Gastrointestinal (GI) tract malignancies constitute a major global health burden and remain among the leading causes of cancer-related morbidity and mortality. Carcinomas arising from the esophagus, gastroesophageal junction (GEJ), stomach, small intestine, colon, rectum, and anal canal account for a substantial proportion of newly diagnosed cancers worldwide. Early diagnosis and accurate histopathological characterization are essential for appropriate therapeutic planning and improved patient survival. The widespread use of flexible gastrointestinal endoscopy has revolutionized the diagnosis of GI diseases by enabling direct visualization of mucosal lesions and targeted biopsy sampling, thereby improving diagnostic accuracy compared with blind tissue sampling. Endoscopic biopsy remains the gold standard for the diagnosis of gastrointestinal neoplasms because it provides adequate tissue for histopathological examination, tumor typing, grading, and ancillary immunohistochemical studies.^[1]

Histopathological evaluation not only confirms malignancy but also determines tumor subtype, degree of differentiation, and other prognostic parameters that guide patient management. However, with the emergence of personalized medicine, molecular biomarkers have become increasingly important in identifying patients who may benefit from targeted therapies. Among these biomarkers, Human Epidermal Growth Factor Receptor-2 (HER2/neu), encoded by the ERBB2 proto-oncogene located on chromosome 17q12, has gained considerable attention. HER2 is a transmembrane receptor tyrosine kinase belonging to the epidermal growth factor receptor family. Gene amplification leads to overexpression of HER2 protein, resulting in activation of intracellular signaling pathways responsible for cell proliferation, differentiation, angiogenesis, invasion, and inhibition of apoptosis.^[2]

HER2 overexpression is well established in breast carcinoma, where targeted therapy with trastuzumab has significantly improved survival. Similar therapeutic benefits have subsequently been demonstrated in gastric and gastroesophageal junction adenocarcinomas exhibiting HER2 overexpression. The landmark ToGA trial established HER2 as a predictive biomarker in advanced gastric and GEJ cancers and demonstrated improved overall survival with trastuzumab combined with chemotherapy in HER2-positive tumors. Consequently, HER2 testing has become a routine recommendation in advanced gastric and GEJ adenocarcinoma.^[3]

Although HER2 expression has been extensively investigated in gastric carcinoma, studies evaluating its expression across the entire spectrum of gastrointestinal carcinomasincluding esophageal, colorectal, duodenal, and anal carcinomashave reported variable frequencies and inconsistent clinicopathological associations. HER2 positivity appears to be more common in intestinal-type gastric adenocarcinoma and gastroesophageal junction tumors than in diffuse gastric carcinoma, while its significance in colorectal and esophageal squamous cell carcinomas remains controversial. Standardized immunohistochemical assessment using the College of American Pathologists (CAP) guidelines has improved reproducibility and clinical interpretation of HER2 expression.^[4]

AIM

To evaluate HER2/neu overexpression in gastrointestinal carcinomas diagnosed on endoscopic biopsy specimens using immunohistochemistry and correlate it with histopathological findings.

OBJECTIVES

1. To study the histopathological spectrum of gastrointestinal carcinomas diagnosed on endoscopic biopsy specimens.
2. To determine HER2/neu expression in gastrointestinal carcinomas using immunohistochemistry.
3. To correlate HER2/neu expression with the histological type, site, and degree of tumor differentiation.

