Immunohistochemical profiles of E-cadherin and p16 in colon carcinomas

Authors

Abstract

Aim Colorectal carcinomas are among the most common cancers worldwide, ranking after skin, lung, and breast cancers, and are the third leading cause of cancer-related mortality. Various histopathological features potentially influencing long-term prognosis after surgical treatment in colorectal carcinomas are under investigation to explain differing clinical courses in patients with similar tumor stages. Defining the histopathological profile of these tumors is crucial for providing patient-specific prognostic predictions. This study aims to evaluate the histopathological and immunohistochemical expression of E-cadherin and p16 in 50 cases diagnosed with colorectal adenocarcinoma.
Materials and Methods Hematoxylin-eosin-stained slides from 50 colorectal adenocarcinoma patients were examined under a light microscope. Suitable paraffin blocks were selected, and immunohistochemical staining for E-cadherin and p16 was performed on 3-4 µm-thick sections.
Results No statistically significant relationship was found between the frequency distributions of E-cadherin and p16 staining (p=0.798). Cross-tabulation revealed that staining scores of 30-60% and above 60% had the highest frequencies (32% and 28%, respectively).
Discussion Our results suggest that E-cadherin and p16 may serve as independent prognostic markers in colorectal adenocarcinoma.

Keywords

Introduction

Colorectal cancer (CRC), a malignant neoplasm arising from the epithelial lining of the colon or rectum, is a significant public health issue and ranks among the top three most commonly diagnosed cancers worldwide. It is also one of the leading causes of cancer-related mortality, particularly in developed countries [1].
According to recent epidemiological estimates, approximately 3.1 million people globally are currently living with colorectal cancer, reflecting both its high prevalence and chronic disease burden [2]. While some countries, particularly in North America and parts of Europe have reported a decline in both the incidence and mortality of CRC (largely attributable to enhanced screening programs, public awareness, and advances in early detection), an alarming upward trend continues in several other regions, including Turkey, China, Venezuela, and Bulgaria [3– 5]. This regional variation underscores the complex interplay between environmental exposures, dietary patterns, healthcare infrastructure, genetic susceptibility, and socioeconomic factors in shaping the global burden of CRC.
The etiology of colorectal cancer is multifactorial, involving a complex cascade of genetic, epigenetic, environmental, and lifestyle-related factors. Key risk factors include age over 50 years, male sex, a diet high in red or processed meats, low fiber intake, obesity, smoking, alcohol consumption, and a sedentary lifestyle [6]. In addition to sporadic cases, hereditary syndromes such as familial adenomatous polyposis (FAP) and Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC) significantly contribute to early-onset CRC cases. The progression of CRC is typically characterized by a well- established adenoma-carcinoma sequence, wherein benign adenomatous polyps gradually accumulate genetic mutations such as APC, KRAS, and TP53 alterations leading to invasive carcinoma over time.
Prognostically, early diagnosis and timely intervention remain the most critical determinants of favorable clinical outcomes for patients with CRC [6]. Therapeutic strategies and long- term survival are predominantly influenced by the tumor stage at diagnosis, which is most commonly assessed using the American Joint Committee on Cancer (AJCC) and Union for International Cancer Control (UICC) Tumor-Node-Metastasis (TNM) staging system [7]. Conventional histopathological features, including tumor depth (T), lymph node involvement (N), distant metastasis (M), tumor grade, lymphovascular invasion, and perineural invasion, are well-established predictors of disease progression and recurrence [8]. However, clinical observation has shown that even among patients with similar TNM stages, prognostic outcomes may vary considerably. Such heterogeneity has prompted a growing interest in identifying additional biomarkers including immunohistochemical and molecular parameters that can supplement conventional staging and facilitate more accurate prognostic predictions and individualized treatment planning [9].
In this context, the evaluation of tumor-specific protein expression patterns through immunohistochemical analysis has emerged as a promising approach for understanding tumor biology and stratifying patients more effectively. Novel biomarkers such as E-cadherin (a transmembrane glycoprotein involved in cell–cell adhesion) and p16 (CDKN2A), a tumor suppressor that regulates cell cycle progression, are increasingly being investigated for their potential prognostic and predictive value in CRC [6, 10]. These markers may not only reflect underlying tumor differentiation and invasiveness but also offer insights into mechanisms such as epithelial–mesenchymal transition (EMT), cellular senescence, and genomic instability. This study aims to contribute to this growing field by evaluating the immunohistochemical expression profiles of E-cadherin and p16 in colorectal adenocarcinoma specimens. The overarching goal is to investigate potential correlations between these markers and established clinicopathological parameters, thereby assessing their prognostic relevance. Such investigations are crucial for refining CRC classification systems, improving risk stratification, and ultimately guiding patient-centered therapeutic decision-making in the era of precision oncology [10, 11].

