Significance of human epidermal growth factor receptor 2 neu immunostain in head and neck squamous cell carcinoma

Ponkhi Doley; Srinivasamurthy Venkataramanappa; Panduranga Chikkannaiah

doi:10.4103/jss.JSS_60_20

ORIGINAL ARTICLE

Year : 2020 | Volume : 47 | Issue : 3 | Page : 180-184

Significance of human epidermal growth factor receptor 2 neu immunostain in head and neck squamous cell carcinoma

Ponkhi Doley, Srinivasamurthy Venkataramanappa, Panduranga Chikkannaiah
Department of Pathology, Employees' State Insurance Corporation Medical College, PGIMSR, Bengaluru, Karnataka, India

Date of Submission	24-Jun-2020
Date of Acceptance	20-Oct-2020
Date of Web Publication	21-Jan-2021

Correspondence Address:
Dr. Panduranga Chikkannaiah
Department of Pathology, Employees' State Insurance Corporation Medical College, PGIMSR, Bengaluru - 560 010, Karnataka
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jss.JSS_60_20

Abstract

Introduction: Squamous cell carcinoma is the most common head and neck cancer. In spite of advances in the diagnosis and treatment, a prognosis still remains poor. Head-and Neck squamous cell carcinoma (HNSCC) is found to express human epidermal growth factor receptor 2 (Her 2) neu marker. Materials and Methods: The present study was conducted for a period of 1 year from January to December 2017. All the cases which have been pathologically diagnosed as HNSCC were included in the study. Results: During the study period, a total of 50 cases were observed. Male: female ratio was 1.7:1. In our study, buccal mucosa was the most common site (26'). Well-differentiated morphology was more common (36'), and 34' cases were positive for Her 2 Neu expression. Conclusion: Her 2 neu expression may not be a reliable molecular prognostic marker for HNSCC.

Keywords: Head and neck cancer, human epidermal growth factor receptor 2 neu, immunohistochemistry, oral cancer

How to cite this article:
Doley P, Venkataramanappa S, Chikkannaiah P. Significance of human epidermal growth factor receptor 2 neu immunostain in head and neck squamous cell carcinoma. J Sci Soc 2020;47:180-4

How to cite this URL:
Doley P, Venkataramanappa S, Chikkannaiah P. Significance of human epidermal growth factor receptor 2 neu immunostain in head and neck squamous cell carcinoma. J Sci Soc [serial online] 2020 [cited 2021 Mar 7];47:180-4. Available from: https://www.jscisociety.com/text.asp?2020/47/3/180/307605

Introduction

Squamous cell carcinoma (SCC) is the most common head and neck cancer. The treatment of SCC has been a challenging because of the high rate of recurrence and its advanced disease at the time of diagnosis. In spite of advances in the diagnosis and treatment of head and neck squamous cell carcinoma (HNSCC), the outcome is still poor. There is an urgent need for the development of targeted therapy in HNSCC to individualize personalized treatment depending on specific receptor expression to improve patient outcome.^[1],[2] Human epidermal growth factor receptor 2 (HER2)/neu is an established molecular prognostic marker in breast, ovarian, and gastric cancers. So far, HER-1 or epidermal growth factor receptor (EGFR) has been the most widely studied receptor in HNSCC. EGFR has been shown to be expressed in the vast majority of HNSCC, and its expression and activation are associated with poor prognosis.^[1] The other biomedical markers such as P53, cyclin D1, P 16 protein angiogenic factors, and metalloproteinases have been shown to be an independent biomarkers of survival among HNSCC patients.^[1] The expression rate of HER 2 Neu in HNSCC as documented in the literature is between 0' and 60'; however, the prognostic significance of the same is still needs to be established.^[3],[4],[5]

Aims and objectives

To study the presence of HER2/neu expression in HNSCC
To correlate Her 2 neu expression with Broder's grading system of squamous cell carcinoma.