Source of Data The data were obtained from all endoscopic gastrointestinal biopsy specimens received in the Department of Pathology, M.R. Medical College, Kalaburagi, attached to Basaveshwar Teaching and General Hospital. Clinical details were obtained from pathology requisition forms, hospital records, and endoscopy reports. Both retrospective and prospective cases fulfilling the eligibility criteria were included. Study Design The study was conducted as a hospital-based retrospective and prospective observational study. Study Location The study was carried out in the Department of Pathology, M.R. Medical College, Kalaburagi, attached to Basaveshwar Teaching and General Hospital, Karnataka. Study Duration The study was conducted over five years, comprising three years of retrospective analysis and two years of prospective analysis, from 1 September 2017 to 31 August 2022. Sample Size A total of 357 endoscopic gastrointestinal biopsy specimens were included in the study. Among these, • 357 endoscopic biopsies underwent routine histopathological evaluation. • 78 histologically confirmed carcinoma cases were subjected to HER2/neu immunohistochemical analysis. Inclusion Criteria • All endoscopic biopsy specimens obtained from the gastrointestinal tract. • Biopsies from patients of all age groups and both sexes. • Histologically adequate biopsy specimens. • Carcinoma cases with sufficient tissue for HER2 immunohistochemistry. Exclusion Criteria • Autolyzed biopsy specimens. • Inadequate or poorly preserved biopsy samples. • Biopsies lacking representative lesional tissue. • Cases with insufficient tissue for immunohistochemical analysis. Procedure and Methodology All endoscopic biopsy specimens received in the pathology department were accessioned and assigned unique laboratory numbers. Clinical information including age, sex, presenting symptoms, anatomical site, and endoscopic findings was recorded. Gross examination was performed to assess specimen number, size, color, consistency, and adequacy. The biopsy specimens were fixed in 10% neutral buffered formalin immediately after receipt. Following fixation, tissues were processed using a routine automated tissue processor, embedded in paraffin wax, and sectioned at 4-5 µm thickness using a rotary microtome. The sections were stained with Hematoxylin and Eosin (H&E) for histopathological examination. Histopathological diagnosis was established according to the WHO classification of gastrointestinal tumors. Tumors were categorized according to anatomical site, histological subtype, and degree of differentiation. All 78 carcinoma cases were subsequently subjected to HER2/neu immunohistochemical evaluation. Antigen retrieval was performed using Tris buffer in a heat retrieval system. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide, followed by incubation with mouse monoclonal primary anti-HER2 antibody. Detection was carried out using a polymer-based detection system and visualization with 3,3'-diaminobenzidine (DAB) chromogen. Counterstaining was performed with hematoxylin. HER2-positive breast carcinoma tissue served as the positive control, while negative controls were processed by omitting the primary antibody. HER2 immunoreactivity was evaluated according to the College of American Pathologists (CAP) gastric HER2 scoring guidelines, and cases were classified as scores 0, 1+, 2+, or 3+ based on membranous staining intensity and distribution. Scores 2+ and 3+ were considered positive/equivocal for analytical purposes. Sample Processing The biopsy specimens were: • Fixed in 10% neutral buffered formalin. • Processed using routine paraffin tissue processing. • Embedded in paraffin wax. • Sectioned at 4-5 μm thickness. • Stained with Hematoxylin and Eosin. • Subjected to HER2 immunohistochemistry for carcinoma cases. • Evaluated independently by experienced pathologists under light microscopy according to CAP guidelines. Statistical Methods The collected data were entered into Microsoft Excel and analyzed using SPSS software (Version 25.0 or later). Continuous variables were expressed as mean ± standard deviation (SD), whereas categorical variables were summarized as frequencies and percentages. Associations between HER2/neu expression and clinicopathological variables were assessed using the Chi-square test or Fisher's exact test, as appropriate. A p-value <0.05 was considered statistically significant. Data Collection Data were collected using a structured proforma containing: • Patient demographics (age and sex) • Clinical presentation • Endoscopic findings • Anatomical site of biopsy • Histopathological diagnosis • Histological subtype • Tumor differentiation • HER2 immunohistochemical score (0, 1+, 2+, 3+) The collected information was compiled, verified, coded, and analyzed to evaluate the relationship between HER2/neu overexpression and various clinicopathological parameters of gastrointestinal carcinomas.

