What’s Known

Tumor budding is linked to invasion, metastasis, and poor prognosis in colorectal carcinoma (CRC). Epithelial-mesenchymal transition (EMT) markers such as β-catenin, E-cadherin, Snail, and Zinc finger E-box-binding homeobox 1 (ZEB1) are frequently altered in CRC. Tumor budding has been associated with distant metastasis and other adverse clinicopathological features, but it is underutilized in standard pathology due to assessment challenges.

What’s New

This study highlights a significant correlation between tumor budding and EMT markers in CRC, providing a deeper understanding of invasion mechanisms. This finding contributes to our understanding of invasion mechanisms in CRC.

Introduction

Colorectal carcinoma (CRC) ranks as the third most frequently diagnosed cancer and is the leading contributor to cancer-related mortality worldwide. It often develops from benign neoplastic lesions, such as adenomatous polyps and serrated polyps. ¹

As the molecular understanding of CRC advances, extensive research is being conducted to determine whether these histological or molecular traits can be used to predict treatment outcomes. Two potential classifications of CRCs have emerged from molecular and genetic investigations, including various gene expression analyses. The first classification is sporadic and unrelated to genetic susceptibility or family history, likely arising from environmental and nutritional factors. ²

Nevertheless, a family history of CRC is present in 20-30% of patients with the disease, and 5% of these cancers arise in the context of conditions with Mendelian heredity. These include nonpolyposis disorders such as cancer familial syndrome (formerly Lynch II) and Hereditary Non-Polyposis colorectal cancer (HNPCC, formerly Lynch I), as well as conditions involving colonic polyps. ^{3 , 4} The Fearon and Vogelstein model has long been accepted as the gold standard for understanding the genetic changes associated with CRC development. ⁵

Given that molecular alterations are the primary cause of cancer, the ability of malignant tumors to invade nearby tissues and even metastasize to distant sites is a secondary factor. While cancer cells frequently exhibit anchorage-independent growth, normal tissue shows a strong correlation between cell adhesion and signaling, as evidenced by their reliance on anchoring for growth. Two of the most intriguing topics in tumor growth and metastasis are the epithelial-mesenchymal transition (EMT) of tumor cells and tumor budding. ⁵

The shifts in adhesion and signaling in malignant cells, leading to metastasis, confirm many established theories in this field. Phenotypic alterations during EMT, including the invasion of the extracellular matrix and the departure of cancer cells from the primary tumor to form distant metastases, support the hypothesis that EMT is pathologically reactivated during malignant transformation. ⁶

Numerous studies have linked tumor budding in CRC to unfavorable outcomes. ^{7 , 8} The International Union Against Cancer has classified tumor budding as an “additional prognostic marker” alongside histological grade, perineural invasion, and tumor boundary. ⁹ However, several factors have made it challenging to incorporate tumor budding assessment into standard pathology reports. ¹⁰ The purpose of the present study is to identify tumor budding in patients with CRC and correlate the results with EMT.

Materials and Methods

Ethical Approval and Case Selection

This study was approved by the Ethics Committee of the University of Duhok, Duhok city, Iraq, and the Duhok Directorate General of Health in Duhok city, Iraq (Approval number: 13062021-7-17). Written informed consent was obtained from all participants.

Evaluation of Tumor Budding and Its Relationship with Clinical Features

In this retrospective observational study, 101 paraffin-embedded, formalin-fixed CRC tissue blocks were collected between January 2017 and May 2023 in Duhok city, Iraq. All relevant resection specimens and hematoxylin and eosin-stained slides were reassessed by other skilled pathologists, blinded to the clinical outcome. The histopathological details of each tumor were obtained from diagnostic records provided by various attending pathologists. CRC cases diagnosed with endoscopic biopsy or treated with neoadjuvant therapy were excluded. A cohort was used to establish a histopathological cutoff for “high” tumor budding and to validate its prognostic significance using an established scoring system. ⁸ According to the tumor budding scoring guidelines from the International Tumor Budding Consensus Conference, ¹¹ cases positive for tumor budding were categorized into three groups: BD1 (1–4 buds/0.785 mm²), BD2 (5–9 buds/0.785 mm²), and BD3 (10 or more buds/0.785 mm²).

