Human Epidermal Growth Factor Receptor 2 (HER2) Expression in Colorectal Cancer and Its Clinical Significance among Jordanian Patients

Abstract

Introduction

The expression of Human Epidermal Growth Factor Receptor-2 (HER2/neu) has shown significant therapeutic implications in malignancies, particularly in breast and gastric cancer. Therefore, the interest in studying HER2 in other malignancies, including colorectal cancer (CRC), has increased. However, the prevalence of HER2 overexpression in CRC remains debatable, with conflicting data. To address this uncertainty, this study aimed to investigate HER2 expression in CRC in Jordanian patients and to assess its association with clinicopathological characteristics, as well as the survival outcome.

Methods

Sixty-nine formalin-fixed, paraffin-embedded tissue blocks of CRC cases were included along with their corresponding patient's clinicopathological data. Tissue-microarray (TMA) were constructed and stained using anti-HER2/neu monoclonal antibody. HER2 scoring was performed using the HERACLES criteria. Fisher's exact test/Chi-squared test were used to investigate the association between HER2 scores and the clinicopathological data. Survival analysis was evaluated using Kaplan–Meier curves.

Results

HER2/neu positivity of any degree was expressed in 30 cases (43.5%). Twenty-five cases (36.2%) were scored 1 + and 4 (5.8%) scored 2+. Only one case (1.5%) was scored as 3+, representing the single positive case. In comparison, 39 cases (56.5%) showed no HER2/neu protein expression and were scored as (0). Two of the four cases that were scored (2+) required FISH testing to confirm the positivity for HER2 overexpression, which was not available. No significant associations were found between HER2/neu expression and clinicopathological parameters or survival rates. However, cases with either 2+ or 3+ HER2/neu scores were associated with worse overall survival (OS) and event-free survival compared to cases with a score of 1+ or no expression (score 0).

Conclusions

The prevalence of HER2/neu overexpression in CRC among Jordanian patients is low, with only 1.5% of the study sample exhibiting HER2/neu overexpression. A trend towards poor outcome was observed in patients with HER2/neu overexpression.

Background

Colorectal cancer (CRC), a common cancer worldwide, develops from the colon and/or rectum. ¹ About 1.9 million new cases of CRC were diagnosed in both males and females combined in 2020, representing nearly 10% of the total cancer cases diagnosed that year. An estimated 935 000 deaths due to CRC were also reported, making it the third most commonly diagnosed cancer and the second cancer associated with death. ² In Jordan, CRC is considered a major health problem. According to the World Health Organization GLOBOCAN 2020 database, CRC ranks as the second most common cancer in Jordan for both genders, accounting for approximately 10.9% of all new cancer cases diagnosed. ³

Despite advancements in detecting and treating CRC, the prognosis for patients, particularly those in advanced stages, remains poor. This highlights the importance of searching for and investigating new diagnostic and prognostic biomarkers to reduce this CRC-related mortality. ⁴

One of the main research targets to enhance the treatment of CRC is human epidermal growth factor receptor 2 (HER2/neu). HER2/neu belongs to the epidermal growth factor family of tyrosine kinase receptors. It includes EGFR (ErbB1, HER1), ErbB2 (HER2, neu in rodents), ErbB3 (HER3), and ErbB4 (HER4), which play an important role in normal cell growth, division, and repair.^5,6 However, in some cancers, including a subset of breast cancers and stomach cancer, amplification of the HER2 gene occurs. This amplification results in an overproduction of the HER2 protein, which leads to uncontrolled cell growth and tumor progression.^5,6 The success of HER2-targeted therapies in improving clinical outcomes for HER2-positive breast and gastric malignancies has led researchers to investigate its potential for managing other malignancies. ⁷ CRC has recently been shown to have HER2 expression in some cases.^8–13

The HER2 receptor is normally expressed on cell membranes of the cells of several organs, including skin and the breast, in addition to the gastrointestinal, reproductive, respiratory, and urinary tracts. ¹⁴ The overexpression of HER2 receptor has been reported in different types of tumors, including ovary, stomach, bladder, and esophageal cancer, as well as in other types of tumors. ¹³