Table 1: Demographic profile and HER2/neu overexpression among gastrointestinal carcinoma cases (N = 78)

Table 1 summarizes the demographic characteristics of 78 patients with gastrointestinal carcinomas and their association with HER2/neu overexpression. The mean age of the study population was 56.97 ± 14.15 years. Patients with HER2-negative tumors had a mean age of 57.52 ± 13.27 years, whereas those with HER2-positive tumors had a slightly lower mean age of 53.64 ± 19.14 years. However, this difference was not statistically significant (Welch's t = −0.65, 95% CI: −16.99 to 9.22 years, p = 0.529). The highest proportion of patients belonged to the 51-60 years age group (22 cases, 28.21%), followed by the 61-70 years age group (21 cases, 26.92%) and 41-50 years (16 cases, 20.51%). HER2 positivity was observed across different age groups without any significant association (Fisher-Freeman-Halton exact test, p = 0.648). Males constituted the majority of the study population (47 cases, 60.26%), while females accounted for 31 cases (39.74%). HER2 positivity was identified in 14.9% of males and 12.9% of females. There was no statistically significant association between sex and HER2 overexpression (OR = 1.18; 95% CI: 0.31-4.43; Fisher's exact test, p = 1.000). Overall, 67 cases (85.90%) were HER2-negative and 11 cases (14.10%) demonstrated HER2 positivity, with the predominance of HER2-negative tumors being highly significant (χ² = 40.21, p < 0.001).

Table 2: Histopathological spectrum of gastrointestinal carcinomas diagnosed on endoscopic biopsy specimens (N = 78)

Table 2 presents the histopathological distribution of gastrointestinal carcinomas diagnosed on endoscopic biopsy specimens. Esophageal carcinoma and gastric carcinoma were the most frequently encountered malignancies, each accounting for 24 cases (30.77%), followed by carcinoma of the colon (14 cases, 17.95%), gastroesophageal junction (7 cases, 8.97%), rectum (6 cases, 7.69%), and isolated cases involving the duodenum, rectosigmoid region, and anal canal (1.28% each). The anatomical distribution of tumors differed significantly from an equal distribution (χ² = 69.28, p < 0.001). Histologically, adenocarcinoma was the predominant tumor type, comprising 53 cases (67.95%), whereas squamous cell carcinoma accounted for 25 cases (32.05%). This predominance of adenocarcinoma was statistically significant (χ² = 10.05, p = 0.002). With regard to tumor differentiation, moderately differentiated carcinomas represented the overwhelming majority (64 cases, 82.05%), followed by poorly differentiated tumors (10 cases, 12.82%) and well-differentiated tumors (3 cases, 3.85%), while differentiation was not specified in one case (1.28%). The distribution of tumor differentiation was highly significant (χ² = 137.69, p < 0.001).

Table 3: HER2/neu immunohistochemical expression among gastrointestinal carcinomas (N = 78)

Table 3 depicts the pattern of HER2/neu immunohistochemical expression among gastrointestinal carcinomas. The majority of tumors demonstrated an IHC score of 0, indicating absence of membranous staining, which was observed in 54 cases (69.23%). An additional 13 cases (16.67%) showed IHC score 1+, representing faint membranous staining and were also considered HER2-negative. Nine cases (11.54%) demonstrated an IHC score of 2+, categorized as equivocal, whereas 2 cases (2.56%) exhibited IHC score 3+, indicating strong complete membranous staining and definitive HER2 positivity. The overall distribution of HER2 IHC scores differed significantly (χ² = 84.56, p < 0.001). When scores were grouped according to HER2 status, 67 tumors (85.90%) were classified as HER2-negative (scores 0 and 1+), while 11 tumors (14.10%) belonged to the HER2 overexpression/equivocal-positive group (scores 2+ and 3+), a difference that was also highly significant (χ² = 40.21, p < 0.001).

Table 4: Correlation of HER2/neu expression with histological type, anatomical site, and tumor differentiation (N = 78)

