Expression of human epidermal growth factor receptor 2/Neu in oral leukoplakia

Ashesh Kumar Jha; Vansh Verma; Karsing Patiri; Nikhil Arora

doi:10.4103/jhnps.jhnps_41_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 10 | Issue : 2 | Page : 162-166

Expression of human epidermal growth factor receptor 2/Neu in oral leukoplakia

Ashesh Kumar Jha¹, Vansh Verma², Karsing Patiri³, Nikhil Arora⁴
¹ Department of General Surgery, AIIMS, Patna, Bihar, India
² Department of General Surgery and Pathology, Dr. BSA Medical College and Hospital, Rohini, Delhi, India
³ Department of Pathology, Dr. BSA Medical College and Hospital, Rohini, Delhi, India
⁴ Department of ENT, PGIMS, Rohtak, Haryana, India

Date of Submission	06-Jul-2022
Date of Decision	02-Sep-2022
Date of Acceptance	04-Sep-2022
Date of Web Publication	15-Dec-2022

Correspondence Address:
Ashesh Kumar Jha
Department of General Surgery, AIIMS, Patna, Bihar
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jhnps.jhnps_41_22

Abstract

Background: Molecular alterations in premalignant lesions of the oral cavity are not well known. Many reports have found increased human epidermal growth factor receptor 2 (HER-2) expression in oral cancer. Overexpression of HER-2 in premalignant lesions may denote its positive contribution in the malignant transformation of these lesions. Materials and Methods: Twenty-three samples of leukoplakia were stained by routine H and E to assess any dysplasia; five samples of normal mucosa were used as control. Immunohistochemical staining for HER-2 was done. ASCO/CAP 2018 guidelines were used for reporting the results. The percentage of lesions expressing cytoplasmic or membranous expression was calculated. Results: One sample of leukoplakia with severe dysplasia expressed focal membranous staining. Cytoplasmic staining was observed in 3/9 (33.33%) dysplastic leukoplakia. Only one nondysplastic leukoplakia expressed cytoplasmic HER-2 staining. Conclusions: Membranous expression in severe dysplasia and higher expression in oral cancer are in concordance with the multistep theory of carcinogenesis.

Keywords: Dysplasia, human epidermal growth factor receptor 2, leukoplakia, premalignant

How to cite this article:
Jha AK, Verma V, Patiri K, Arora N. Expression of human epidermal growth factor receptor 2/Neu in oral leukoplakia. J Head Neck Physicians Surg 2022;10:162-6

How to cite this URL:
Jha AK, Verma V, Patiri K, Arora N. Expression of human epidermal growth factor receptor 2/Neu in oral leukoplakia. J Head Neck Physicians Surg [serial online] 2022 [cited 2023 Jun 4];10:162-6. Available from: https://www.jhnps.org/text.asp?2022/10/2/162/363925

Introduction

Oral cancer accounts for approximately 30% of all malignancies in India.^[1] It is usually preceded by a premalignant lesion, and approximately 60% appear as white keratotic lesions.^[2],[3] The overall prevalence of precancerous lesions in India has been reported to be as high as 8.4%.^[4] In India, the prevalence of leukoplakia varies from 0.2% to 5.2% making it the most commonly encountered lesion. The risk of malignant transformation of these lesions is variable, ranging from 0.3% to 10% depending upon the grade of dysplasia, location, clinical type, and molecular changes.^[5] Identification of high-risk premalignant lesions and appropriate interventions at an early stage is desirable to ensure a favorable outcome. Hence, it seems prudent to look for molecular changes in leukoplakia to predict their malignant potential.