HER2 overexpression or amplification occurs in 2%-4% of patients with metastatic colorectal cancers, indicating a potential therapeutic target. Moreover, HER2 alterations in CRC have been associated with resistance against EGFR-targeted therapies like cetuximab and panitumumab, for the treatment of RAS wild-type metastatic CRC. Nonetheless, HER2-positive metastatic CRC is rare, but assessing HER2 levels is important. Anti-HER2 therapies demonstrated a non-toxic profile and high efficacy in cases where chemotherapy was ineffective. ¹⁵

Numerous studies have attempted to analyze HER2 expression in CRC, as shown in Table 1. The results have varied significantly, likely due to differences in the scoring algorithms used, as well as population heterogeneity, which includes genetic, environmental, and demographic differences. Additionally, both intra-tumour and inter-tumour heterogeneity may contribute to these discrepancies.^16,17 Consequently, the prevalence of HER2 overexpression in CRC remains a topic of debate, with conflicting data emerging from various studies. To address this uncertainty, the current study aimed to investigate HER2 expression in CRC among a group of Jordanian patients and to evaluate its association with multiple clinicopathological characteristics, as well as the survival outcomes.

Table 1.

HER2/neu Protein Expression in CRC in Different Countries.

Author/Year	Study Population	Number of Samples	HER2 Scoring Method	HER2 Prevalence (%)
				HER2/neu (0, 1+) Score %	HER2/neu (2+, 3+) Score %
Schell et al, 2006 9	Austria	77	IHC using the HercepTest Kit; Score 0: no staining at all or membrane staining in < 10% of tumour cells. Score 1+: faint/barely perceptible membrane staining in > 10% of tumour cells. 2+: weak to moderate staining of the entire membrane in > 10% of the tumour cells. 3+: strong staining of the entire membrane in > 10%.	96%	4%
Shabbir et al, 2016 10	Pakistan	95	IHC with α-Her-2 antibody; quantified based on three criteria: staining pattern, intensity, and percentage of tumor cells stained (Pattern of expression: Membranous/cytoplasmic/membranous + cytoplasmic; intensity of staining: weak/moderate/strong; percentage of cells stained: 10%-40% = score 1+ 40%-70% = score 2+ >70% = score 3+)	31%	69%
Huang et al, 2021 18	China	1240	IHC used HER2/ErbB2 (29D8) rabbit monoclonal antibody. Score 0 to 1 + is HER2-negative. Score 2 is equivocal. Score 3 + is HER2-positive. FISH test done if equivocal.	95.4%	4.6%
Torabizadeh et al, 2015 8	Iran	50	anti c-erb 2 (anti HER-2/neu) biogenexe. Scoring based on system used to evaluate Hercep Test: Score 0 (negative or <10% of tumor cells). Score 1+ (Negative) faint weak positivity in >10% of tumor cells. Score 2+ (Weak positive) weak to moderate complete membrane positivity in >10% of tumor cells. Score 3+ (Positive) with moderate to strong complete >10% of tumor cells.	60%	40%
Mustafa and Jalal, 2018 19	Iraq	103	Scoring of the HER2/neu IHC was performed according to the criteria for breast carcinoma scoring.	47%	53%
Kılıçarslan et al, 2018 20	Turkey	100	The rabbit monoclonal antibody 4B5 for Her2/neu was used. Membranous and cytoplasmic staining were scored. HER2/neu membranous staining was scored 0 to 3 according to the recommendations for gastric cancer. Scores of 3 + are positive, scores of 2 + are equivocal, and scores of 0 or 1 + are negative. Cytoplasmic staining was defined as negative (score of 0), weak (score of 1), and strong (score of 2).	88%	12%
Current study, 2023	Jordan	69	The scoring algorithm used in this study was based on the previously published HERACLES criteria 19	92.8%	7.2%