Table 4 evaluates the association between HER2/neu expression and various clinicopathological characteristics of gastrointestinal carcinomas. Among adenocarcinomas, 45 cases (84.9%) were HER2-negative and 8 cases (15.1%) were HER2-positive, whereas 22 squamous cell carcinomas (88.0%) were HER2-negative and 3 cases (12.0%) were HER2-positive. Although HER2 positivity was numerically higher in adenocarcinomas (15.09%) than squamous cell carcinomas (12.00%), the difference was not statistically significant (OR = 1.30; 95% CI: 0.31-5.40; Fisher's exact test, p = 1.000). Analysis according to anatomical site revealed the highest HER2 positivity in gastroesophageal junction carcinomas (28.57%), followed by colonic carcinomas (21.43%), rectal carcinomas (16.67%), gastric carcinomas (12.50%), and esophageal carcinomas (8.33%), whereas no HER2-positive tumors were observed in the duodenum, rectosigmoid region, or anal canal. Nevertheless, none of these site-specific differences reached statistical significance, and the overall association between tumor site and HER2 expression was not significant (Fisher-Freeman-Halton exact test, p = 0.724). With respect to tumor differentiation, HER2 positivity was observed in 20.00% of poorly differentiated tumors, 14.06% of moderately differentiated tumors, and in none of the well-differentiated tumors. Although poorly differentiated carcinomas demonstrated the highest proportion of HER2 positivity (OR = 1.64; 95% CI: 0.30-8.98), this association was also not statistically significant (p = 0.626), and the overall association between HER2 expression and tumor differentiation remained non-significant (p = 1.000). These findings indicate that, despite a trend toward increased HER2 expression in gastroesophageal junction, colonic, and poorly differentiated tumors, HER2 overexpression did not show a statistically significant correlation with histological type, anatomical site, or tumor differentiation in the present study.

