The retrospective study included 366 patients diagnosed with primary LSCC who underwent radical surgical resection of LSCC and cervical lymph node dissection from 2004 to 2021 in Beijing Tongren Hospital and Beijing Luhe Hospital. The exclusion criteria included patients with a history of other malignancies; patients with a histologic type other than squamous cell carcinoma; patients who had received neoadjuvant therapy before surgery; and patients with incomplete clinical information and follow-up data. According to the inclusion and exclusion criteria, 268 patients were included in our study cohort. Informed consent was obtained from patients. The approval of our study was granted by the Ethics Committee. Our study has been carried out in accordance with the Code of Ethics of the Word Medical Association (Declaration of Helsinki) for experiments involving humans.

All Hematoxylin and Eosin (HE) stained sections of the 268 LSCC patients and their histological features were reevaluated under a Nikon microscope by two experienced head and neck pathologists who were blinded to the patients’ clinicopathological information, including tumor histologic grade, tumor cell nest size, tumor cell mitotic activity, tumor stroma ratio, stromal tumor Infiltrating Lymphocytes (TILs), TB activity, pathological-T (pT) stage, pathological-N (pN) stage, angiolymphatic invasion, and perineural invasion. Any differences of opinion between the two pathologists were discussed under a multi-head microscope until a consensus was reached.

Two sections were taken from one tumor tissue block at 50 microns interval, and about 10 sections were used for histologic evaluation in each laryngeal cancer case. The modified version of Broders’ grading system used for evaluating the histologic grade. 0%‒25% of undifferentiated tumor cells and 75%–100% of differentiated tumor cells were well-differentiated tumors; 50 %–75 % of differentiated tumor cells and 25%‒50% of undifferentiated tumor cells were moderately differentiated tumors; more than 50% of undifferentiated tumor cells were poorly differentiated tumors. The overall tumor cell nest size was evaluated in tumor areas containing the smallest tumor cell nests based on Boxberg's methods,14 rather than in the front of the tumor invasion, to avoid confusion with TB. The tumor cell nests consisting of less than 5 tumor cells were called small cell nests, 5–15 tumor cells were called intermediate cell nests, and more than 15 tumor cells were called large cell nests. High mitotic count was defined as greater than 15 mitotic counts in 10 consecutive High Power Fields (HPFs), and low mitotic count was defined as less than or equal to 15 in 10 HPFs.15 The tumor stroma ratio was divided into two groups using a cutoff of less than 50% for the low group and greater than or equal to 50% for the high group.16 In addition, the stromal TILs were divided into 0%‒10% TILs, 11%‒50% TILs, and greater than 50% TILs.17

TB was evaluated based on the Colorectal Cancer Tumor Budding Report recommendations of the International Tumor Budding Consensus Conference (ITBCC) 201618 and was assessed using a single 10× objective microscope at the hot spot areas showing the maximal budding at the front of the tumor invasion. Then, a 20× objective was used with a 22 mm field diameter, and the results were divided by the normalization factor (1.210 based on a 22 mm eyepiece field diameter) to obtain the standardized budding count. Finally, pathological staging of LSCC was performed according to the eighth edition of the American Joint Committee on Cancer (AJCC) staging system.

The 268 LSCC patients were followed up for 10–192 months, and the end outcomes were Overall Survival (OS) and Disease-Free Survival (DFS). DFS was assigned as the period of clinical, radiological, or pathological metastasis or recurrence after surgery or the period until the last follow-up, and OS was defined as the time from surgery to the date of death or final follow-up.

All statistical analysis was performed using Statistics Package for Social Sciences (SPSS) software, version 21.0 (IBM Corporation, NY, USA). In addition, a Receiver Operating Characteristics (ROC) curve analysis was performed to determine the cutoff point of TB grouping. Associations between clinicopathological parameters and TB were assessed with Chi-Square tests, and Logistic regression analysis was used to analyze the clinicopathological risk factors related to TB. The survival curves were plotted using the Kaplan-Meier method, and the Log-rank test assessed the survival difference. Univariate and Multivariate survival analysis were also performed using a Cox proportional hazard model; p-values lower than 0.05 were considered to indicate statistically significant results for all statistical tests.