Methods

Ethical consideration: An Institutional Review Board (IRB) approval was obtained from the King Hussein Cancer Centre (KHCC) in Amman, Jordan. As this was a retrospective study, consent was not required. The archives of the pathology departments at KHCC were reviewed for histopathological cases of confirmed colorectal cancer (CRC) in patients who had not yet started chemotherapy, between the years 2013 and 2019. A total of 120 formalin-fixed paraffin-embedded (FFPE) CRC-confirmed tissue blocks and their corresponding Hematoxylin and Eosin (H&E) slides were retrieved. Out of the initial cases, 22 were excluded due to reasons such as the absence of clinical data in the medical record information system or the presence of only very small biopsies. This left 98 cases that were initially included in the study. An expert pathologist (M.S.) reviewed the H&E slides to evaluate the presence and adequacy of tumor areas corresponding to their respective blocks. As a result, 15 cases were excluded due to insufficient or absent tumor areas. For the remaining 83 cases, the tumor area was determined and marked on each case for tissue microarray (TMA) block. During TMA processing, 14 samples were lost, ending up with 69 CRC cases. Data regarding the patient's age, sex, clinical stage, survival, and status were then obtained from the medical records. All patients were de-identified, as a master-linking log was developed, and each patient was assigned a unique study number.

Three Tissue Microarray (TMA) blocks were prepared; each block contained 30 tissue cylinder cores. Two 4μm sections were created for each block: the first was stained with H&E stain to evaluate the sectioning for the slide, and the second was for IHC staining. HER2 (Clone 4B5, Ventana Medical Biosystems, Tucson, AZ) was used. The IHC method was applied using an automated IHC system using BenchMark ULTRA (VENTANA /Roche Diagnostics, Tucson, AZ) The staining procedure for HER2 expression followed the manufacturer's recommendations. ²¹

The scoring algorithm used in this study was based on the previously published HERACLES criteria. ²² This algorithm accounts for the intensity and completeness of membranous HER2 staining similar to breast scoring methods but with more stringent criteria. The HERACLES diagnostic criteria include HER2 IHC scoring from 0 to 3 + based on staining intensity, pattern, and the percentage of positive cells. Scores of 0 (no staining) and 1+ (faint segmental or granular staining) are considered negative. A moderate (2+) intensity in less than 50% of cells is also negative, whereas 2 + staining in 50% or more of cells is deemed equivocal and requires confirmation by FISH. Intense (3+) staining in 10% or fewer cells is negative, while 3 + staining in more than 10% of cells is considered positive, with no FISH testing needed.

The corresponding author, a senior pathologist with extensive experience in HER2 scoring and based in a cancer center performed scoring. The nature of the specimens being TMA may be inaccurate, as it does not entirely account for tumor heterogeneity. Additionally, we did not address interobserver variability in this study, as it is a retrospective analysis rather than a clinical trial.

Statistical analysis: The association between clinicopathological features and HER2 status was assessed by the Chi-square test or Fisher's exact test, as appropriate. Kaplan–Meier analysis and the Log-rank test were performed to evaluate the relationship between HER2 expression and the survival outcome. All statistical analyses were performed using a two-tailed approach, with P-values less than or equal to .05 considered statistically significant. SPSS Statistics software was used for all statistical analyses.

Results

Patients’ Clinicopathological Characteristics

This study included 69 patients diagnosed with CRC at our Center between 2013 and 2019. Among the patients, 66.7% were male, and 33.3% were female, indicating that there were twice as many males as females in the sample. The average age of the cohort was 53 years, ranging from 62 to 83. In terms of tumor locations, 63.6% had left-sided colon cancer, while 36.4% had right-sided colon cancer. Additionally, 8.7% of the tumors were in the rectum and rectosigmoid region. The predominant histological subtype was conventional adenocarcinoma, which accounted for 82.6% of cases. Other subtypes included mucinous adenocarcinoma (13.0%) and signet ring cell carcinoma (4.3%). Regarding histological grade, most cases were low grade (G1, G2), well to moderately differentiated, compromising 80%, and the remaining were poorly-differentiated tumors (G3) 20%.