The present study evaluated HER2/neu immunohistochemical expression in 78 gastrointestinal carcinomas diagnosed on endoscopic biopsy specimens and correlated the findings with age, sex, anatomical site, histological type, and tumor differentiation. HER2/neu overexpression, defined for the study as an IHC score of 2+ or 3+, was identified in 11 cases (14.10%). However, according to contemporary gastroesophageal HER2-testing criteria, only an IHC score of 3+ is independently considered positive, while an IHC score of 2+ is equivocal and requires confirmation by in-situ hybridization. Therefore, the combined 2+/3+ rate in the present study should be interpreted as immunoreactive or potentially HER2-positive rather than definitively amplified HER2-positive disease. Bartley et al. (2017)[2] similarly recommended reflex in-situ hybridization for IHC 2+ gastroesophageal adenocarcinomas to ensure accurate selection for HER2-targeted therapy. Demographic profile and HER2/neu expression The mean age of the study participants was 56.97±14.15 years. Patients with HER2-negative tumors had a mean age of 57.52±13.27 years, compared with 53.64±19.14 years among HER2-positive patients. The mean difference was not statistically significant (Welch’s t=−0.65; p=0.529). Most patients belonged to the 51-60-year (28.21%) and 61-70-year (26.92%) age groups, indicating that gastrointestinal carcinomas predominantly affected middle-aged and older adults. This pattern is consistent with the established epidemiology of gastrointestinal malignancies, which shows increasing incidence with advancing age, although variations occur according to tumor site, population structure, and regional risk factors. Arnold et al. (2020)[1] demonstrated that cancers of the esophagus, stomach, colorectum, liver, and pancreas together constitute a major global cancer burden and occur predominantly in older populations. The absence of a significant association between age group and HER2 expression in the present study (p=0.648) agrees with several investigations in which HER2 status was not independently related to age. Heidarpour et al. (2020)[5], while evaluating HER2 expression in esophageal squamous cell carcinoma, reported substantial variability in HER2 immunoreactivity but did not establish age as a consistent predictor of expression. Similarly, Benli Işık and Barut (2020)[7] found no significant relationship between HER2 expression and most clinicopathological characteristics in colorectal carcinoma, except for distant metastasis. Males constituted 60.26% of the study population, producing a male-to-female ratio of approximately 1.5:1. HER2 positivity was marginally higher among males than females14.9% versus 12.9%but the association was not statistically significant (OR=1.18; 95% CI: 0.31-4.43; p=1.000). Male predominance is commonly observed in upper gastrointestinal malignancies, particularly esophageal and gastroesophageal-junction cancers, owing to differences in exposure to tobacco, alcohol, dietary carcinogens, obesity, and gastroesophageal reflux. Nagaraja et al. (2015)[3] noted that HER2-positive gastric and esophageal tumors were reported more frequently in men in some studies, but the association was inconsistent across populations. The absence of a significant sex-based difference in the present study may reflect the relatively small number of HER2-positive cases and the inclusion of carcinomas from multiple gastrointestinal sites with differing biological profiles. Histopathological spectrum of gastrointestinal carcinomas Esophageal and gastric carcinomas were the most frequent malignancies, each accounting for 24 cases (30.77%). These were followed by colonic carcinoma in 14 cases (17.95%), gastroesophageal-junction carcinoma in seven cases (8.97%), rectal carcinoma in six cases (7.69%), and one case each involving the duodenum, rectosigmoid region, and anal canal. The site-wise distribution was significantly unequal (χ²=69.28; p<0.001), demonstrating a clear predominance of upper gastrointestinal carcinomas in the endoscopic biopsy material. The high representation of esophageal and gastric tumors may be attributable to the hospital’s referral pattern, regional exposure to risk factors, and frequent use of upper gastrointestinal endoscopy in patients presenting with dysphagia, upper abdominal pain, vomiting, weight loss, anemia, or gastrointestinal bleeding. Arnold et al. (2020)[1] emphasized that the burden of individual gastrointestinal cancers differs markedly between regions. Consequently, hospital-based biopsy distributions may not precisely reflect population-level incidence but are useful for describing the diagnostic workload and histological spectrum encountered in clinical practice. Adenocarcinoma was the predominant histological type, accounting for 53 cases (67.95%), whereas squamous cell carcinoma constituted 25 cases (32.05%). The predominance of adenocarcinoma was statistically significant (χ²=10.05; p=0.002). This finding was expected because adenocarcinoma represents the principal malignant epithelial tumor of the stomach, gastroesophageal junction, small intestine, colon, and rectum. In contrast, squamous cell carcinoma primarily arises in the esophagus and anal canal. The inclusion of gastric and colorectal carcinomas therefore increased the overall proportion of adenocarcinoma. Most tumors were moderately differentiated (82.05%), followed by poorly differentiated (12.82%) and well-differentiated carcinomas (3.85%). This distribution was highly significant (χ²=137.69; p<0.001). Moderately differentiated morphology is commonly encountered in endoscopic biopsy specimens because many gastrointestinal carcinomas retain partial glandular or squamous differentiation despite architectural and cytological atypia. However, biopsy-based grading can be affected by sampling limitations and intratumoral heterogeneity, as a small biopsy may not represent the most poorly differentiated component of the entire tumor. HER2/neu immunohistochemical expression In the present study, 54 tumors (69.23%) had an IHC score of 0, while 13 (16.67%) had a score of 1+, nine (11.54%) had a score of 2+, and two (2.56%) had a score of 3+. Thus, 67 cases (85.90%) were negative by IHC scores 0/1+, while 11 cases (14.10%) were placed in the combined 2+/3+ immunoreactive group. The distribution of IHC scores was significantly skewed toward score 0 (χ²=84.56; p<0.001). The overall 14.10% immunoreactivity rate falls within the broad range reported for gastrointestinal carcinomas. Nagaraja et al. (2015)[3] found substantial variability in HER2 prevalence across gastric and esophageal studies, resulting from differences in tumor site, histological subtype, antibodies, tissue type, scoring criteria, and confirmatory testing. Their review showed that HER2 plays an important role in a subset of gastric and esophageal cancers, although the reported association with prognosis remained inconsistent. Only two cases in the present study demonstrated strong IHC 3+ positivity, corresponding to a definitive immunohistochemical positivity rate of 2.56%. The remaining nine immunoreactive cases were IHC 2+ and should ideally have undergone reflex fluorescence or silver in-situ hybridization. Bartley et al. (2017)[2] recommended that gastroesophageal adenocarcinomas with IHC 2+ expression be tested by in-situ hybridization, while IHC 3+ tumors may be reported as HER2-positive without additional confirmation. Therefore, failure to perform in-situ hybridization may overestimate the clinically