Human epidermal growth factor receptor 2 (HER-2)/neu (c-erbB-2 oncogene) belongs to the erbB family of receptors. Upon ligand binding, the receptors can homodimerize or heterodimerize with other members of this family, resulting in autophosphorylation and downstream signaling, these proteins are expressed in most epithelial cell layers and have a key role in development. HER-2/neu shares sequence homology to epidermal growth factor receptor (EGFR), which is overexpressed in oral squamous cell carcinoma, oral premalignant, and dysplastic lesions of the oral cavity.^[6] HER-2/neu plays a central role in cell proliferation and is known to inhibit cell death, as a result, it contributes to oncogenesis in several malignancies. A sequential increase in EGFR and HER-2 expression starting at precancerous stages in an animal model for oral carcinogenesis has also been reported.^[7] The PI3K pathway is implicated in premalignant lesions' pathogenesis, and HER-2 acts through the same leading to increased cellular proliferation and survival.^[8],[9]

Reported expression levels in oral squamous cell carcinoma have been diverse, ranging from 0% to 47%.^{[10],[11],[12],[13]} According to Xia et al., HER-2 expression was the most significant factor in predicting disease outcome, similarly Cavalot et al. concluded that HER-2/neu expression was an independent predictor of disease-free survival in same cancer.^[9],[12]

Previous studies done regarding the expression of HER-2 in premalignant lesions of the oral cavity have shown a variable expression ranging from 0 to as high as 80% in severe dysplasia. Fong et al. in Taiwan reported 25% premalignant samples expressing HER-2 using immunohistochemistry (IHC) but did not divide the staining pattern into membranous or cytoplasmic.^[14] Seifi et al. in Iran reported 11% premalignant samples expressing membranous HER-2, on the other hand, Werkmeister et al. reported chromosome 17 deletions in approximately 15% of lesions.^[15],[16] Rautava et al. reported 35% premalignant samples showing slightly increased staining and 13% lesions with markedly increased staining.^[17] Kobayashi et al. in Japan took only nondysplastic leukoplakias and found no samples with increased expression, this was contrary to Wilkman et al. who found increased HER-2 expression in some samples with epithelial hyperplasia and dysplasia, they reported 57% premalignant lesions positive for HER-2.^[18],[19] Hou et al. so considered cytoplasmic HER-2 expression as positive and reported 13%, 71%, and 80% of samples with mild, moderate, and severe dysplasia, respectively, expressing HER-2.^[20]

The key molecular players associated with oral leukoplakia are TIMP1, Bcl-2, transforming growth factor β, and HER-2 interacts with the abovementioned either in a binary manner or through signal transducers; thus HER-2 is a potential candidate gene in leukoplakia pathogenesis.^[21],[22] Since oral malignancies have shown HER-2 expression, overexpression of the same in leukoplakia may denote its positive contribution in the malignant transformation of these lesions. Available literature shows conflicting and divergent results concerning the overexpression of HER-2/neu in such lesions. Hence, this study was undertaken to evaluate the expression of HER-2 in oral leukoplakia.

Materials and Methods

This prospective observational study was conducted for 2 months in the department of surgery, otolaryngology, and pathology. During this period, all consenting patients with premalignant lesions of the oral cavity were included in the study. Ethical clearance was obtained from the institution ethics committee. Biopsy was taken from the premalignant lesions of the study subject. Moreover, it was processed with routine H and E staining. Control was taken from the adjacent healthy mucosal tissue upon the patient's consent. A histological diagnosis was made to confirm the clinical diagnosis. Dysplasia, if the present was be graded according to the WHO 2017 system for grading of dysplasia. Further, unstained sections were taken for immunohistochemical staining.

Immunohistochemical expression of HER-2/neu was determined on 4-micron thick paraffin sections; all steps were carried out in moist and humid containers. The results were recorded as per CAP/ASCO 2018 guidelines [Table 1]. The percentage of positive cells was determined along with intensity; the results were tabulated. The results from IHC were analyzed; patients were classified depending upon the type of premalignant lesion. Statistical analysis – the percentage of lesions expressing HER-2 was calculated using Excel.