The ages of the 268 patients ranged from 35 to 86 years old, with a median of 62 and an average age of 61.96 ± 9.117. According to the median age, 142 patients (53.0%) were less than 62 years old, and 126 patients (47.0%) 62 years old or older. There were 240 males (89.6%) and 28 females (10.4%). Additionally, well-differentiated tumors were present in 64 of the cases (23.9%), moderately differentiated tumors in 142 cases (53.0%), and poorly differentiated tumors in 62 cases (23.1%). Small tumor cell nests were observed in 47/268 cases (17.5%), intermediate cell nests in 162/268 (60.5%), and large cell nests in 59/268 (22.0%). Next, high mitotic figures occurred in 106/268 cases (39.6%) and low mitotic figures in 162/268 cases (60.4%). There were 153 cases (57.1%) with tumor stroma ratios ≤50% and 115 (42.9%) with tumor stroma ratios >50%. 77 cases (28.7%) were pT1 stage, 82 (30.6%) were pT2 stage, 70 (26.1%) were pT3 stage, and 39 (14.6%) were pT4 stage. Furthermore, 214 cases (79.9%) were pN0 stage, and 54 cases (20.1%) were pN1+N2 stage. There was angiolymphatic invasion in 49 cases (18.3%) and perineural invasion in 23 cases (8.6%). Finally, stromal TILs were divided into TILs 0%‒10% (80/268, 29.9%), TILs 11%‒50% (137/268, 51.1%), and TILs >50% (51/268, 19.0%).

TB was observed in 226/268 (84.3%) cases, and the TB activity was 0–19/0.785 mm2, with an average of 6.46 ± 3.866/0.785 mm2 and a median of 7/0.785 mm2. ROC curve analysis showed that 7/0.785 mm2 was the optimal cutoff point to predict both the recurrence (sensitivity = 70.0%, specificity = 70.0%) and death (sensitivity = 72.5%, specificity = 71.8%) of the LSCC patients (Fig. 1). Thus, we divided the TB results into low-grade TB (0–6/0.785 mm2) (Fig. 2A and B) and high-grade TB (≥7/0.785 mm2) (Fig. 2C and D). Of the 268 cases, low-grade TB accounted for 81 (30.2%), and high-grade TB accounted for 187 (69.8%). In the low-grade TB group, there were 42 cases without TB, and 39 cases with 1 to 6 TB.

Figure 1.

Tumor budding grouping by Receiver operating characteristics curve. Receiver operating characteristics curve showed the best cutoff point for association of numbers of tumors budding with recurrence (A) and death (B). The best cutoff point was seven buddings in 0.785 square millimeters.

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Figure 2.

Examples of LSCC cases with low- and high-grade tumor budding. (A) An example with low-grade tumor budding (<7 buddings), where an invasive front arranged in large nests of cells with occasional presence of tumor budding is observed (Hematoxylin and eosin staining, ×100). (B) Low-grade budding (black arrow) under 200× magnification (Hematoxylin and Eosin staining). (C) Example with high-grade tumor budding (≥7 buddings), where the invasive front of the tumor is observed on the left where in the area of invasion there are abundant individual cells infiltrating the stroma (Hematoxylin and Eosin staining, ×100). (D) High-grade budding (black arrow) under 200× magnification (Hematoxylin and Eosin staining).