actionable HER2-positive rate when IHC 2+ tumors are included as positive. The predominantly negative staining pattern may also reflect the biological heterogeneity of HER2 expression in gastrointestinal tumors. HER2 staining is often heterogeneous in gastric and gastroesophageal-junction adenocarcinomas, particularly in small endoscopic biopsies. A tumor may contain both strongly positive and completely negative areas; consequently, limited tissue sampling may cause either underestimation or overestimation of expression. This emphasizes the importance of obtaining multiple biopsy fragments from different regions of a suspected tumor and carefully evaluating basolateral or lateral membranous staining according to gastroesophageal-specific criteria. Correlation with histological type HER2 immunoreactivity was observed in eight of 53 adenocarcinomas (15.09%) and three of 25 squamous cell carcinomas (12.00%). Although adenocarcinoma showed a slightly higher positivity rate, the difference was not statistically significant (OR=1.30; 95% CI: 0.31-5.40; p=1.000). The wide confidence interval indicates uncertainty due to the limited number of positive cases. The observed 12.0% positivity among squamous cell carcinomas is broadly consistent with the variable expression reported in esophageal squamous cell carcinoma. Egebjerg et al. (2021)[6], in a systematic review and meta-analysis of 18 studies involving 1,505 patients, estimated the prevalence of IHC 3+ HER2 overexpression at 6%, IHC 2+ expression at 10%, and overall HER2 positivity after appropriate confirmatory testing at 8.6%. These pooled findings are close to the present study’s combined immunoreactivity in squamous tumors and demonstrate that HER2-positive esophageal squamous cell carcinoma represents a small but potentially relevant subset. Rong et al. (2020)[4] reported a relatively low frequency of HER2 overexpression in a large series of Chinese esophageal squamous cell carcinomas, supporting the concept that strong HER2 positivity is uncommon in this tumor type. Heidarpour et al. (2020)[5] also demonstrated that HER2 expression in esophageal squamous cell carcinoma varies widely and that its relationship with clinicopathological parameters and survival remains uncertain. Differences between studies may reflect the use of breast versus gastric scoring systems, varying thresholds for positivity, inclusion or exclusion of IHC 2+ cases, and lack of confirmatory gene-amplification analysis. Correlation with anatomical site The highest HER2 immunoreactivity was observed in gastroesophageal-junction carcinomas (28.57%), followed by colonic (21.43%), rectal (16.67%), gastric (12.50%), and esophageal carcinomas (8.33%). No positive expression was observed in the single duodenal, rectosigmoid, or anal-canal cases. However, the overall association between anatomical site and HER2 status was not statistically significant (Fisher-Freeman-Halton p=0.724). The relatively high proportion in gastroesophageal-junction tumors is consistent with reports that HER2 overexpression is more frequent in gastroesophageal-junction and proximal gastric adenocarcinomas than in distal or diffuse-type gastric tumors. Nagaraja et al. (2015)[3] observed wide variation in reported gastric and esophageal HER2 rates but noted the biological and therapeutic importance of HER2-positive gastroesophageal tumors. Bartley et al. (2017)[2] consequently recommended HER2 assessment in patients with advanced gastroesophageal adenocarcinoma who may be candidates for HER2-directed therapy. The 21.43% immunoreactivity rate in colonic carcinomas was higher than the approximately 4% definitive HER2 positivity reported by Yagisawa et al. (2021)[8] among 370 patients with metastatic colorectal cancer. In that study, HER2 positivity was defined more stringently as IHC 3+ or IHC 2+ with positive fluorescence in-situ hybridization. Thus, the higher rate in the present study may largely reflect the inclusion of unconfirmed IHC 2+ tumors. Benli Işık and Barut (2020)[7] reported IHC 3+, 2+, and 1+ expression in 13%, 21%, and 16% of 123 colorectal carcinomas, respectively. They found that HER2 positivity was associated with distant metastasis but not with most other clinicopathological variables. Their comparatively high expression rate illustrates the influence of methodology and scoring criteria on reported prevalence. Achalla et al. (2022)[9], in a review of HER2 in colorectal carcinoma, similarly concluded that published frequencies vary greatly and that standardized testing is required before inter-study comparisons can be considered reliable. Jang et al. (2023)[11] identified HER2-positive tumors in 9.3% of 108 patients with metastatic colorectal carcinoma and found no significant difference in response to standard chemotherapy, progression-free survival, or overall survival between HER2-positive and HER2-negative groups. These findings support the present observation that HER2 positivity may occur in colorectal carcinoma without necessarily showing a strong relationship with routine clinicopathological variables. Correlation with tumor differentiation HER2 immunoreactivity was observed in 20.00% of poorly differentiated carcinomas and 14.06% of moderately differentiated carcinomas, while none of the three well-differentiated tumors was positive. Despite the numerically higher rate among poorly differentiated tumors, no statistically significant association was found (OR=1.64; 95% CI: 0.30-8.98; p=0.626). The overall association between HER2 expression and differentiation was also non-significant. Published results regarding HER2 expression and tumor grade are inconsistent. Some gastric and gastroesophageal studies have shown higher HER2 expression in well or moderately differentiated intestinal-type adenocarcinomas, whereas other studies have reported increased expression in high-grade or aggressive tumors. Nagaraja et al. (2015)[3] noted that HER2-positive gastric tumors were more commonly intestinal-type and relatively well differentiated, although heterogeneity between studies was substantial. In contrast, some esophageal studies have associated increasing HER2 expression with higher grade, invasion, or nodal metastasis. These inconsistent observations suggest that the relationship between HER2 and differentiation is influenced by anatomical site and histological subtype and should not be generalized across all gastrointestinal carcinomas. The lack of significant clinicopathological correlations in the present study may be explained by the small number of HER2-immunoreactive tumors, unequal distribution of cases across anatomical sites, and inclusion of biologically distinct squamous cell carcinomas and adenocarcinomas in a single analysis. Furthermore, most tumors were moderately differentiated, limiting the statistical power to detect differences between grades. Despite its low prevalence, identifying HER2-positive gastrointestinal carcinoma has important therapeutic implications. Siena et al. (2018)[10] described HER2 as an actionable oncogenic driver in a molecularly selected subgroup of colorectal cancers. Siena et al. (2021)[12] subsequently demonstrated clinically meaningful activity of trastuzumab deruxtecan in previously treated HER2-positive metastatic colorectal cancer. More broadly, Meric-Bernstam et al. (2024)[13] reported durable responses to trastuzumab deruxtecan across several HER2-expressing solid tumors, supporting biomarker-directed treatment beyond conventional organ-specific classifications.