Immunohistochemistry

The following EMT markers were tested: Snail, Zinc finger E-box-binding homeobox 1 (ZEB1), E-cadherin, and β-catenin. The tissue sections were incubated with the following primary antibodies at room temperature and the indicated dilutions: anti-β-catenin (1:200, Dako, Denmark), anti-E-cadherin (1:100, Dako, Denmark), anti-ZEB1 (1:150, Abcam, UK), anti-Snail (1:500, GeneTex, USA), and anti-vimentin (1:100, Dako, Denmark). Immunohistochemical staining was performed using the DAKO Kit system (DAKO, Denmark) along with a peroxidase/DAB Kit (DAKO).

The reactivity was assessed based on the percentage of positive cells and staining intensity. Staining intensity was classified into four levels: negative (0), weak (1), moderate (2), and high (3). Five groups were established based on the percentage of positively stained cells: 0-5% (0), 6-25% (1), 26-50% (2), 51-75% (3), and 76-100% (4). A staining index score between 0 and 12 was calculated by multiplying the staining intensity score by the percentage of positive cells. A staining index score between 6-12 indicated positive protein expression, while a score between 0–6 indicated negative protein expression. The subcellular localization of the staining (nucleus, cytoplasm, and membrane) was independently evaluated for β-catenin and E-cadherin. Aberrant expression of E-cadherin and β-catenin was indicated by ectopic staining in the cytoplasm or nucleus and the absence of membrane staining. ^{12 , 13}

Statistical Analysis

All statistical analyses were performed using IBM SPSS Statistics software, version 26.0 (IBM Corp., Armonk, NY, USA). Categorical data were presented as numbers and percentages (n, %). The Chi square test and Fisher’s exact test were used to assess associations between categorical variables. A P value <0.05 was considered statistically significant.

Results

Clinicopathological Characteristics of CRC Patients

A total of 101 patients (48 women and 53 men) diagnosed with CRC enrolled in the study. The patients’ ages ranged from 18 to 83 years, with a mean age of 53.90 years. Tumor grade data showed that moderately differentiated tumors were the most prevalent, accounting for 76.2% of cases, while well-differentiated and poorly differentiated tumors accounted for 15.8% and 7.9%, respectively. Conventional adenocarcinoma was the most common histological type, found in 88 cases (87.1%), while mucinous carcinoma and signet ring cell carcinoma were found in 12 cases (11.9%) and one case (1.0%), respectively. Stage III was the most frequent, present in 37 cases (36.6%), while stage IV was the least common, present in eight cases (7.9%). More than half of the tumors (57.4%) were located in the left colon (58 cases). Data are shown in table 1.

Tumor Budding among CRC Patients

Out of 101 patients, 64 cases (63.4%) exhibited tumor budding. Table 2 summarizes all the relevant data. The most prevalent category was low (BD1), while the least common category was high (BD3). Figures 1 and 2 illustrate the tumor budding categories. Tumor budding and advanced tumor stage, tumor invasion, vascular invasion, perineural invasion, and desmoplasia were significantly associated. The link between regional lymph node metastases and tumor budding was highly significant (P=0001). Table 3 shows that N1 revealed 23/26 (88.5%) cases, and N2 revealed 17/17 (100%) cases affected by tumor budding.

Figure 1. Histological images show tumor budding in colorectal carcinoma: (A) typical architecture of moderately differentiated adenocarcinoma (20×); (B) BD2 with 5–9 buds (red arrows) (40×); and (C) BD3 with ≥10 buds (red circles) (40×). BD2: Budding 2; BD3: Budding 3

Figure 2. Histological images show mucinous carcinoma with malignant epithelial cells in mucin pools (red arrows) and high-grade tumor budding (BD3) (red circles), indicating poor prognosis (40×).

