HER2 immunohistochemical expression and its association with clinicopathological features of gastric adenocarcinoma in Uganda

Abstract

Objective: Despite the remarkable improvement in gastric adenocarcinoma treatment modalities, the prognosis of gastric adenocarcinoma remains poor. The purpose of this study was to determine the prevalence of HER2 immunohistochemical expression and its association with clinicopathological features of patients with gastric adenocarcinoma.

Material and Methods: This was a cross-sectional study which was conducted at the department of pathology. A total of 86 formalin fixed paraffin embedded tissue blocks of the patients who were confirmed histologically with gastric adenocarcinoma from January 2009 to December 2019 were included in the analysis. Laboratory requisition form and patients’ files were used to extract the clinical and pathological data of the cases. Immunohistochemistry to assess HER2 overexpression was done using monoclonal (SP3 clone) rabbit anti-HER2/neu (Thermo Fisher Scientific-USA). Chi-square statistical test was used to determine the association of the clinicopathological characteristics with HER2 expression. P< 0.05 was considered statistically significant.

Results: Mean age of the patients included in the study was 58.5 ± 14.3 years, and over half 54.7% (n= 47) of the patients were males. Poorly cohesive non-signet ring types contributed most (47.7%) (n= 41) of the cases, and diffuse/mixed histological subtypes were more prevalent (57%) (n= 49) subtypes. Poorly differentiated cases accounted for the majority (66.3%) (n= 57) of the cases. The prevalence of HER2 immunohistochemical expression was 8.1% (n= 7). None of the clinicopathological characteristics were associated with HER2 expression.

Conclusion: This study has shown almost every one in 10 patients with gastric adenocarcinoma may express HER2 when using immunohistochemistry test. However, the HER2 in this study was not associated with age, sex, tumor location, the nature of biopsy, histological subtypes, and tumor grade.

Keywords: Gastric adenocarcinoma, HER2 overexpression, prognosis, clinicopathological characteristics

Introduction

Gastric adenocarcinoma is the fifth most common malignancy globally accounting for 5.7% of all adenocarcinomas with an incidence of 11.4 per 100.000 population annually (1). It ranks third among the causes of adenocarcinoma-related deaths in both sexes worldwide, accounting for 8.2% of adenocarcinoma deaths with a rate of 8.45 per 100.000 population annually (1,2). There is a geographical variation in the incidence of gastric adenocarcinoma globally, and over 50% of the cases occur in developing countries (2,3). The highest incidence is in Eastern Asia, and the lowest in Central Africa (3). In Uganda, the incidence of gastric adenocarcinoma has increased by more than 11-fold from 0.8 per 100.000 population reported in the 1960s to nine per 100.000 population reported in 2014 (4,5). It is now one of the main causes of cancer-related deaths in Uganda with a mortality rate of 8.7 per 100.000 population (5). In most countries including Uganda, gastric adenocarcinoma is usually diagnosed at an advanced stage when curative surgical resection is no longer possible, which contributes to poor prognosis (6). Several poor prognostic factors such as diffuse histological subtype, high grade, and overexpression of proteins like human epidermal receptor 2 (HER2) among many others have been identified by independent studies (7,8).

HER2 protein, which is involved in various aspects of tumor cell biology, has previously been shown to be an independent poor prognostic factor in gastric adenocarcinoma (9). HER2 positive tumors behave more aggressively and have higher chances of recurrence than HER2 negative tumors (7,8). Moreover, HER2 protein overexpression in gastric adenocarcinoma is also considered a predictive biomarker for response to HER2 targeted therapy (7,10). The prognosis of HER2 positive gastric adenocarcinoma has been relatively improved by the use of the targeted therapies against HER2 (11). Nonetheless, a wide range of variation in the frequency of HER2 protein overexpression in gastric adenocarcinoma, ranging from 8% to 53.4%, which also with different populations, histological subtypes of gastric adenocarcinoma and tumor grade has been demonstrated by previous studies (12-14). The wide range of HER2 overexpression is attributed to many factors including geographical differences, tumor biology and heterogeneity, and methodological differences (14,15).

In Uganda, the prevalence of HER2 protein overexpression in gastric adenocarcinoma remains unknown. Therefore, this study aimed to determine the immunohistochemical expression of HER2 protein and its association with demographic and pathologic features of gastric adenocarcinoma.

Material and Methods

Study Design and Setting

This was a cross-sectional study design which was conducted at the department pathology, Makerere College of Health Sciences (MakCHS) in Kampala, Uganda. The college is located in the same area with Mulago hospital. Therefore, the department of pathology receives most of samples from Mulago National Hospital and also from other health facilities which perform surgery mainly within the central region where Kampala is located. The department serves the roles of teaching, research, as well as offering diagnostic services for histological, cytological and autopsy services within Kampala, other parts of the country, and other areas of the neighboring countries such as Kenya, Rwanda, and Democratic Republic of Congo.