The present study demonstrated that HER2/neu overexpression was identified in a minority of gastrointestinal carcinomas diagnosed on endoscopic biopsy specimens. Among the 78 carcinoma cases, 67 (85.90%) were HER2-negative with IHC scores of 0 or 1+, while 11 (14.10%) showed IHC scores of 2+ or 3+. Strong HER2 positivity with an IHC score of 3+ was observed in only 2 cases (2.56%), whereas 9 cases (11.54%) showed equivocal 2+ staining. Adenocarcinoma was the predominant histological type, and most tumors were moderately differentiated. Esophageal and gastric carcinomas were the most frequently encountered lesions.

HER2/neu immunoreactivity was numerically higher in gastroesophageal-junction, colonic, rectal, and poorly differentiated carcinomas; however, no statistically significant association was found between HER2 expression and age, sex, anatomical site, histological type, or degree of tumor differentiation. These findings indicate that HER2 overexpression cannot be reliably predicted from routine demographic or histopathological parameters alone. Therefore, direct immunohistochemical assessment is necessary for identifying potentially eligible patients for HER2-targeted therapy.

Although only a small proportion of cases demonstrated definite or equivocal HER2 expression, its detection remains clinically important because HER2-directed treatment may improve outcomes in selected patients. Cases with IHC score 2+ should undergo confirmatory in-situ hybridization before being classified as definitively HER2-positive. Standardized testing, appropriate quality control, adequate biopsy sampling, and adherence to validated scoring criteria are essential for accurate HER2 evaluation in gastrointestinal carcinomas.