The distribution of clinical stages among the patients indicated that advanced stages (III and IV) comprised the majority of the cohort, with 69.6% classified as stage III and 26.1% as stage IV. Stage I was identified in only 1.4% of patients, while stage II accounted for 2.9%. Regarding nodal status, 28 patients (40.6%) were negative for metastasis, whereas the majority, 41 patients (59.4%), showed positive nodal involvement. The prevalence of HER2 in CRC:

The overall rate of HER2 protein expression evaluated by IHC in this study was 43.5% (n = 30) regardless of the assigned positive score given to each case; either 1+, 2+, or 3 + . In contrast, the rest of the patients 56.5% (n = 39) were negative and scored as (0) for the presence of HER2 expression (Table 2). In this study, the HERACLES criteria were adopted as described before for HER2 scoring, where the positive cases for HER2 expression only counted for the ones scored as 3 + and to the 2 + with moderate staining in ≥ 50% of cells conditionally coupled with a confirmed positivity by in situ hybridization (ISH) test, which was not available at our center. Figure 1 shows five microscopic sections of different cases representing examples of the range of HER2 protein expression scores.

Table 2.

Distribution of HER2 Protein Expression Scores by Immunohistochemically Testing among Jordanian Patients (n = 69).

HER2 Score	No. of Cases	Percentage	Interpretation
0	39	56.5%	HER2-negative
1+	25	36.2%	HER2-negative
2+	4*	5.8%	HER2-borderline (Equivocal)
3+	1	1.5%	HER2-positive: Over-expression of HER2 in 70-year-old male patient presented with stage III CRC adenocarcinoma originating from the ascending right colon.

*Two of the four cases required ISH testing for status confirmation, which was unavailable; therefore, these two cases were excluded.

Figure 1.

Representative Cases of Different HER2 Protein Expression in Colorectal Cancer Detected by Immunohistochemistry. A (3+): Intense Membranous Staining in ≥50% of Tumor Cells. B (2+): Moderate Membranous Staining in ≥50% of Tumor Cells. C (2+): Moderate Membranous Staining in <50% of Tumor Cells. D (1+): Incomplete, Faint/Weak Membranous Staining of HER2. E (0): No Staining. All Images Were Obtained at 40× Magnification.

Out of a total of 69 samples presented in Table 2, 56.5% (n = 39) scored as 0, 36.2% (n = 25) scored as 1+, 5.8% (n = 4) scored as 2+, and 1.5% (n = 1) scored as 3+. Of the four cases with a HER2 IHC score of 2+, two showed moderate staining in ≥50% of tumor cells and were therefore classified as equivocal according to the HERACLES Diagnostic Criteria; these would have required FISH confirmation, which was unavailable, and were excluded. The remaining two cases with 2 + staining involved <50% of tumor cells and were classified as negative without the need for FISH testing. Consequently, the expression rate for the entire cohort was 1.5%, represented by the single identified case that scored as 3+out of the 67 cases considered. This particular case involved a 70-year-old male patient with stage III CRC. The tumor primarily originated from the ascending colon of the right side and was classified as moderately differentiated adenocarcinoma. The patient underwent standard chemotherapy; however, the cancer progressed, and he passed away within one year of diagnosis.

HER2 Expression and Clinicopathological Parameters

The study investigated the correlation between HER2 expression and various clinicopathological parameters. Due to the limited number of positive cases, the cohort was divided into two groups to identify any potential correlations. The first group consisted of negative cases, which included scores of 0 and 1+, while the second group included positive cases with a score of 3 + as well as equivocal cases with a score of 2+ (Table 3). No significant associations were found between HER2 expression and the clinicopathological parameters examined, such as age, gender, tumor location, and others.

Table 3.

Correlations of HER2 Expression with Clinicopathological Factor.