Table 1: ASCO/CAP 2018 guidelines

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Results

Of the 23 patients taken for this study, 21 were males, and 2 were females, the male-to-female ratio was 11:1. Age range observed is 28–60 years; the average age of the patients is 43 years. All patients in this study were tobacco users. Clinical characterization – 23 patients in the study were characterized as having a white patch, clinical diagnosis of leukoplakia was made [Table 2]. Site distribution of lesions – of the 23 lesions, 16 were located on the buccal mucosa, 5 were located on the tongue, and 1 each was located on the lower lip and gingiva. Of the 23 leukoplakia cases, 9 cases demonstrated dysplasia and 6 cases had hyperplasia. Thus, 39.13% samples of leukoplakia samples had dysplasia. HER-2 staining can be observed in a membranous/cytoplasmic fashion, however, 2+ is considered equivocal, and 3+ is considered positive as per the ASCO/CAP guidelines. Focal membranous positivity (2+) [Figure 1] was seen in one case of leukoplakia with severe dysplasia, i.e., 4.34% of leukoplakia samples in this study.

Table 2: Lesions encountered in study subjects

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Figure 1: Focal membranous staining (2+) for HER-2-neu in leukoplakia sample with severe dysplasia (×400). HER-2: Human epidermal growth factor receptor 2

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Cytoplasmic staining was observed in three cases of leukoplakia [Figure 2] with mild dysplasia, i.e., 13.04% of samples in this study. In cases of leukoplakia with dysplasia, 33.33% of lesions expressed cytoplasmic staining. Cytoplasmic staining for HER-2 was also observed in one case of hyperplasia [Table 3].

Figure 2: Cytoplasmic staining for HER-2-neu (×400). HER-2: Human epidermal growth factor receptor 2

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Table 3: Staining characteristics of dysplastic samples

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Discussion

In this study, males outnumbered the females, this is in concordance with higher tobacco consumption in men according to societal norms. Tobacco consumption has been regarded as the primary etiological factor for the development of premalignant lesions, all patients in this study were tobacco users. Buccal mucosa was the most common site accounting for 69.56% of cases, followed by 21.74% of cases in the tongue and 4.34% cases each in the lip and gingiva.

HER-2 staining can be observed as membranous or/and cytoplasmic, however, ASCO/CAP 2018 guidelines consider only membranous expression (2+ and 3+) as positive staining for breast cancer. Fluorescent in situ hybridization is recommended for 2+ cases as per CAP/ASCO 2018 guidelines.

In our study, 1 out of 23 leukoplakia samples demonstrated focal membranous and cytoplasmic staining and 3 samples demonstrated cytoplasmic staining.

Taking into account the dysplastic samples, 1 out of 2, i.e., 50% samples of severe dysplasia expressed focal membranous with cytoplasmic staining and 2 mildly dysplastic lesions expressed cytoplasmic expression, no sample of moderate dysplasia stained for HER-2. HER-2 staining in oral premalignant lesions has been reported to be between 0% and 80% in the existing literature.

We report 4.34% leukoplakia expressing focal membranous with cytoplasmic positivity, our expression rate is lower as compared to studies elsewhere, Seifi et al. reported 11% samples having membranous positivity, and Wilkman et al. reported 60% samples having membranous staining.^[15],[19] For studies which did not divide staining patterns as membranous or cytoplasmic, such comparison is not possible. Our findings are in contrast to the two previously done studies in India which did not report membranous staining, they did not quantify the percentage of samples showing cytoplasmic staining.^[23],[24] However, we report that 13.04% of leukoplakia samples having cytoplasmic staining.

Fong et al. and Rautava et al. considered both membranous and cytoplasmic staining as positive and reported 25% and 35% of samples showing increased HER-2 expression.^[14],[17] If we consider both cytoplasmic and membranous staining, we get 4/23 samples showing increased expression, i.e., 17.3%.