Limitations of the Study

1. The study was conducted at a single tertiary-care centre, which limited the generalizability of the findings to other populations and geographical regions.
2. The number of gastrointestinal carcinoma cases subjected to HER2 immunohistochemistry was relatively small, particularly within individual anatomical and histological subgroups.
3. The distribution of cases across different gastrointestinal sites was unequal, with very few cases from the duodenum, rectosigmoid region, and anal canal.
4. Endoscopic biopsy specimens were used, and these small samples might not have represented the entire tumor because of intratumoral heterogeneity.
5. HER2 expression in gastrointestinal carcinomas may be heterogeneous; therefore, limited biopsy material could have resulted in underestimation or overestimation of HER2 status.
6. Cases with IHC score 2+ were included in the combined immunoreactive group, but confirmatory fluorescence or silver in-situ hybridization was not performed.
7. The study did not assess HER2 gene amplification or molecular alterations using in-situ hybridization or other molecular techniques.

Clinical stage, lymph-node involvement, distant metastasis, treatment response, recurrence, and survival outcomes were not evaluated.  

1. Arnold M, Abnet CC, Neale RE, Vignat J, Giovannucci EL, McGlynn KA, et al. Global burden of 5 major types of gastrointestinal cancer. Gastroenterology. 2020;159(1):335-349.e15.
2. Bartley AN, Washington MK, Colasacco C, Ventura CB, Ismaila N, Benson AB 3rd, et al. HER2 testing and clinical decision making in gastroesophageal adenocarcinoma: guideline from the College of American Pathologists, American Society for Clinical Pathology, and American Society of Clinical Oncology. Arch Pathol Lab Med. 2017;141(10):1345-63.
3. Nagaraja V, Eslick GD. HER2 expression in gastric and oesophageal cancer: a meta-analytic review. J Gastrointest Oncol. 2015;6(2):143-54.
4. Rong L, Liu Y, Hui Z, Zhao Z, Zhang Y, Wang B, et al. HER2 expression and relevant clinicopathological features in esophageal squamous cell carcinoma in a Chinese population. Diagn Pathol. 2020;15:27.
5. Heidarpour M, Taheri M, Akhavan A, Goli P, Kefayat A. Investigation of HER-2 expression and its correlation with clinicopathological parameters and overall survival of esophageal squamous cell carcinoma patients. Iran J Pathol. 2020;15(4):274-81.
6. Egebjerg K, Garbyal RS, Hasselby JP, Baeksgaard L, Mau-Sørensen M. Prevalence of HER2 overexpression and amplification in squamous cell carcinoma of the esophagus: a systematic review and meta-analysis. Crit Rev Oncol Hematol. 2021;161:103339.
7. Benli Işık C, Barut SG. Evaluation of the prevalence of HER-2 expression and its relationship with prognostic parameters in colorectal carcinoma. Istanbul Med J. 2020;21(3):207-12.
8. Yagisawa M, Sawada K, Nakamura Y, Fujii S, Yuki S, Komatsu Y, et al. Prognostic value and molecular landscape of HER2 low-expressing metastatic colorectal cancer. Clin Colorectal Cancer. 2021;20(2):113-120.e1.
9. Achalla LSV, Shinde RK, Jogdand S, Vodithala S. Review of the role of HER2/neu in colorectal carcinomas. Cureus. 2022;14(5):e25409.
10. Siena S, Sartore-Bianchi A, Marsoni S, Hurwitz HI, McCall SJ, Penault-Llorca F, et al. Targeting the human epidermal growth factor receptor 2 oncogene in colorectal cancer. Ann Oncol. 2018;29(5):1108-19.
11. Jang JY, Kim S, Kim JH, Lee J, Park SH, Park JO, et al. Effect of human epidermal growth factor receptor 2 overexpression in metastatic colorectal cancer on standard chemotherapy outcomes. J Gastrointest Oncol. 2023;14(5):2130-40.
12. Siena S, Di Bartolomeo M, Raghav K, Masuishi T, Loupakis F, Kawakami H, et al. Trastuzumab deruxtecan in patients with HER2-expressing metastatic colorectal cancer: primary results from the DESTINY-CRC01 multicentre, open-label, phase 2 trial. Lancet Oncol. 2021;22(6):779-89.
13. Meric-Bernstam F, Makker V, Oaknin A, Oh DY, Banerjee S, González-Martín A, et al. Efficacy and safety of trastuzumab deruxtecan in patients with HER2-expressing solid tumors: primary results from the DESTINY-PanTumor02 phase II trial. J Clin Oncol. 2024;42(1):47-58.