taken and stained with hematoxylin and eosin stain. The microscopic features were assessed and the diagnosis was rendered accordingly.

Immunohistochemical Determination of HER2/neu

All the sections from the tumor tissue were subjected to immune-histochemical (IHC) staining. The Rabbit Anti-Human c-erb-2 Oncoprotein Antibody DAKO Kit was used to demonstrate the HER2/neu expression. By using the peroxidase-antiperoxidase method, IHC was done.

Staining Procedure

NBF-fixed paraffin embedded tissue sections of 4 µm were taken on poly-L-lysine-coated slides. The slides were fixed in an incubator at 600 °C for 20 min. Deparaffinization was done. Hydration was done in running tap water then with distilled water for 5 min. Antigen retrieval process was done with citrate buffer (pH 6.0–6.8) using microwave oven at 950 °C for 2 cycles of 10 min each. Then slides were brought to room temperature and washed with distilled water. Slides were then treated with endogenous peroxidase block for 10 min and then washed in wash buffer (phosphate), 3 times for 3 min. Treated with power block for 10 min, the solution was allowed to drain. Primary antibody was applied for an hour then washed with wash buffer, 3 times for 3 min. The super-enhancer was added and left for 20 min. The secondary antibody was applied for 30 min and then washed with wash buffer, 3 times for 3 min. Diaminobenzidine chromogen was applied for 5 min and then it was washed with distilled water to stop chromogen reaction. Counterstaining was done with Hematoxylin for 2 min and then finally washed with tap water.

Staining Pattern

The staining pattern was compared with control slides. Positive controls were sections of a previously found breast carcinoma previously found to be positive for Her2/neu (with score 3+). Negative controls were duplicate sections of the same sample in which the primary antibody had been excluded and replaced with PBS.

Immunohistochemical Analysis

Representative fields were selected in each immunohistochemistry stained section. The intensity of staining of Her2/neu positive cells per field was calculated.

• • “3+”: Complete and intense membrane staining of > 10% tumor cells.

• • “2+”: Complete but moderate staining of > 10% cells.

• • “1+”: Weak and incomplete staining in > 10% cells.

• • “0”: No membrane staining or staining in < 10% cells.

Score 0 was considered negative and Score 1, 2, and 3 were positive.

Statistical Analysis

Data were analyzed by using SPSS version 20.0 software (IBM Corp., Armonk, NY, USA) licensed to the institute. Descriptive data are presented as frequency distribution. Odds ratio and 95% confidence interval were used to express the association. Independent samples Mann–Whitney U test (as the data was not normally distributed) was applied to find the association between depth of invasion and HER2/neu expression. A p value of < 0.05 was considered statistically significant.

Results

Out of the 74 patients, there were 39 non-operated cases. Their clinical-stage, grade of differentiation and HER2/Neu expression is described in Table 1. Majority of the cases were poorly differentiated (84.6%, n = 33), belonged to clinical-stage IV-A (51.3%, n = 20) and HER2/Neu expression was found to be negative (82.1%, n = 32).

Table 1.

Association of HER2/neu expression among 39 non-operated cases of OSCC

Parameters	N (%)	HER2 + ve	Odds ratio (confidence interval)	p value
Clinical stage
Stage I and II	15 (38.5%)	5 (33.3%)	0.182 (0.030–1.102)	0.062
Stage III, IV AandB	24 (61.5%)	2 (8.3%)
Grade
WD	5 (12.8%)	3 (60.0%)	0.089 (0.011–0.705)	0.032
MD and PD	34 (87.2%)	4 (11.8%)

The association of clinico-pathological parameters like Lymphovascular invasion (LVI), Perineural invasion (PNI), the grade of differentiation, clinical and pathological stage of the tumour with HER2/neu expression in case of 35 operated cases is shown in Table 2. Due to a smaller number of cases, stages I and II, and III and IV were pooled together in case of clinical and pathological staging. It was observed that tumors with no LVI or PNI, which were moderate to poorly differentiated and in the clinical and pathological stages I and II were associated with higher HER2/neu expression as compared to their counterparts. However, it was statistically not significant.

Table 2.

Association of HER2/neu Expression among 35 operated cases of OSCC

Parameters	N (%)	HER2 + ve	Odds ratio (confidence interval)	p value
LVI
Positive	9 (25.7%)	3 (33.3%)	0.68 (0.14–3.34)	0.636
Negative	26 (74.3%)	11 (42.3%)
PNI
Positive	7 (20.0%)	2 (28.6%)	0.53 (0.88–3.24)	0.214
Negative	28 (80.0%)	12 (42.9%)
Grade
WD	17 (48.6%)	5 (29.4%)	2.40 (0.596–9.670)	0.214
MD and PD	18 (51.4%)	9 (50.0%)
Clinical stage
Stage I and II	8 (22.9%)	4 (50.0%)	0.59 (0.120–2.887)	0.511
Stage III, IVA andIVB	27 (77.1%)	10 (37.0%)
Pathological stage
Stage I and II	14 (40.0%)	8(57.1%)	0.30 (0.073–1.241)	0.091
Stage III and IV	21 (60.0%)	6 (28.6%)

Similarly, there was no statistically significant association of the depth of invasion of the tumour with HER2/neu expression (p = 0.21).

Discussion

The head and neck cancers specifically oral squamous cell carcinoma (OSCC) span 8–10% of all known cancers till date and are considered to be one of the commonest causes of mortality due to cancer [10]. The conventional treatment of HNSCC by surgery and adjuvant radiotherapy with or without chemotherapy has failed to improve survival beyond a certain limit. Poor survival is usually related to delayed diagnosis and frequent disease recurrence. Though clinical staging and grading do help in planning the treatment and estimation of prognosis, still it fails to predict information at the onset of the disease for early detection. Therefore, the search for more reliable prognostic markers is still required. Her-2/neu oncoprotein enhances the metastatic potential of the tumour, by promoting invasion through several stages and the metastatic cascade, thus, plays an important role in carcinogenesis [6]. In the present study, the expression of Her-2/neu oncoprotein was analyzed by IHC technique.

Giatromanolaki et al. observed 89 HNSCC cases with cytoplasmic staining but failed to exhibit any correlation between Her-2/neu expression, clinico-pathological parameters. HER-2/neu expression was significantly higher in advanced stage IV cases than the stage I–III cases. There was no significant correlation between HER-2/neu expression and clinico-pathological parameters in HNSCC patients [11]. In our study, similar results were found with no significant correlation between HER-2/neu positivity and the clinical/pathological stage, the grade of differentiation, LVI, PNI and depth of invasion. Similar results have also been reported in a study by Khan et al.[12]. There was no apparent correlation in HNSCC and HER-2/neu level with clinico-pathological variables except early and advanced tumour stage (p value = 0.017) reported by Dhuria et al. [13]. However, Cavalot et al. [14] and Xia et al. [15] reported a correlation between the HER2/neu positivity and the stage, grade or lymph node status of the patient, which was not seen in our study. Our observations also project that HER-2/neu expression in HNSCC and its clinico-pathological parameters are not significantly associated.

Conclusion

From this study, it can be concluded that HER2/neu positivity has no association with pathological staging, grade, depth of invasion, lymphovascular invasion (LVI) and perineural invasion (PNI) in the oral squamous cell carcinoma. Hence, our findings indicate that HER-2/neu may not be having any prognostic significance and clinical implications in OSCC patients.

Data Availability

All the data were purely obtained from patients.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Consent to Participate

All participants were informed about the study and informed written consent was obtained.

Consent for Publication

All authors have given their consent for the publication.

Ethical Approval

Ethical clearance has been done.