Clinicopathological Factor		No. of Samples (N = 69)	Negative HER2 Score (0, 1)	Positive HER2 Score (3+) and Equivocal HER2 score (2+)	P-Value
			% (No.)	% (No.)
Age	≤ 60	48	93.8% (45)	6.2% (3)	.636 Chi-square test
	> 60	21	90.5% (19)	9.5% (2)
Gender	Female	23	91.3% (21)	8.7% (2)	1.000 Fisher's exact test
	Male	46	93.5% (43)	6.5% (3)
Histological Differentiation	G1,G2 (well and moderately differentiated)	55	90.9% (50)	9.1% (5)	.575 Fisher's exact test
	G3 (poorly Differentiated)	14	100% (14)	0% (0)
Tumor primary site	Colon	67	94.0% (63)	6.0% (4)	.141 Fisher's exact test
	Rectum	2	50.0% (1)	50.0% (1)
Location in colon (N = 67)	Left	43	93.0%	7.0%	1.000 Fisher's exact test
	Right	24	95.8% (23)	4.2% (1)
Histological subtype	Conventional Adenocarcinoma	57	91.2% (52)	9.8% (5)	.323 Fisher's exact test
	Mucinous Adenocarcinoma	9	100% (9)	0% (0)
	Signet ring cell carcinoma	3	100% (3)	0% (0)
TNM Stage: *					Chi-square test
Tumor	T1-T2	3	66.7% (2)	33.3% (1)	.205
	T3-T4	66	93.9% (62)	6.1% (4)
Nodes	N0	28	96.4% (27)	3.6% (1)	.642 Chi-square test
	N1-N2-N3	41	90.2% (37)	9.8% (4)
Metastasis	M0	58	91.4% (53)	8.6% (5)	.585 Fisher's exact test
	M1	11	100% (11)	0% (0)

*TNM, Tumor Node Metastasis. TNM staging was performed according to Mulla et al. ²³

HER2 Expression and Survival Outcome

The prognostic value of HER2 expression was statistically assessed for both overall survival (OS) and event-free survival (EFS). According to the two groups that were arranged before, a Kaplan – Meier analysis was conducted, which showed that the OS for the patient group that scored as (2+, 3+) was slightly worse than those who scored (0, 1+) (Figure 2). Moreover, it was even worse for EFS (Figure 2). OS was defined as the time from diagnosis to death from any cause. EFS was defined as the time from diagnosis to the first occurrence of disease recurrence, progression, or death.

Figure 2.

A: Event Free Survival (EFS) and B: Overall Survival (OS) for (0, 1+) HER2 Scored Results VS the (2+, 3+) Results: Kaplan – Meier Analysis.

However, the difference in both OS and EFS for the two groups was found statistically insignificant (log-rank P = .7845 and P = .1582, respectively). The median survival time for the (2+, 3+) group was significantly shorter than the (0, 1+) group in both OS and EFS. For OS, the median was 10.6 months for the (2+, 3+) group compared to 130.9 months for the (0, 1+) group. Similarly, the EFS group median was 11.3 months for the (2+, 3+) group compared to 71.9 months for the (0, 1+) group.

Discussion

The HER2 gene, when abnormally active or amplified, contributes to aggressive breast cancer by promoting uncontrolled cell growth. ²⁴ This discovery revolutionized breast cancer management by enabling a better understanding and classification of the disease. Researchers developed targeted therapies to block the HER2 protein, culminating in the FDA's 1998 approval of trastuzumab (Herceptin), the first HER2-targeted therapy. This breakthrough significantly improved outcomes, prognosis, and survival rates for metastatic HER2-positive breast cancer patients. ²⁵

Several studies^8–10,19 investigated the prevalence and potential exploitation of HER2 in CRC, referencing its established role in HER2-positive breast cancer. In recent years, studying HER2 receptor overexpression in CRC has been a hot research area. Various studies have reported differing prevalence rates of HER2 protein in CRCs and its implications across different populations. Therefore, this study focused on evaluating the expression of HER2 in CRC among Jordanian patients, evaluating the possibility of using HER2 expression rate as a biomarker for anti-HER2 therapies and investigating the potential correlation with the assigned clinicopathological factors.