Immunohistochemical Stains

Aberrant Epithelial Markers (β-catenin and E-cadherin) in CRC Patients

As shown in table 4 and figures 3 and 4, out of the 101 samples examined, 82 (81.2%) showed aberrant β-catenin localization, whereas 65 (64.4%) showed aberrant E-cadherin. In addition, table 5 demonstrates the relationship between the aberrant subcellular localization of E-cadherin and β-catenin and the clinicopathological characteristics of CRC patients. The study revealed a positive correlation between aberrant β-catenin expression and histological types (P=0.049), tumor invasion (P=0.004), and perineural infiltration (P=0.023). The correlation between clinicopathological characteristics and E-cadherin was not statistically significant.

Figure 3. Immunohistochemical staining shows normal membranous β-catenin in tumor cells (A) and aberrant cytoplasmic/nuclear β-catenin (B), reflecting Wnt/β-catenin pathway activation and EMT promotion (40×).

Figure 4. Immunohistochemistry shows normal membranous E-cadherin (A) and aberrant cytoplasmic/nuclear E-cadherin (B), indicating reduced adhesion and EMT-associated mesenchymal shift (40×).

Expression of Mesenchymal Markers (Snail and ZEB1) in CRC Patients

Snail and ZEB1 are typically expressed abnormally and are localized within the cytoplasm of tumor cells, as presented in figures 5 and 6. Out of the 101 cases, Snail reactivity was found in 55 cases (54.5%) and ZEB1 reactivity in 32 cases (31.7%). Tables 6 and 7 illustrate the correlation between Snail and ZEB1 expressions and the clinicopathological characteristics of CRC patients; was not statistically significant.

Figure 5. Immunohistochemistry reveals Snail absence in tumor cells with stromal cell presence (A) (20×) and positive Snail expression in tumor cells (B), suggesting EMT-associated transcriptional changes (40×).

Figure 6. Immunohistochemistry shows cytoplasmic ZEB1 expression in tumor cells, indicating EMT activation and increased tumor invasiveness.

Association between Epithelial and Mesenchymal Markers (EMT)

The association between aberrant β-catenin and E-cadherin with mesenchymal marker expression (Snail and ZEB1) in CRC patients is shown in table 8. Aberrant β-catenin displays a significant association with ZEB1 (P=0.005).

Relationship between Tumor Budding and EMT Expression

The relationship between tumor budding and the expression of EMT markers, β-catenin, E-cadherin, Snail, and ZEB1 is presented in tables 9 and 10. The data showed that there is a significant correlation between tumor budding and Snail (P=0.002), while other EMT markers were not statistically significant.

Discussion

In this study, we evaluated the pathological and clinical significance of tumor budding TB and its association with EMT markers in CRC. Tumor budding was observed in 63.4% of cases and significantly correlated with advanced stage, tumor invasion, distant metastasis, vascular invasion, perineural invasion, and desmoplasia. Immunohistochemistry revealed frequent aberrant expression of β-catenin and E-cadherin and positivity for Snail and ZEB1. Although EMT markers were commonly expressed alongside tumor budding, no statistically significant association was found, suggesting that tumor budding may independently serve as a stronger indicator of tumor aggressiveness in CRC.

Despite intensive efforts to elucidate the mechanisms behind tumor development and migration, they remain elusive and contentious. Since Stephen Paget’s groundbreaking 19^th-century work introduced the concept of seed and soil, a significant amount has been written and accomplished in the field of cancer metastasis. ¹⁴

There is a good possibility of forecasting the tumor’s course and the tumor management approach by considering the dynamics of tumor progression and metastasis. Tumor budding is a histological phenomenon that has been observed in several tumors characterized by the presence of individual or tiny clusters of malignant cells in the tumor stroma. Tumor budding is a real tumor growth pattern that can include zero buds to many buds. It is classified based on the number of buds present. However, it is debatable if the cutting artifact is the cause of a small number of tumor buds per defined area. ¹⁵

This study confirmed that CRC patients had a significant tumor budding rate. Tumor budding was discovered in 64 cases (63.4%) out of 101 patients. It revealed that 21 and 27 patients (26.7%, 20.8%) had low and intermediate tumor budding, respectively, and that 16 patients (15.8%) had significant tumor budding. These results were consistent with those of Pyo JS and others, ¹⁶ who found that 135 (50.8%) and 32 (12.0%) of the patients had low and high tumor budding, respectively.

