The Role of Tumor Budding in Early‐Stage Laryngeal Cancer Treatment Selection

ABSTRACT

Objective

Laryngeal cancer is a prevalent malignant neoplasm, with early‐stage glottic tumors treated with surgery or radiotherapy. Radioresistance significantly complicates treatment, highlighting the necessity for early detection methods. This study aimed to evaluate the relationship between tumor budding and radiotherapy outcomes in affected patients.

Methods

This investigation encompassed patients diagnosed with early‐stage squamous cell carcinoma of the glottic larynx who received radiotherapy and had a minimum follow‐up of 1 year. Pathological specimens collected at diagnosis were reassessed for tumor budding. The data were analyzed to determine the best cut‐off value for tumor budding to predict radioresistance and differences in survival based on tumor budding cut‐off value.

Results

The study cohort comprised 49 patients who met the pre‐established criteria. The radioresistant group comprised 14 patients. All except one patient were male (mean age: 64 years). The best cut‐off value for tumor budding was determined as 2. Patients categorized into high risk (tumor budding ≥ 2) had a significantly higher chance of radiotherapy failure than those with low risk (tumor budding < 2, AUC = 0.696; p = 0.034; sensitivity = 78.6%; specificity = 62.9%). The high risk group also had a significantly reduced 5‐year disease‐free survival compared to the low risk group (p = 0.008). Overall survival was similar.

Conclusion

The prognosis for early‐stage glottic laryngeal cancer is generally favorable, yet a standardized risk stratification tool for treatment selection remains lacking. Tumor budding assessment in routine histopathology could help identify patients at higher risk of recurrence, guiding decisions on radiotherapy intensity and post‐treatment surveillance. Integrating tumor budding into clinical practice may support more personalized treatment strategies, ultimately improving patient outcomes.

Level of Evidence: 3.

The search for treatment choices in the treatment of early‐stage laryngeal cancer is still ongoing. In this group with high treatment rates, it is important to be able to predict the unsuccessful group in advance. Tumor budding shows promising results. The failure rate after radiotherapy is high in patients with tumor budding of 2 or more. We think it would be useful to take these values into account when choosing treatment.

1. Introduction

Laryngeal cancer represents one of the most prevalent forms of head and neck tumors. Despite being the second most prevalent head and neck tumor globally, it remains the most common head and neck tumor in Turkiye [1]. The treatment options for early‐stage glottic tumors are evaluated in two main groups: surgical options and radiotherapy. Although these two treatment options for glottic cancers have been demonstrated to have both advantages and disadvantages, numerous studies have indicated that there is no significant difference in average survival rates between the two [2]. The reported range for this rate in early‐stage glottic cancers treated with radiotherapy is between 65% and 90%.

The early stages of glottic laryngeal cancer can be effectively treated with radiotherapy and conservative laryngeal surgery. Given these elevated rates, it is of paramount importance to be able to identify radioresistant tumors prior to the commencement of treatment. A substantial body of research has been conducted with the objective of detecting radioresistant tumors in advance [3, 4]. However, no reliable molecular predictor of radioresistance in laryngeal carcinoma has been established. While early studies suggested potential markers, later investigations with larger cohorts failed to validate them [5]. More recent research has linked hypoxia‐related pathways to radiotherapy failure, showing that targeting Glut‐1 and HIF‐1α can enhance radiosensitivity [6]. However, clinical translation remains limited due to tumor heterogeneity, variability in immunohistochemical assessment, and the lack of prospective validation studies. Additionally, evidence suggests that molecular markers alone may be insufficient, as treatment‐resistant tumor microenvironments contribute significantly to radiotherapy failure [7]. Given the limitations of molecular markers, alternative histopathological features may offer practical prognostic value. Tumor budding, extensively studied in various malignancies, has emerged as a potential indicator of tumor aggressiveness. This study aimed to evaluate whether tumor budding could serve as a predictive marker for radiotherapy outcomes in early‐stage glottic laryngeal cancer.