Materials and Methods

It is a retrospective study conducted in the department of pathology at a tertiary care teaching hospital and a medical college. The present study was conducted for a period of 1 year from January to December 2017. Demographic profile of the patients such as age, gender, location of the tumor, and history of tobacco habits was obtained from medical records. The hematoxylin and eosin-stained slide was retrieved from the files, if the H and E slide is not available, paraffin block were retrieved and 5μ thickness section were cut and stained by H and E as per the standard protocol. All the slides were reviewed to confirm the diagnosis and separately graded by two pathologists by using Broder's grading system. For immunohistochemical stain, 4 μm sections were cut and staining for Her 2 neu was carried out using monoclonal antibody anti-Her 2 neu antibody (ErbB 2 antibody ab2428, DAKO Corporation, Denmark).

Inclusion criteria

All the cases which have been pathologically diagnosed with HNSCC were included in the study.

Exclusion criteria

Improper fixed specimens.

Specimens from female patients with known history of treatment of breast carcinoma.

Immunohistochemical procedure

NBF-fixed paraffin-embedded tissue sections of 3–4 μm were taken on coated slides. The slides are incubated at 58°C–60°C in an incubator. Deparafinization was done in xylene. Hydration was done in running tap water, and then changed to distilled water for 5 min. Antigen retrieval was done with ethylenediaminetetraacetic acid buffer using pressure cooker. Slides were brought to the room temperature and washed with distilled water. Slides were then treated with endogenous peroxidase block for 20 min. Further, slides were washed in wash buffer (tris-buffered saline [TBS]), two times for 3 min. Primary antibody was applied for 1½ h then washed with TBS wash buffer, two times for 5 min. Secondary antibody was applied for 30 min and then washed with wash buffer, two times for 5 min. Diaminebenzidine chromogen was applied for 5–10 min, and then, it was washed with buffer to stop chromogen reaction. Counter staining was done with hematoxylin for 2 min and then finally washed with tap water. Dehydrate with alcohol and xylene and mount with dibutyl phthalate in xylene. Her 2 neu gives a membrane staining. The positive control was previously reported breast carcinoma with 3 + positivity.^[6]

Immunohistochemical slides were graded by two pathologists, separately, who scored the immunostainings twice to decrease intra-observer variability, in a blinded fashion. In unmatched cases, slides were evaluated again by both pathologists. Randomly ten fields were chosen for each section and the total numbers of positive cells for all 10 examined fields were counted and the percentage of staining was calculated.

Immunohistochemistry (IHC) was scored according to the American Society of Clinical Oncologists/College of American.

Pathologists (ASCO/CAP) guidelines for Her2/neu testing in breast cancer, as described by Lebeau et al.^[7] 2001:

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.

The results were tabulated.

Statistical analysis

The Chi-square test was used for the analysis of contingency tables, with P < 0.05 as the criterion of statistical significance. Her2/neu overexpression was correlated with Broder's grading system.

Results

During the study period, a total of 50 cases were observed. Out of 50 cases, 32 were male and 18 were female patients with a male-to-female ratio of 1.7:1. Patients ranged in age from 35 to 80 years with maximum number of cases in the age group of 51–60 years (36') followed by 61–70 years (28') [Table 1]. Twenty-five patients were smokers, and all were male, 40 were tobacco chewers, whereas 10 patients had both habits. In our study, buccal mucosa was the most common site (26') followed by the pharynx and pyriform fossa (18') [Table 2]. Among 50 cases of HNSCC 24 (48') cases were well differentiated, 18 (36') cases were moderately differentiated, and 8 (16') cases were poorly differentiated [Figure 1]a,[Figure 1]b,[Figure 1]c.

Table 1: Age and sex distribution among head and neck squamous cell carcinoma patients

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Table 2: Location of head-and-neck squamous cell carcinomas with percentage of occurrence

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Figure 1: (a) Microphotograph of squamous cell carcinoma well-differentiated showing keratin peals (H and E, ×40). (b) Microphotograph showing clusters of malignant squamous cells (moderately differentiated squamous cell carcinoma) (H and E, × 40). (c) Microphotograph of poorly differentiated squamous cell carcinoma showing individual dyskeratotic squamous cell (H and E, ×40). Microphotograph showing Immunohistochemistry showing Her 2 neu positivity 1+ grading (d), 2+ grading (e), and 3+ grading (f). (IHC, ×40 anti Her 2 Neu Biogenix)

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Immunohistochemical analysis

Out of 50 cases, 17 (34') cases were positive for Her 2 neu expression [Table 2] and [Figure 1]d,[Figure 1]e,[Figure 1]f. Site wise Her 2 neu positivity is shown in Table 2. Correlation was carried out among the Broder's grading system for each site with the Her 2 neu expressivity and P value was found to be more than 0.05 for the all site and is insignificant [Table 3].