Study Population

The study analysed archived formalin-fixed paraffin-embedded (FFPE) tissue blocks of patients who were diagnosed with various histological types of gastric adenocarcinoma from January 2009 to December 2019. Laboratory requisition forms and patients’ files were used to select the required cases. Cases with available clinical information and confirmed histological diagnosis and also available FFPE tissue blocks with good quality were retrieved and included in the study. However, cases with poorly fixed or processed FFPE tissue blocks, those with necrotic tissue blocks, cases with missing patients’ files, and those with prior history of chemotherapy and/or radiotherapy al were excluded from the analysis.

Sample Size Estimation

The sample size was determined using the Kish Leslie formula in which the sample size was calculated as n= Z² P (1-P)/e² ; n= sample size, z= standard normal deviate at a 95% confidence interval corresponding to 1.96, and e= margin of error of 5% (16). The p= prevalence of HER2 protein overexpression in gastric adenocarcinomas was assumed to be 42.2% from a study done in Kenya (17). Therefore, n= (1.96)² × 0.422 (0.578)/ (0.05)² = 0.9370/0.0025= 374.81. The possible population size for cases with gastric adenocarcinoma was estimated to be around 110. Therefore, the calculated sample size of approximately 375 was not possible which necessitated to apply the finite population correction formula so as to reduce the calculated sample size to an achievable sample size as follows: n= (calculated sample size x population size)/(calculated sample size) + (population size - 1). n= (374.81 x 110)/(374.81 + (110-1)). Therefore, n= 85.2. The obtained minimum sample size required for this study was approximately 86 (18).

Sampling Method

The required number of the cases were sampled using convenience sampling method, and all eligible number of FFPE tissue with confirmed histological diagnosis of gastric adenocarcinoma were retrieved consecutively from the archive of FFPE tissue blocks in the department of pathology. Retrieval of the FFPE tissue blocks was done by a laboratory technician using the inclusion criteria.

Haematoxylin and Eosin Staining

Each retrieved FFPE tissue block was processed and stained with haematoxylin and eosin (H,E) as it was previously done for re-confirmation of the previous diagnosis (19). Histological classification and grading of the tumors were done in accordance with the Lauren and World Health Organization 2010 report classification systems using an Olympus CX23 microscope (20,21). The evaluation of the microscopic tissue slides was done by two independent experienced pathologists.

Immunohistochemistry Staining

Immunohistochemistry (IHC) staining was performed to detect HER2 protein using a previously optimized protocol by a qualified technician (22). Monoclonal SP3 clone rabbit antiHER2/neu (Thermo Fisher Scientific, USA) antibody was used. A known HER2 positive breast adenocarcinoma case was used as positive control while omitting the primary antibody served as the negative control. The percentage of tumor cells with HER2 positivity was recorded along with the strength of staining as done in a previous study (23). Tumors with score 0 or score 1+ in which there was no membranous staining or weak staining detected in only one part of the membrane were considered as negative. Cases with moderate or weak complete or basolateral membranous staining were regarded equivocal cases and were given score of 2+. Tumors with score 3+ in which there was strong complete or basolateral membranous staining were considered positive for HER2 protein. Background staining and cytoplasmic staining were considered as nonspecific staining. Only complete or basolateral membranous staining was considered as true positive. The cut-off points of at least five cohesive, unequivocal tumor cells in endoscopic biopsies, and at least 10% of tumor cells in resection biopsies were used to evaluate HER2 over-expression.

Data Analysis

Data analysis was performed using STATA programme version 15.0. Continuous and categorical variables were presented in mean ± standard deviation and proportions, respectively. Pearson Chi-square statistical test was used to assess the association of the clinicopathological characteristics with HER2 immunohistochemical expression. P< 0.05 was considered to be statistically significant.

Results

Clinicopathological Characteristics of the Patients

Table 1 presents the clinicopathological characteristics of the patients. Mean age of the patients with gastric adenocarcinoma was 58.5 ± 14.3 years, with an age range of 28-87 years. Majority of the patients (74.4%) (n= 64) were older than 50 years, and over half (54.7%) (n= 47) of the patients were males, and the male to female ratio was 1.2:1. Most of the cases of gastric adenocarcinoma (52.3%) (n= 45) were non-cardia tumors and endoscopic biopsies dominated the specimens (62.8%) (n= 58). Regarding Lauren and WHO classification of the cases, 57.0% (n= 49) and 47.7% (n= 41) were of diffuse type and poorly non-cohesive signet ring type, respectively. Majority (66.3%) (n= 57) of the tumors were poorly differentiated adenocarcinomas. Various histopathological variants of gastric adenocarcinoma are shown in Figure 1.