2. Materials and Methods

This study evaluated patients diagnosed with early‐stage glottic laryngeal squamous cell carcinoma of the larynx and treated with radiotherapy between January 2013 and January 2022 at the Otorhinolaryngology Clinic of Rize Recep Tayyip Erdoğan University Training and Research Hospital. The study included patients who had been followed for at least 1 year after the conclusion of their treatment. The patients were divided into two groups, comprising those with and without recurrence following radiotherapy treatment. The paraffin block and hematoxylin and eosin (H&E) stained preparations of the biopsies taken at the time of diagnosis were re‐examined blindly by a pathologist experienced in head and neck cancer using an Olympus BX51 light microscope to evaluate the tumor buds located at the border of tumor parenchymal infiltration.

A tumor budding is a cell or cell group with epithelial‐mesenchymal plasticity, independent of the central tumor mass at the border of tumor surrounding tissue infiltration, consisting of a single cell or a cluster of less than five dedifferentiated cells (Figure 1). We evaluated the tumor budding in hematoxylin–eosin (H&E) sections, the most infiltrative area containing the most tumor budding, in one high‐power field (HPF) area (×20) to be applied more practically, inexpensively, and widely in routine pathology under the guidance of the International Tumor Budding Consensus Conference 2016 [8]. H&E sections containing the tumor area of each case were scanned by two pathologists (O.O., Ç.O.) with a 10 × objective (Olympus, BX‐51) independent of the clinical and pathological data of the patients. The most infiltrative area where the tumor budding is highest was determined. The tumor budding was counted in a single HPF area with a 20×objective (Olympus, BX‐51, ocular 22 mm, field size 0.950 mm2). PanCK immunohistochemical stain was applied to evaluate the tumor budding in cases with intense inflammation [9]. Inter‐observer agreement for tumor budding assessment was evaluated using kappa statistics. Two pathologists independently assessed tumor budding, and cases with discrepant scores were re‐evaluated under a double‐headed microscope to reach a consensus. Out of 49 assessments, three discrepancies were identified and resolved through joint review. Inter‐observer agreement for tumor budding assessment yielded a kappa value of 0.925 (p < 0.001), demonstrating a high level of concordance between the two pathologists.

FIGURE 1.

Laryngeal squamous cell carcinoma, high tumor budding, H&E 200×. [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]

Prior to the commencement of the study, approval was obtained from the Recep Tayyip Erdoğan University Faculty of Medicine, Non‐Interventional Clinical Research Ethics Committee (Decision No: 2023/222).

Data collected in the study were analyzed using the IBM SPSS data analysis package version 23 and R (version 4.2.2; R Foundation for Statistical Computing, Vienna, Austria). Mean, standard deviation, median, minimum, and maximum descriptive values were reported for continuous data. Normality of variables was assessed using the Kolmogorov–Smirnov test. Confounding factors were checked with a logistic linear model with post‐radiotherapy recurrence as a dependent parameter, and age and tumor budding count as non‐dependent parameters. ROC analysis was performed to determine the cutoff point of tumor budding count to identify post‐radiotherapy recurrence (cutpointr package, version 1.1.2, maximize_metric function). Pearson chi‐square or Fisher's exact test was used to compare categorical variables. The Kaplan–Meier test was used to assess whether there was a difference between tumor budding and overall and disease‐free survival, and survival curves were drawn. Results were considered statistically significant if the p‐value was less than 0.05.

3. Results

The study cohort comprised 49 patients who met the pre‐established inclusion criteria. The patients were divided into two groups, one comprising those who experienced recurrence following radiotherapy and the other comprising those who did not. The radioresistant group comprised 14 patients (28.6%), while the radiosensitive group consisted of 35 patients (71.4%). A single female patient was included in the study. A total of 48 patients (98%) were male. The mean age of the participants was 64 years (range 40–83 years).

A binomial logistic regression model assessed the association between post‐radiotherapy recurrence and two predictors: age and tumor budding in 48 patients, excluding a single female patient due to high standard deviation with the gender parameter. The intercept (−0.2847, p = 0.9046) was not significant, indicating no baseline effect on recurrence. Age was not a significant predictor (p = 0.4374), suggesting it does not influence recurrence risk. Tumor budding was significant (p = 0.0289), with a negative coefficient (−0.2336), implying an unexpected inverse association—higher tumor budding correlated with a lower likelihood of recurrence.