Materials and Methods

Study Population and Specimen Selection
The study included fifty colorectal cancer resection specimens collected over a ten-year period (2012–2022) from patients who underwent surgery at the General Surgery Department of Kafkas University Faculty of Medicine Hospital. Inclusion criteria were histopathologically confirmed colorectal adenocarcinoma cases. To ensure homogeneity and reduce variability, cases with special histological variants such as mucinous carcinoma, signet-ring cell carcinoma, or neuroendocrine differentiation were excluded from the study.
Tissue Preparation and Slide Selection
Hematoxylin and eosin (H&E) stained slides from each case were re-evaluated by an experienced pathologist to confirm the diagnosis and select representative tumor areas. Corresponding formalin-fixed, paraffin-embedded (FFPE) tissue blocks containing sufficient tumor tissue were chosen for immunohistochemical analysis.
Immunohistochemical Staining Procedure
From the selected paraffin blocks, 3–4 µm thick tissue sections were cut and mounted on adhesive slides to ensure tissue adherence during staining. Sections were deparaffinized using xylene and rehydrated through a graded ethanol series to distilled water. Heat-induced epitope retrieval was performed by boiling slides in 10% citrate buffer (pH 6.0) for antigen unmasking. Endogenous peroxidase activity was quenched using 10% hydrogen peroxide to prevent nonspecific background staining.
Non-specific protein binding was blocked by incubation with a commercial blocking reagent (W Block, ThermoFisher) for 5 minutes at room temperature. Primary antibodies targeting E-cadherin and p16 were applied and incubated for 60 minutes at room temperature, allowing specific binding to target antigens. After washing, slides were incubated sequentially with biotinylated secondary antibodies and streptavidin-peroxidase complexes to amplify the signal. Visualization was achieved by applying DAB chromogen, which produces a brown precipitate at sites of antibody-antigen binding. Slides were counterstained with Mayer’s hematoxylin to highlight cellular nuclei, then dehydrated, cleared, and coverslipped for microscopic examination. Evaluation and Scoring of Immunostaining Two independent pathologists blinded to clinical data evaluated the immunohistochemical staining. Cytoplasmic staining was considered positive for both E-cadherin and p16. The extent of staining was semi-quantitatively scored based on the percentage of positively stained tumor cells: 1 (0-30%), 2 (30-60%), and 3 (>60%). Discrepancies between observers were resolved by consensus. Statistical Analysis Data were entered into IBM SPSS Statistics, version 26. Normality and variance homogeneity were checked using Kolmogorov-Smirnov and Levene’s tests, respectively. Parametric comparisons between groups were conducted using Independent Samples T-test. Associations between categorical variables and staining scores were analyzed with Fisher’s exact test. Correlations between protein expression and clinicopathological parameters were evaluated using Kendall’s tau-b test. Statistical significance was set at p < 0.05.
Immunohistochemistry Protocol
Non-specific protein binding was blocked by incubating the tissue sections with a commercial blocking reagent (W Block, ThermoFisher) for 5 minutes at room temperature. Primary antibodies targeting E-cadherin and p16 were applied and incubated for 60 minutes at room temperature to allow specific binding to the target antigens. After thorough washing, the slides were sequentially incubated with biotinylated secondary antibodies followed by streptavidin–peroxidase complexes to amplify the signal. Visualization was achieved using the DAB chromogen, which produces a brown precipitate at the sites of antibody-antigen interaction. The slides were counterstained with Mayer’s hematoxylin to highlight cell nuclei, then dehydrated, cleared, and coverslipped for microscopic examination.
Evaluation and Scoring of Immunostaining Immunohistochemical staining was independently evaluated by two pathologists who were blinded to the clinical data. Cytoplasmic staining was considered positive for both E-cadherin and p16. The extent of staining was semi- quantitatively scored based on the percentage of positively stained tumor cells as follows:
Score 1: 0–30%,
Score 2: 31–60%,
Score 3: >60%.
Any discrepancies between observers were resolved by consensus.
Statistical Analysis
Data were analyzed using IBM SPSS Statistics version 26. Normality of data distribution and homogeneity of variances were assessed using the Kolmogorov–Smirnov and Levene’s tests, respectively. Parametric comparisons between groups were performed using the Independent Samples T-test. Associations between categorical variables and staining scores were analyzed using Fisher’s exact test. Correlations between protein expression and clinicopathological parameters were assessed using Kendall’s tau-b test. A p-value of less than 0.05 was considered statistically significant.
Ethical Approval
This study was approved by the Ethics Committee of the Faculty of Medicine, Kafkas University (Approval Date: 2021-01-07, No: 07). Patient confidentiality was strictly maintained throughout the study.