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High-grade TB was associated with gender, histologic grade, tumor cell nest size, mitotic count, tumor stroma ratio, pT stage, pN stage, stromal TILs, and angiolymphatic invasion (p <  0.05). Logistic regression analysis further showed that intermediate (OR = 2.644, 95% CI 2.071–4.029, p < 0.001) and small tumor cell nest (OR = 2.829, 95% CI 1.119–5.135, p =  0.001), higher pT stage (OR = 1.571, 95% CI 1.059–2.040, p =  0.040), stromal TILs 0%‒10% (OR = 2.120, 95% CI 1.512–3.980, p <  0.001) and stromal TILs 11%‒50% (OR = 2.785, 95% CI 1.914–3.960, p = 0.001) were the risk factors for high-grade TB, while moderate tumor differentiation (OR = 0.341, 95% CI 0.129‒0.900, p =  0.030) and tumor stroma ratio ≤50% (OR = 0.228, 95% CI 0.081‒0.639, p =  0.005) were protective factors for high-grade TB. The relationships of TB to the various clinicopathological parameters are shown in Table 1 and Fig. 3.

Figure 3.

Logistic regression forest plot. Logistic regression forest plot showed small and intermediated tumor cell nest, higher pT stage, stromal TILs 0%‒10% and 11%‒50% were the risk factors (**) for high-grade budding. Moderately differentiated tumor and tumor stromal ratio ≤50% were the protective factors (*) for high-grade budding.

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The average follow-up period was 48.77 ± 26.44 months (range 10–192 months), and the median period was 44 months. During follow-up, 76 (28.4%) patients had tumor recurrence and 80 (29.8%) patients died.

Among the patients with no TB, 1–6 TB, and more than 6 TB (high-grade TB), univariate Cox survival analysis indicated that there was no statistical difference in DFS and OS between the patients with no TB and those with 1–6 TB (p >  0.05); there were statistically significant differences in DFS and OS between the more than 6 TB (high-grade TB) and the no TB, and between the more than 6 TB (high-grade TB) and the 1–6 TB (p <  0.05) (Supplementary Table and Supplementary Fig. 5A‒B).

In the low-grade TB and high-grade TB, univariate Cox survival analysis indicated that poorly differentiated tumors, small and intermediate tumor cell nest, high mitotic count, tumor stroma ratio ≥50%, higher pT stage, stromal TILs 0%‒10%, and high-grade TB were associated with poor DFS (p <  0.05) (Table 2 and Fig. 4A). Multivariate Cox survival analysis revealed that high-grade TB (HR = 7.566, 95% CI 2.529–22.638, p <  0.001) was independent risk factors for DFS (Table 2).

Figure 4.

The effect of tumor budding on the survival of patients with LSCC illustrated by Kaplan-Meier curves: (A) disease-free survival; (B) overall survival; (C) disease-free survival in patients with T1-2N0 LSCC; (D) overall survival in patients with T1-2N0 LSCC.

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In addition, univariate Cox survival analysis revealed that moderately and poorly differentiated tumors, small and intermediate tumor cell nest, higher pT stage, angiolymphatic invasion, stromal TILs 0%‒50%, and high-grade TB were associated with poor OS (p <  0.05) (Table 3 and Fig. 4B). Multivariate Cox survival analysis showed that high-grade TB (HR = 4.279, 95% CI 1.474–12.422, p =  0.008), moderate tumor differentiation (HR = 2.766, 95% CI 1.087–7.040, p =  0.033), and poor tumor differentiation (HR = 3.909, 95% CI 1.381–11.061, p =  0.010) were found to be independent risk factors for OS (Table 3).

In 141 cases of T1-2N0 LSCC, 63 (44.7%) had low-grade budding, and 78 (55.3%) had high-grade budding. Multivariate Cox survival analysis showed that high-grade TB (HR = 13.052, 95% CI 1.161–146.732, p =  0.037) (Fig. 4C) and small tumor cell nest (HR = 11.962, 95% CI 1.083–132.156, p =  0.043) were independent prognostic factors for DFS in T1-2N0 LSCC (Table 4). In addition, high-grade TB (HR = 7.202, 95% CI 1.637–31.685, p =  0.009) (Fig. 4D), moderate tumor differentiation (HR = 2.844, 95% CI 1.074–7.534, p =  0.035), and angiolymphatic invasion (HR = 6.339, 95% CI 1.886–21.301, p =  0.003) were found to be independent prognostic factors for OS in T1-2N0 LSCC (Table 4).