The optimal tumor budding cut‐off value was determined as 2, based on receiver operating characteristic (ROC) curve analysis (Figure 2). Using this threshold, patients were classified into low‐risk (tumor budding < 2) and high‐risk (tumor budding ≥ 2) groups. Among 49 patients with early‐stage laryngeal cancer (T1‐2, N0) who underwent radiotherapy, 14 patients (28.6%) experienced recurrence, while 35 did not. Patients in the high‐risk tumor budding group had a recurrence rate of 45.8%, compared to 12% in the low‐risk group (p = 0.009). Of the 14 patients who experienced recurrence following radiotherapy, 11 exhibited high‐risk tumor budding (Table 1). The ROC analysis demonstrated moderate discriminative ability in predicting recurrence, with an area under the curve (AUC) of 0.696. At the optimal threshold of tumor budding ≥ 2, the model achieved 78.6% sensitivity and 62.9% specificity, indicating that tumor budding is a clinically relevant predictor of post‐radiotherapy recurrence.

FIGURE 2.

Receiver operating characteristic (ROC) curve illustrating the predictive value of tumor budding for post‐radiotherapy recurrence. The optimal cutoff point was determined using Youden's Index, calculated as (sensitivity + specificity—1). The maximum Youden's Index was observed at a tumor budding count ≥ 2, yielding a sensitivity of 78.6% and specificity of 62.9%. This point is marked by the red dot on the curve. The area under the curve (AUC) was 0.696, indicating a moderate discriminative ability. [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]

TABLE 1.

Relationship between tumor budding and RT failure.

		TMB
		< 2	≥ 2	p
		n (%)	n (%)
RT failure	No	22 (88)	13 (54.2)	0.009
	Yes	3 (12)	11 (45.8)

Abbreviations: RT, radiotherapy; TMB, tumor budding.

The impact of tumor budding on disease‐free survival following radiotherapy was evaluated. Patients with low tumor budding exhibited a 5‐year survival rate of 88%, whereas those with high tumor budding demonstrated a significant decline in survival, with a 54.2% reduction. A comparison of the disease‐free survival of patients with high and low tumor budding revealed a statistically significant difference (p = 0.008) (Figure 3).

FIGURE 3.

Kaplan–Meier disease‐free survival (DFS) curves for tumor budding. The blue line represents patients with tumor budding < 2, while the red line represents those with tumor budding ≥ 2. Censored data points, indicated by “+” symbols, represent cases where patients were lost to follow‐up or remained disease‐free at the last follow‐up. The log‐rank test (p = 0.008) indicates a statistically significant difference in DFS between the two groups. [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]

The impact of tumor budding on overall survival following radiotherapy was evaluated, and no statistically significant discrepancy was identified (Figure 4).

FIGURE 4.

Kaplan–Meier overall survival (OS) curves for tumor budding. The blue line represents patients with tumor budding < 2, while the red line represents those with tumor budding ≥ 2. Censored data points, indicated by “+” symbols, represent cases where patients were lost to follow‐up or remained alive at the last follow‐up. The log‐rank test (p = 0.659) indicates no statistically significant difference in OS between the two groups. [Color figure can be viewed in the online issue, which is available at www.laryngoscope.com.]

4. Discussion

Early‐stage glottic laryngeal cancer (EGC) is defined as stage 1 (T1N0M0) and stage 2 (T2N0M0). In EGCs, median survival rates have been reported to range from 70% to 90% [10]. The primary objective of treatment is to utilize a single modality and larynx‐sparing procedures, thereby avoiding the adverse effects associated with such interventions. The results of surgical methods and radiotherapy are comparable in terms of survival outcomes [11]. In early‐stage glottic laryngeal cancers, patients treated with surgery had an overall survival rate of 85% and a disease‐specific survival rate of 94.4%, whereas those who received radiotherapy had an overall survival rate of 71.5% and a disease‐specific survival rate of 87.3% [12]. It has been documented that cancers affecting the glottic region demonstrate superior survival rates compared to other regions in early‐stage laryngeal cancers [13]. In our study, the mean survival rate was 67.3%, and the disease‐free survival rate was 71.4% in patients who received radiotherapy for early‐stage glottic laryngeal cancers. It should be noted, however, that the aforementioned data do not include all patients who received radiotherapy. Consequently, they cannot be considered to represent the success results of radiotherapy treatment.