The current study investigated the prevalence of HER2 overexpression in CRC among Jordanian patients, presenting a low rate of 1.5% using the HERACLES scoring system. This finding aligns with the low prevalence reported in multiple studies across different populations, suggesting a potential low prevalence for HER2 overexpression. Several studies^9,12,18 also demonstrated low HER2 prevalence, aligning with our findings. For example, a study from Austria with 77 participants reported a 4% prevalence. In addition, a German study with a large cohort of primary CRC (n = 1645) reported 1.6%. A Chinese study with 1240 patients reported a prevalence of 4.6%. Despite differences in population size and ethnic background, all these studies reported relatively low HER2 positivity, suggesting that HER2 overexpression in CRC is uncommon globally. Our rate is even slightly lower than those reported elsewhere, which may reflect population-specific genetic factors in the Jordanian cohort. Two out of the four (2+) scored cases reported in the current study showed moderate staining in more than or equal % to 50 of the cells (equivocal according to the HERACLES system), which requires a FISH test to confirm its positivity. Suppose both cases were tested and turned out to be positive. In that case, HER2 prevalence in this study may rise to 4.3%, nearly matching the findings of some of the previously reported articles. Nevertheless, this was not done, and the current study found a 1.5% prevalence of HER2 overexpression. Contrary to our findings, Torabizadeh et al performed a study that highlighted the impact of scoring criteria applied on the HER2 prevalence, reporting a prevalence of 40%.This 40% prevalence rate was reported by encompassing both “2+” and “3+” as positive, with 38% scoring 2 + and only 2% scoring 3+. ⁸ The inclusion of 2 + unconditionally may be the reason for the elevated prevalence reported, whereas this contrasts with our HERACLES system's stricter criteria, which require more stringent conditions for a “2+” score to be considered positive. This difference in scoring likely explains their significantly higher prevalence compared to our study.

Shabbir et al reported a significantly higher rate of HER2 expression at 78.9% compared to our any positive rate of 43.5% rate. ¹⁰ This discrepancy likely arises from their scoring criteria, which incorporate the cytoplasmic staining of Her-2/neu expression in addition to intensity of staining, and the percentage of stained cells, which is different from ours. Their study details this staining pattern which shows that out of the 75 (78.9%) positive cases, 20 (26.6%) showed membranous Her-2/neu expression, 36 (48%) cases showed cytoplasmic expression, and 19 (25.3%) cases showed membranous plus cytoplasmic expression. Opposite to our approach, they considered the cytoplasmic expression of Her-2/neu staining along with the membranous expression, which may inflate their overall reported rate. Moreover, the differences in HER2 expression rates could be attributed to several potential factors, including differences in sample size, scoring criteria, patient populations, and methodologies used for HER2 detection.

Moreover, this study investigated the association between HER2 expression and clinicopathological parameters. The results of this study indicated no statistically significant association between HER2 expression and any of the parameters evaluated, including age, gender, tumor stage, primary tumor site, histological differentiation, and histological subtypes. Several studies^9,19,26 have reported similar findings, suggesting no statistically significant association between HER2 expression and the examined clinicopathological parameters. In contrast to the current study findings, Ingold Heppner et al reported that HER2/neu positivity was significantly associated with more advanced tumor stages (P < .020). There was a significant correlation between HER2/neu positivity and higher TNM stages when analyzed as four groups (I/II/III/IV: P < .017) and as two groups (I/II vs III/IV: P < .017). Also, HER2/neu positivity significantly correlated with lymph node metastases (P < .033). When analyzed as two groups (N0 vs N+, P < .029), the association between HER2/neu positivity and lymph node metastases indicated a significant association. ¹² A meta-analysis by Sun et al (2016) also indicated that HER2 expression levels are associated with clinicopathological features in CRC, as high HER2 expression was linked to lymph node metastasis and more advanced Dukes stages. This suggests that HER2 may serve as a potential biomarker for the diagnosis and prognosis of CRC. ²⁷

HER2 overexpression activates potent oncogenic signalling cascades, particularly the PI3 K/AKT/mTOR and MAPK pathways. These pathways drive cell proliferation, survival, and resistance to apoptosis, which directly affect patient prognosis independently of traditional factors such as stage, grade, or tumor site. ²⁸ Future studies, including molecular assessments of downstream pathway activity, functional assays, and evaluations of treatment responses, will provide valuable mechanistic insights.

Overall, the small sample size and the small number of positive cases imposed some limitations on this study especially for the finding of any definite association with clinicopathological variables.