Figure 1. A. The above micrograph shows sheets of neoplastic epithelial cells with marked cytological atypia diffusely invading a desmoplastic stroma. B. Photomicrograph showing well differentiated papillary/intestinal adenocarcinoma under H&E stain (x100). C. Photomicrograph showing cohesive signet ring carcinoma under H&E stain (x400), and D. photomicrograph showing moderately differentiated mucinous/intestinal adenocarcinoma under H&E stain (x100).

Age Distribution According to Sex of the Patients with Gastric Adenocarcinoma

The frequency of the gastric adenocarcinoma cases was not linear for both age and sex of the patients. There was a kind of trend to some extent, and there was an increase (35-39 years) and decrease (40-44 years) of the frequency of the cases with age and sex of the patients in the study. Moreover, at the age group of 55-59 years, there was a steep decrease (one case) in the frequency of cases among males whereas a significant increase (five cases) in the frequency of cases among females was noticeable. Conversely, at the age group of 65-69 years, there was a decrease (three cases) in the frequency of cases among females whereas a significant increase (eight cases) in the frequency of cases among males was observed (Figure 2).

Figure 2. Age distribution according to sex of the patients with gastric adenocarcinoma.

Prevalence of HER2 Immunohistochemical Overexpression

The variation of HER2 overexpression for the cases of gastric adenocarcinomas is presented in Figure 3. The prevalence of HER2 immunohistochemical overexpression was 8.1% (n= 7) (Figure 3a), all the other 91.9% (n= 79) of the cases were negative (both 0 and 1+ scores) (Figures 3b-d). There was intratumoral heterogeneity of HER2 over-expression in the current study, which occurred in 57% of cases. No equivocal cases were found in this series of tumors regarding HER2 expression.

Figure 3. A. Photomicrograph showing HER2 positive score 3+ of a poorly cohesive non-signet ring adenocarcinoma case (HER2 immunohistochemistry antibody x400), B. photomicrograph showing HER2 negative score 1+ of a poorly cohesive non-signet ring adenocarcinoma case (HER2 immunohistochemistry antibody, x200), C. photomicrograph showing heterogeneous HER2 negative score 1+ in a poorly cohesive non-signet ring adenocarcinoma case (HER2 immunohistochemistry antibody, x200), and D. photomicrograph showing HER2 negative score 0 of a well differentiated tubular adenocarcinoma case (HER2 immunohistochemistry antibody, x100).

Association of HER2 Overexpression with Clinicopathological Characteristics of the Patients

Table 2 shows the association of HER2 overexpression with clinicopathological characteristics. HER2 overexpression was higher in patients younger than or of 50 years of age (13.6%, 3/22), than in patients older than 50 years (6.3%, 4/64), although the difference was not statistically significant (p= 0.274). Four out of fortyseven (8.5%) gastric adenocarcinoma tumors from males overexpressed HER2 compared to 3/39 of tumors (7.7%) in females (p= 0.890). Tubular adenocarcinoma had the highest prevalence of HER2 positivity (17.4%, 4/23) compared to other histological subtypes, and we considered the difference was insignificant (p= 0.253). Considering Lauren classification, intestinal subtype of gastric adenocarcinomas had a higher rate of HER2 overexpression (13.5%, 5/37) than diffuse or mixed subtype (4.1%, 2/49), and the difference was also not statistically significant (p= 0.113). Regarding tumor grade, the moderately differentiated tumors had more HER2 protein overexpression (13.3%, 2/15) compared to either well differentiated (7.1%, 1/14,) or poorly differentiated cases (7.0%, 4/57) but the difference was not significant (p= 0.720). Also, the overexpression of HER2 protein was not associated with tumor location (p= 0.658) although all four tumors from the cardiac location were negative.

Discussion

This study aimed to determine the prevalence of HER2 immunohistochemical overexpression among gastric adenocarcinoma cases and to establish its association with clinicopathological features of gastric adenocarcinoma. The prevalence of HER2 overexpression in gastric adenocarcinoma in the present study was 8.1% which is in agreement with previous studies (15,24). A wide range of HER2 immunohistochemical expression of 8%-53.4% among cases of gastric adenocarcinoma has been reported in previous studies (24,25).

The reasons for such a wide range of HER2 expression reported in previous studies and extended by the current study could be due to the nature of biopsies, type of antibody clone, scoring system, and tumor heterogeneity (14,26). Among these, tumor heterogeneity could be the leading factor contributing to the wide variation in HER2 prevalence. Previously, tumor heterogeneity of about 30% of tumor cells exhibiting reactivity or only focal staining of tumor cells in at least 30% of HER2 positive cases has been reported in gastric adenocarcinoma (27). Differences in sensitivity and specificity among the antibodies might have contributed to the variation of HER2 protein overexpression (28). Also, previous studies considered only cases with score 3+ as positive, similar to our study, which provided a positivity ranging from 6% to 10.5% (29,30). However, studies where tumors even with scores 2+ were considered positive reported relatively higher HER2 immunohistochemical overexpression rates. For instance, a study by Barros-Silva et al. showed 9.3% HER2 positivity after combining 3.9% which were score 2+ cases and 5.5% for score 3+ cases (31). Likewise, Van Trung reported 11.7% of HER2 positivity after combining 6.9% which were score 2+ cases with 4.8% score 3+ cases (32).