The occurrence of recurrence following treatment is attributed to a number of factors. The continuation of smoking following radiotherapy treatment has been linked to a number of adverse outcomes, including reduced local control, decreased average survival, elevated risk of second primary tumors, and impaired voice quality [2]. In a meta‐analysis conducted by Eskiizmir et al., it was determined that male gender and low hemoglobin levels were associated with an increased risk of radiotherapy failure [14]. In addition, a substantial body of molecular‐based research has been undertaken with the objective of predicting the occurrence of failure following radiotherapy. In a study that analyzed Bcl‐2, Ki‐67, and EGFR, only Ki‐67 was identified as an independent predictor of disease‐free survival [3]. In a separate study, patients who had experienced a relapse following radiotherapy or chemoradiotherapy were examined. It was found that p16, p53, EGFR, CD31, and vimentin were not associated with survival. In the same study, the status of CD8 and CD4 tumor‐infiltrating lymphocytes was found to be associated with an improvement in disease‐free survival and disease‐specific survival in patients who had experienced a relapse. Nevertheless, no correlation was identified between median survival and the aforementioned factors [15]. In a recent study, Schrijvers et al. identified hypoxia‐inducible factor 1 alpha subunit (HIF1a), carbonic anhydrase IX (CA‐IX) and Fas‐associated death domain (FADD) molecules as potential prognostic markers for predicting local recurrence in early‐stage laryngeal cancer treated with radiotherapy [16, 17].

Wildeman et al. conducted a comprehensive study to identify molecular markers predictive of radiotherapy resistance in laryngeal carcinoma. They evaluated 13 biomarkers, including those related to hypoxia (CA9, HIF‐1α), apoptosis (Bcl‐2, Bcl‐xL, p53), cell cycle regulation (p16, p21, p27, cyclin D1, pRB), proliferation (Ki‐67), and inflammation (COX‐2, EGFR), using immunohistochemistry on pre‐treatment biopsy samples [5]. While the initial analysis suggested that CA9 (hypoxia) and COX‐2 overexpression were the strongest candidates for predicting recurrence, neither marker reached statistical significance after adjustment for clinical variables. No single biomarker or combination of markers formed a robust predictive profile, highlighting the challenges in identifying molecular predictors of radioresistance.

In contrast, tumor budding was found to be a statistically significant predictor of post‐radiotherapy recurrence in our analysis (p = 0.0253). The logistic regression model demonstrated that higher tumor budding was associated with an increased risk of recurrence, and its predictive performance was further supported by the ROC analysis at an optimal cutoff of 2. In contrast, molecular markers such as hypoxia (CA9) and COX‐2 overexpression showed only borderline associations and failed to form a robust predictive profile.

Beyond its statistical performance, tumor budding assessment offers clear practical advantages over molecular markers. Unlike immunohistochemical panels, which require costly antibodies, specialized equipment, and standardized protocols, tumor budding can be evaluated using routine hematoxylin and eosin (H&E) staining, making it a cost‐effective and widely accessible biomarker. Its histopathological assessment is readily integrable into routine pathology workflows, ensuring greater clinical feasibility without the added complexities of molecular assays.

The disease‐free survival rate was found to be significantly lower in patients with metastasis‐associated colon cancer 1 (MACC1) positivity [18]. No statistically significant difference was observed between the CD133 level and mean survival or disease‐free survival [19]. A substantial body of literature exists about markers. Nevertheless, it is evident that further investigation is required for these to be employed as routine prognostic factors, alongside established indicators such as age, stage, and anterior commissure involvement. Furthermore, the necessity for additional contrast agents for these molecules presents a challenge in terms of cost and accessibility.