The prognostic value of HER2 protein and its association with survival were investigated for different malignancies. Breast cancer, for example, is known to display the highest overexpression of HER2 protein among the different malignancies, in which HER2-positive phenotype breast cancer patients are significantly associated with shorter OS, displaying a poor prognosis. ²⁴ This subset of breast cancer patients showed an increase in OS after treatment with anti-HER2 drugs, which highlighted the prognostic value of HER2 protein. ²⁵ From this perspective, the value of HER2 protein overexpression and exploiting targeting it by blocking therapies has elevated the focus on it in other malignancies. In the current study, the differences in OS and EFS were studied for HER2 expression in two groups, the (0, 1+) group and the (2+, 3+) group, which were statistically insignificant. However, it has been noticed that group (2+, 3+) HER2-scored patients displayed a worse OS than (0, 1+) HER2-scored patients. In addition, the (2+, 3+) group tended to have poorer EFS than the (0, 1+) HER2 scored group.

Ingold Heppner et al performed a retrospective study of 1645 primary CRC cases that revealed HER2 positivity had no significant impact on the OS; however, similar to our findings, the HER2-positive tumors showed a likelihood of poorer prognosis. ¹² A meta-analysis of 11 published studies from various ethnicities investigated the effect of HER-2 overexpression on CRC survival in order to evaluate its prognostic value. The analysis of collected data for the studies suggests that HER-2 overexpression has little effect on CRC survival. ²⁹ Furthermore, a large study enrolled 1240 CRC patients, by which a survival analysis for this cohort was investigated, which divided the cohort into four groups: stage I-III group, stage III stage I-II, and stage IV group, whereby a comparison in overall survival (OS) and disease-free survival (DFS) between HER2 positive and HER2 negative were investigated in order to assess HER2 prognostic role in each group separately. The results showed there is a significant difference in OS between HER2 positive and HER2 negative patients in the stage I-III (P = .040) group, stage III group (P = .012), and stage IV (P = .012) subsets, implicating the role of HER2 positivity in the disease's poor prognosis. However, no significant difference in OS was found in the stage I-II group (P = .940). Additionally, the Stage III group was the only group of the divided subsets showing a significant difference in DFS and OS between HER2-positive and HER2-negative patients. In contrast, both Stage I-III and I-II groups did not reveal any significant difference in DFS. ¹⁸

Limitations of the study: This study faced limitations due to a small sample size, and FISH testing was not performed on two equivocal (2+) results to confirm or deny their positivity for HER2 overexpression.

Recommendations: Future research should involve a larger sample size to enhance statistical power and yield more definitive insights into the prognosis of HER2 expression and its association with clinicopathological parameters. Comparative studies utilizing various testing methods are strongly recommended. Molecular testing methods offer potentially higher accuracy and could provide valuable insights into the true prevalence of HER2 overexpression in CRC.

Conclusion

HER2 protein overexpression was observed in 1.5% of CRC patients in the Jordanian cohort, which aligns with the low rates reported in multiple studies from various countries. Notably, significant differences in HER2 prevalence were observed among populations of the same and different ethnicities. These variations may be attributed to different methodologies, particularly the scoring criteria used, which significantly influenced the reported results. No significant association was found between HER2 expression and clinicopathological variables or survival outcomes. However, patients with higher HER2 scores were linked to poorer overall and event-free survival. Despite the limited number of HER2-positive cases identified, this study represents the first investigation into HER2 protein expression prevalence in CRC among Jordanian patients. Furthermore, while the proportion of HER2 overexpression cases is low, identifying these patients could provide valuable insights for alternative treatments, as they may potentially benefit from anti-HER2 therapies, similar to those used and observed in breast cancer.

Abbreviations

CRC

Colorectal cancer

DFS

Disease-free survival

EGFR

Epidermal growth factor receptor

EFS

Event free survival

FFPE

Formalin-fixed paraffin-embedded

FISH

Fluorescence in situ hybridization

H&E

Hematoxylin and eosin

HER

Human epidermal growth factor receptor

HER2

Human epidermal growth factor receptor 2

IHC

Immunohistochemistry

IRB

Institutional review board

ISH

In situ hybridization

LIS

laboratory information system

MMR

mismatch repair

OS

overall survival

TMA

Tissue microarray