There was a significant association between tumor budding and aggressive tumor behavior, including stage, tumor invasion, regional lymph node metastasis, vascular invasion, perineural invasion, and desmoplasia. Tumor budding was also shown to be an independent prognostic factor linked to overall survival in CRC. ^{17 - 19}

Tumor budding was found to be an independent prognostic factor linked to lymph node metastases in numerous published studies. ^{19 - 21} Out of 43 patients in the current study identified with lymph node metastases, 40 (93%) demonstrated a correlation with tumor budding.

The subcellular distribution and loss of membranous β-catenin and E-cadherin in CRC tumor samples revealed a high percentage of aberrant β-catenin and E-cadherin expression, 81.2% and 64.4%, respectively, in an immunohistochemical analysis conducted to examine markers related to cell adhesion. In contrast, the mesenchymal markers ZEB1 and Snail exhibited high frequency, 32 (31.7%) and 55 (54.5%), respectively. These results were consistent with previous research. ^{22 , 23}

Aberrant β-catenin and E-cadherin expression patterns and clinicopathological parameters showed a significant association, particularly with histological type, tumor invasion, and perineural invasion. These results agreed with Gao and others, ²⁴ Hussein and colleagues, ²⁵ and Bruun and others, ²⁶ respectively.

Another study stated that there was no significant link between the prognosis and accumulation of β-catenin and E-cadherin in the cytoplasm and/or nucleus. ²⁷

Furthermore, the current study confirmed a feature of EMT: many cases showed mesenchymal transitional characteristics while maintaining epithelial characteristics. For instance, 16 cases (29.1%) had positive E-cadherin and Snail while β-catenin was normal, and 10 cases (18.2%) had positive Snail. ZEB1 and E-cadherin demonstrated a similar correlation, with nine cases (28.1%) showing positive ZEB1 and normal E-cadherin. ZEB1 expression was the lone exception, showing aberrant β-catenin in 31 cases (96.9) and normal β-catenin in one case (3.1). The sequences of tumor transition from epithelial to mesenchymal features and subsequent tumor invasion highlighted the same correlation. ²⁸

Tumor budding and EMT markers have a high frequency association. In 53/82 (64.6%), 39/65 (60.0%), 30/55 (54.5%), and 21/32 (65.6%) cases, tumor budding was concurrently present with abnormal β-catenin, E-cadherin, ZEB1, and Snail. Although this discovery did not have statistical significance, it did indicate a high rate of connection and suggested that the presence of EMT markers may not be a key concurrent factor in determining tumor budding status. The current study included 66 individuals (65.3%) with a high proportion of desmoplasia.

This study had certain limitations. The results were dependent on a particular sample set while leaving out important variables. Because of its relatively small sample size, the study methodology fell short of forecasting long-term outcomes, particularly the prognosis and survival rate, underlining the necessity of conducting thorough research to explore the clinical implications of tumor budding in CRC.

Conclusion

In repetitive histological sections of CRC, tumor budding is prevalent and can be measured independently. It significantly correlates with the metastasis of lymph nodes in staging parameters. In CRC, A significant correlation was found between aberrant β-catenin, E-cadherin, and expression of ZEB1 and Snail, which reflects that EMT markers are frequently altered and linked to tumor invasion and perineural infiltration. EMT has a high correlation with tumor sprouting. When compared to individuals with any level of tumor budding, EMT is less indicative.

Acknowledgment

I am incredibly grateful to everyone who helped complete this manuscript. I want to express my sincere gratitude to my histology department colleagues.

Authors’ Contribution

The authors conceived the study, performed the research, analyzed the data, and wrote the manuscript. The authors read and approved the final manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Conflict of Interest

None declared.

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