With regard to dysplasia, we report 1/9 samples expressing membranous positivity, our findings are in contrast to Wilkman et al. who reported HER-2 membranous expression in 57% of dysplasia.^[19] Hou et al.^[20] considered cytoplasmic staining as positive and reported 13%, 71%, and 80% samples expressing HER-2 in mild, moderate, and severe dysplasia, respectively. In our study, we did not find any sample with moderate dysplasia expressing HER-2. Recent studies have reported less HER-2 positivity as compared to the ones done previously, this could be possibly due to differences in methods and antibodies used.

Similarly, in oral squamous cell carcinoma, various studies have reported expression between 0% and 47%.^{[10],[11],[12],[13]} The major difficulty in comparing immunohistochemical studies is the variety of antibodies, procedural variations such as incubation time, antigen retrieval, and lack of standardization in scoring may cause variation.

The significance of cytoplasmic staining is a widely debated topic, in colorectal and oral cancer, few studies have shown cytoplasmic association to be linked with prognosis, indicating a role in pathogenesis.^[9],[12] A plausible explanation could be the homodimerization of cytoplasmic domains of HER-2 leading to tyrosine kinase activation and PI3K overexpression further regulating cell proliferation.

Polysomy of chromosome 17 is a well-known step in oral carcinogenesis, a study reported that 15.3% of lesions with pure cytoplasmic staining had chromosome 17 polysomy, whereas all samples with moderate membranous with cytoplasmic staining had chromosome 17 polysomy, even in non-HER-2 amplified breast cancer, HER-2 immunohistochemical expression has been found to be linked with polysomy of chromosome 17. It has been suggested that this genetic aberration may result in a significant increase of HER-2 gene copies in the tumor cells and an increased HER-2 protein production to the level that could be demonstrated by IHC as overexpressed.^[25] In breast cancer, it was suggested that cytoplasmic HER-2 could be a different or a truncated 155 kD protein as compared to the 185 kD protein expressed on the membrane, however, in colorectal cancer, Osako et al. reported 185 kD protein in both the fractions and reported no 155 kD peptide, indicating an activated intracellular HER-2 receptor. Some authors suggest that cytoplasmic HER-2 expression is an artifact or occurs due to cross-reactivity possibly with keratin, others propose that it may reflect true protein expression and incomplete receptor degradation.

For other types of cancer, it has been reported that HER-2 overexpression can occur through mechanisms other than gene amplification, such as through increased levels of promoter-binding proteins as in gastric cancer where elevated levels of binding proteins to the TATA box were observed, which led to HER-2 overexpression. Theoretically, mutations in downstream targets of HER-2, like KRAS, might also influence the expression of HER-2 through feedback processes. These mechanisms could play a role in the observed cytoplasmic HER-2 overexpression without any gene amplification. However, their significance in oral cancers is uncertain.^[25]

HER-2 expression can have a significant impact on the pathogenesis of premalignant lesions and oral cancer, EGFR is known to be expressed in oral premalignant lesions, and HER-2 can form heterodimers with EGFR and activate tyrosine kinase pathway leading to PI3K overexpression.

Trastuzumab targets only membranous HER-2 receptors, however, neratinib, a relatively newer drug recently approved for breast cancer patients target intracellular tyrosine kinase and may possibly be used for premalignant/malignant lesions showing cytoplasmic HER-2 staining. For completely unraveling the role of HER-2 in oral premalignant and malignant lesions, follow-up studies with treatment and simultaneous measurement of HER-2 levels will be needed. Moreover, no clear-cut guidelines for HER-2 immunohistochemical expression reporting exist, this is in contrast to breast and gastric cancer where clear-cut guidelines exist. This leads to irregularities in reporting results.

Conclusions

Membranous expression in severe dysplasia and higher expression in oral cancer are in concordance with the multistep theory of carcinogenesis.

Disclosure

This material has never been published and is not currently under evaluation in any other peer-reviewed publication.

Ethical approval

The permission was taken from Institutional Ethics Committee before starting the project. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Financial support and sponsorship

This work was supported by Indian Council of Medical Research through its short term studentship scheme (STS number 2019-05752).

Conflicts of interest

There are no conflicts of interest.

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