Results

Demographic and Clinical Characteristics
Among the 50 patients included in the study, 40% were female (n=20) and 60% were male (n=30). There was no statistically significant difference in mean age between the genders. Most surgical specimens were obtained through right hemicolectomy procedures, accounting for 40% of the cases (n=20). The most common tumor location was the cecum, observed in 30% of patients (n=15). In terms of macroscopic morphology, the majority of tumors exhibited an ulcerovegetative pattern (n=31, 62%). Pathological staging revealed that most tumors were classified as pT3 (n=28, 56%). According to TNM nodal staging, half of the cases were staged as pN0 (n=25, 50%), indicating absence of regional lymph node metastasis. Histopathological grading showed that most tumors were moderately differentiated (n=34, 68%). Additionally, tumor perforation was absent in the majority of patients (n=45, 90%), and distant metastasis was not observed or not assessed (pMX) in 94% of cases (n=47), with confirmed distant metastasis (pM1b) found in only 6% (n=3) (Table 1).
Abbreviations and Descriptions: pT2–pT4b: Pathological tumor stages according to TNM classification, pN0–pN2a:
Pathological lymph node involvement stages
Immunohistochemical Findings
E-cadherin expression was low in the majority of cases, with most tumors exhibiting 0–30% positively stained cells. (Table 2) In contrast, p16 expression was high in a significant portion of cases, with 42% demonstrating more than 60% positivity. There was no statistically significant direct correlation between E-cadherin and p16 expression levels. (Figure 1)
However, further analysis revealed significant associations between protein expression and tumor grade. E-cadherin expression showed a negative correlation with tumor grade, suggesting decreased expression in poorly differentiated tumors. Conversely, p16 expression positively correlated with tumor grade, indicating higher levels in more aggressive tumors. Age-related analysis revealed that younger patients (≤60 years) had a higher frequency of poorly differentiated tumors, whereas older patients (>60 years) more often had well-differentiated tumors. E-cadherin expression showed a weak, non-significant increase with age. p16 expression did not differ significantly by age but showed a tendency toward higher expression in younger patients (Table 3).