Tumor budding can be defined as the presence of tumoral cells in an area distant from the tumor tissue, although this definition is not entirely precise. Although the concept was first introduced to the literature in the mid‐twentieth century, its current form was established by Hase et al. [20, 21]. In 2016, the International Tumor Budding Consensus Conference reached a consensus on a classification system for the utilization of tumor budding in clinical practice [8]. In recent years, there have been numerous studies conducted on tumor budding in head and neck cancers, as well as in other regions. Tumor budding in early oral cavity tumors has been shown to be associated with a poor prognosis, both in terms of regional control and disease‐free survival [22]. Another study demonstrated a significant association between tumor budding and overall survival, whereas no correlation was observed with disease‐free survival [23]. The incorporation of tumor budding into novel histomorphological scoring systems highlights its growing clinical significance [24]. The impact of tumor budding on prognosis and therapeutic applications has been demonstrated in many studies [25, 26]. A review of the literature on tumor budding in laryngeal cancers reveals that the first study was conducted by Sarıoğlu et al. 14 years ago. This study demonstrated the relationship between tumor budding and distant metastasis in laryngeal cancers and established tumor budding as a prognostic factor [27]. Another study showed that high tumor budding significantly decreased overall survival and disease‐free survival in laryngeal cancer [28]. Similarly, Abd Raboh et al. concluded that tumor budding was correlated with poor prognostic clinicopathologic indicators in laryngeal squamous cell carcinoma and recommended the use of pancytokeratin immunohistochemistry for evaluating tumor budding, particularly in ambiguous cases [29]. In line with these findings, Zang et al. identified total tumor budding as an independent prognostic parameter for 5‐year recurrence‐free survival (RFS) and overall survival (OS) in laryngeal squamous cell carcinoma, suggesting that tumor budding assessment should be incorporated into routine histopathological reports for larynx SCC [30]. Furthermore, Boxberg et al. demonstrated that budding activity was a significant prognostic factor for overall survival (OS), disease‐specific survival (DSS), and disease‐free survival (DFS) [31]. Subsequently, the number of analogous studies has risen steadily over time. In these studies, an attempt was made to determine a cut‐off value. Reported cut‐off values in previous studies range from 3 to 15. It should be noted, however, that these cut‐off values are typically derived from studies on prognosis. In the present study, the objective was to determine a comparable cut‐off value, which was intended to be utilized for the purpose of determining the selection of treatment, as opposed to prognosis. In this study, a cut‐off value of 2 was determined.

It is our contention that tumor budding represents a valuable tool in the treatment selection process for early‐stage laryngeal cancers, offering a potentially beneficial avenue for determining the optimal surgical or radiotherapy approach. The prediction of the radioresistant group may facilitate the selection of an appropriate treatment, the reduction of recurrence and morbidity. This may facilitate the introduction of additional treatment modalities, such as chemotherapy or novel targeted molecular agents, into surgical or radiotherapy regimens, with the objective of reducing the incidence of recurrence following radiotherapy.

It should be noted that the present study is not without limitations. It should be noted that this study is retrospective in nature. Consequently, this may result in a degree of bias and potentially compromise the validity of the findings. The restricted number of cases from a single centre may not be representative of other regions or of all patients. It is evident that future studies would benefit from multicentre investigations incorporating a greater number of cases.

5. Conclusions

The selection of an appropriate treatment for early‐stage laryngeal cancers has historically presented a significant challenge for clinicians. Post‐radiotherapy recurrence has been associated with reduced overall survival and increased risk of serious complications that may impair quality of life. In the present study, we have described a new method that can be applied and compared with minimal expense, guiding clinicians about the risk of post‐radiotherapy recurrence at the time of incisional biopsy. Given its statistical significance, predictive value, and ease of implementation, tumor budding represents a promising and practical biomarker for identifying patients at higher risk of recurrence after radiotherapy.

Consent

All included patients were provided with detailed information about the aims and methods of the study and signed informed consent forms.

Conflicts of Interest

The authors declare no conflicts of interest.

Birinci M., Gül O., Okcu O., et al., “The Role of Tumor Budding in Early‐Stage Laryngeal Cancer Treatment Selection,” The Laryngoscope 135, no. 9 (2025): 3222–3228, 10.1002/lary.32218.