Table 3: Comparison of differentiation characteristics of head and neck squamous cell carcinoma Broder's grades between hematoxylin and eosin and human epidermal growth factor receptor 2 stains

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Discussion

HNSCC is the most common carcinoma in India. The current treatment modality is surgery followed by radiotherapy and chemotherapy. Even with this modality of treatment survival beyond a certain limit is not possible which triggers the researchers to search for the better, reliable prognostic factor.

In the present study, male outnumbered the female. The same has also been observed in the literature. The male predominance of HNSCC is due its association with smoking and alcohol consumption.^[1],[2]

The common sites of HNSCC show a geographic variations, oral cavity is the most common site in India. The national cancer registry has identified gingivo-buccal cancer as Indian cancer because of keeping tobacco in the sulcus for long time. In the present study, we observed buccal mucosa as the common site (26') followed by the pharynx (18'), and our findings are in consistent with Vats et al.^[1] In an another Indian study by Dhuria et al.^[5] observed larynx as the common site. Xia et al.^[8] in their study in China also observed the oral cavity as the most common site. Whereas, the larynx was the most common site in Italy and France.^[9],[10]

With respect to grading of tumor, well-differentiated carcinoma is most common followed by moderately and poorly differentiated and our findings are in consistent with the literature.^[1],[5]

Various authors have identified different percentage of Her 2 neu expressivity; it ranges from 2' to 60'. Bernardes et al.^[11] recorded lowest expressivity of 2.2', Sardari et al.^[12] observed expressitivity of 60'. Seifi et al.^[13] and Khan et al.^[14] observed 17', Vats et al.^[1] observed 20', Cavalot et al.^[9] observed 39', Guerry et al.^[15] observed 45.2', Xia et al.^[8] observed 51.3', and Papavasileiou et al.^[16] observed 59.45. Our finding of 34' of Her 2 neu expressivity is within the range.

The broad range of positivity may be due to many factors, different authors have used different method of IHC (direct vs. indirect) and difference in the antigen retrieval technique. Few have used monoclonal antibody while others have used polyclonal antibody. Different methods of grading IHC slide and variation in the frequency of occurrence of HNSCC.^[1],[5] In the present study, we have used monoclonal antibody and membrane positivity more than 10' was considered positive, and we have used ASCO/CAP guidelines for grading IHC slides.

In the present study, tumors of the buccal mucosa exhibited maximum positivity for Her 2 Neu followed by the pharynx. The region of maximum percentage of expression varies among different studies.^[1],[5],[9] In the present study, there was no statistical significance was recorded among Her 2 neu expressivity with region of the tumor and grade of the tumor. Similar observation was also recorded by Khan et al.^[14] Vats et al.^[1] observed no statistical association between stage, grade and lymph node status, and stage of tumor. In an another study by Dhuria et al.^[5] observed no statistical significance with Her 2 neu expressivity with age, sex, grade of tumor, tobacco use, alcohol consumption, family history, tumor site, lymph node involvement, treatment modality, and recurrence of tumor. However, they found statistical significance with Her 2 neu expressivity and stage of the tumor. Cavalot et al.^[9] and Xia et al.^[8] found positive correlation between Her 2 neu expression and nodal status while Guerry et al.^[15] observed decreased risk of metastasis in Her 2 neu positive cases.

Conclusion

It is evident from the observation of the present study and review of literature that the oral cavity is the most common site of HNSCC. There was no statistical significance of Her 2 neu positivity with other prognostic markers of HNSCC. Hence, Her 2 neu expression may not be a reliable molecular prognostic marker for HNSCC as documented in carcinoma of the breast and stomach.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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