Discussion

Colorectal cancer (CRC) remains a major global public health concern, ranking as the third most commonly diagnosed malignancy and the second leading cause of cancer-related death worldwide. In 2020, an estimated 1.9 million new cases and 930,000 deaths were reported to CRC, underscoring its substantial clinical and societal impact [12]. Histologically, over 98% of CRCs are adenocarcinomas, with a predominance in industrialized countries due to lifestyle, dietary, and environmental factors [13].
Although colon adenocarcinoma is considered treatable when diagnosed at an early stage, prognosis declines significantly in advanced disease. Early detection is critical to therapeutic success and long-term survival [14]. Alarmingly, the incidence of CRC in individuals under 50 is rising. According to WHO data, early-onset CRC (EOCRC) has increased by 2.1% annually from 1992 to 2024, reflecting a troubling epidemiological shift [12]. This trend has been linked to the rising prevalence of obesity, Westernized diets, sedentary lifestyles, and hereditary syndromes such as Lynch syndrome and familial adenomatous polyposis [15, 16].
While CRC has historically been considered a disease of older adults, recent evidence highlights a shifting age distribution. In our cohort, 65% of patients were over 60 years old; however, a substantial 35% were younger than 60. Poorly differentiated tumors were more common in younger patients, whereas well- differentiated tumors predominated in older individuals. This finding supports the hypothesis of biological differences in tumor behavior across age groups, consistent with reports that EOCRC more frequently presents with mucinous differentiation, signet-ring cell morphology, and high proliferative indices [17, 18].
Gender distribution was skewed slightly toward males (60%), aligning with data suggesting higher CRC risk in men, possibly due to lifestyle factors such as red meat consumption, alcohol use, and smoking [19, 20]. However, no significant prognostic differences between genders were observed in our study (p = 0.248). Tumor staging, based on the AJCC TNM system, remains the cornerstone of prognostic evaluation. In our series, 56% of tumors were classified as pT3, indicating extension through the muscularis propria into pericolorectal tissue. This is consistent with global patterns in which many patients present at intermediate or advanced stages due to vague initial symptoms [21]. Although survival data were unavailable due to lack of follow-up, the literature shows a marked decline in 5-year survival with advancing stage from approximately 90% in stage I to below 20% in stage IV [22].
Tumors were fairly evenly distributed across colon segments, with a slight predominance of left-sided lesions, particularly in the sigmoid colon. Left-sided tumors are more likely to present with obstructive symptoms and altered bowel habits, whereas right-sided tumors often manifest subtly with anemia or weight loss [23]. Ulcerovegetative and annular morphologies were the most common macroscopic patterns. Perforation was rare and not statistically associated with other parameters.
Lymph node status is a key prognostic factor in CRC. In our cohort, 50% of patients were node-negative (N0), while the remaining cases showed varying degrees of nodal involvement. A significant correlation was observed between lymph node metastasis and distant metastasis (p < 0.001), reaffirming the link between nodal spread and systemic disease progression [24].
Tumor differentiation is another vital histological parameter. Moderately differentiated tumors were most common (68%), followed by poorly differentiated ones (20%). Although this was not statistically significant, literature consistently indicates that poorly differentiated tumors are associated with worse clinical outcomes due to their invasive nature and resistance to therapy [6].
At the molecular level, we investigated the immunohistochemical expression of p16 and E-cadherin. p16 (CDKN2A), a tumor suppressor gene, encodes a cyclin-dependent kinase inhibitor that regulates the G1/S checkpoint. Altered p16 expression due to methylation, deletion, or mutation is common in various malignancies, including CRC. Overexpression of p16, as seen in immunohistochemistry, may represent a compensatory response to upstream oncogenic stimuli [15]. In our study, p16 was more prominently expressed in patients aged ≤60, suggesting unique molecular features in EOCRC.
E-cadherin, encoded by the CDH1 gene, is a calcium-dependent transmembrane glycoprotein essential for epithelial integrity and suppression of tumor invasion and metastasis. Loss of E-cadherin is a hallmark of epithelial–mesenchymal transition (EMT), a process integral to cancer progression [6]. Our findings showed an inverse relationship between E-cadherin expression and tumor grade, with lower expression associated with poorer differentiation. However, no statistically significant correlation was found between E-cadherin and p16 expression levels (p = 0.798), indicating that these markers may act independently in CRC pathogenesis rather than in a coordinated or directly related manner.
These results support the potential utility of p16 and E-cadherin as independent prognostic markers in colorectal adenocarcinoma. Their differential expression across tumor grades and patient ages may provide additional insights into tumor behavior and help refine prognostic stratification. Nonetheless, further studies with larger sample sizes and prospective designs are necessary to validate these findings.

Limitations

This study has several limitations that should be acknowledged. First, the sample size was relatively small (n=50), which may limit the statistical power of the findings and restrict the generalizability of the results to broader populations. Second, the study was conducted retrospectively using archived pathology materials from a single center, which may introduce selection bias and limit external validity. Third, immunohistochemical evaluation was semi-quantitative and subject to interobserver variability, despite efforts to standardize scoring. Additionally, potential confounding clinical factors such as treatment regimens, genetic profiles, and comorbidities were not analyzed in depth. Future studies with larger, multi-center cohorts and prospective design are needed to validate and expand upon these findings.

Conclusion

Colorectal adenocarcinoma continues to present a major clinical challenge due to its high incidence, increasing prevalence in younger populations, and frequent diagnosis at advanced stages. While traditional prognostic markers such as tumor stage, nodal involvement, and histological grade remain crucial, incorporating molecular biomarkers like p16 and E-cadherin may enhance prognostic accuracy. These findings underscore the promise of personalized oncology guided by molecular profiling as a pathway toward more effective, targeted therapeutic strategies in colorectal cancer management.

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