The lack of association between HER2 immunohistochemical overexpression with all demographic and pathologic characteristics (age, sex, tumor site, grade, and histological subtype) in this study has also been reported in previous studies, though other studies have reported association of HER2 expression with some of the demographic and pathologic characteristics (33,34). This is likely because in the study of Li et al. it was also found that HER2 protein overexpression which is involved in tumor progression was not associated with age of the patients (35). Although the prevalence of HER2 expression was slightly higher in males compared to females, the difference was not statistically significant, similar to other studies (27,33,36,37). On the contrary, a study done in India found an association between HER2 overexpression and sex of the patients (27). The prevalence of HER2 immunohistochemical overexpression varied with WHO histological subtypes, in which tubular adenocarcinoma showed the highest HER2 overexpression, but the difference was not statistically significant, contrary to the findings in the previous studies (36,37). The intestinal subtype that was least frequent in the current study had a higher HER2 overexpression than the diffuse/mixed subtypes as reported by previous studies (36,38). This could partly explain the low prevalence of HER2 immunohistochemical overexpression in this study compared to previous studies which reported a higher proportion of the intestinal subtype of gastric adenocarcinoma than the diffuse/ mixed subtypes (24,33,39). Unlike the finding in the current study, Kataoka et al. and Takehana et al. reported a positive association between histological type and HER2 expression (36,37).

Regarding association of tumor grade with HER2 expression, in this study, moderately differentiated adenocarcinomas showed a higher prevalence of HER2 positivity than poorly differentiated adenocarcinomas, though the difference was not significant similar to previous studies (27,36). However, some studies have shown an association between HER2 overexpression and tumor grade (36,40). This is probably due to the different antibody clones used for HER2 testing. Out of the seven cases with positive HER2 protein overexpression, three were noncardia, while none of the cardia tumors showed reactivity. Compared with a previous study that showed a significant association between HER2 overexpression and tumor location, the current study is in agreement with another studies, which found no significant association (27,41). HER2 overexpression was more prevalent among endoscopic biopsy specimens (11.1%) than surgical biopsies (3.1%) This was consistent with the findings of previous studies (27,42), which also suggested that since small biopsy specimens fix much quicker than surgical biopsies with less ischemic time, antigens are preserved better resulting in higher positivity (41). Consistent with previous studies, there was no significant association between HER2 overexpression and nature of biopsy, probably because of the similarity of small sample size compared to some studies that showed significant association (27,41).

Study Limitations

We faced difficulty in obtaining a large number of FFPE tissue blocks from the tissue blocks repository due to poor storage despite the identification of potential cases from the laboratory requisition forms and patients’ files. Incomplete information because of inadequately filled request forms with some important variables lacking like tumor location, incomplete clinical history on including a history of previous chemotherapy or radiotherapy contributed to failure to associate such factors with HER2 overexpression. Also, inappropriate tissue fixation might have affected antigen retrieval during IHC staining. Endoscopic biopsies contained one or two samples per patient which were not enough to represent the tumor.

Conclusion

The present study shows that almost one in 10 patients with gastric adenocarcinoma tumors in Uganda are more likely to have HER2 expression, and for that reason, they may benefit from targeted therapy that specifically targets inhibition of HER2. Moreover, HER2 immunohistochemical overexpression in this study was not significantly associated with age, sex, tumor location, nature of biopsy, histological subtype, and tumor grade. Larger scale prospective studies should be conducted to evaluate HER2 immunohistochemical overexpression in gastric adenocarcinoma cases in Uganda to obtain more generalizable results.

Cite this article as: Kahiye M, Yahaya J, Kalungi S, Nalwoga H. HER2 immunohistochemical expression and its association with clinicopathological features of gastric adenocarcinoma in Uganda. Turk J Surg 2024; 40 (4): 328- 335.

This study was approved by Makerere University College of Health Sciences School of Biomedical Sciences Research and Ethics Committee (Decision no: SBS-713, Date: 30.01.2020).

Externally peer-reviewed.

Concept - MK; Design - MK; Supervision - HN, SK; Fundings - MK; Materials - JY; Data Collection and/or Processing - MK, HN; Analysis and/or Interpretation - JY, SK; Literature Search - MK; Writing Manuscript - JY; Critical Reviews - All of authors.

The authors have no conflicts of interest to declare.

The authors declared that this study has received no financial support.

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