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The prognostic value of tumor budding in laryngeal squamous cell carcinoma

Background The predictive value of tumor budding in several cancers is of the essence. The 5-year survival of laryngeal squamous cell carcinoma patients is decreasing despite the improvement of therapy. In order to help improve the prognosis of LSCC patients, we aimed to investigate the value of tumor budding on the prognosis in laryngeal squamous cell cancer (LSCC) by the pathological characteristics of the surgical cases. Methods Archival clinical specimens of 51 patients diagnosed with LSCC were included in the research. On the basis of hematoxylin and eosin (H&E)-stained slides, tumor-stroma ratio (TSR), tumor budding and nuclear features were assessed. Correlation between clinical data and histologic characteristics was carried out using the Kaplan-Meier method and the Cox proportional hazards regression model, respectively. Results Total tumor budding was independent prognostic parameter of 5-year recurrence-free survival (RFS) and overall survival (OS) in LSCC. The evaluation of tumor budding can be as a part of the routine histopathological report in LSCC. Conclusions Tumor budding can be an independent factor of prognosis of LSCC patients and should be as a part of routine histopathologic report for LSCC cases.

metastasis may lead to better stratify patients with LSCC and provide better prognostic outcome. It has been reported that cancer cells that are located in the invasive tumor front are more aggressive (3). Tumor budding is identified as isolated small tumor clusters composed of less than five non-glandular cancer cells scattered in the invasive tumor front (3). The presence of tumor budding is regarded as characteristic of aggressive cancer and has been previously considered as a new histopathologic marker in several malignancies, including lung squamous cell carcinoma (6), esophageal squamous cell carcinoma (7), colorectal cancer (8), breast cancer (9), and pancreatic ductal adenocarcinoma (10).
Tumor tissues consist of epithelial cells and stroma originated from normal tissues. The stroma may serve as a barrier to constrain cell proliferation and migration in normal tissues while they facilitate tumor progression in tumor tissues. The tumor stroma is composed of inflammatory cells, capillaries, fibroblasts and extracellular matrix (11). Fibroblasts that surround and infiltrate the tumor are also named as cancer associated fibroblasts (CAFs). CAFs can secrete chemokine and growth factor to enhance cells migration and invasion and promote angiogenesis (12). It was reported that tumor-stroma ratio (TSR) has a predictor value in lymph nodes. TSR combined with lymph nodes has been documented to be a significant prognostic parameter in breast cancer (13).
Herein, we evaluated the prognostic significance of tumor budding and TSR on the grounds of slides stained with hematoxylin and eosin (H&E). Our research found that tumor budding and T and N status may serve as reliable independent histologic prognostic markers, and tumor budding should be used in routine histologic diagnosis in LSCC.

Patients
The patients in this research were diagnosed with LSCC and went surgical resection between 2010 and 2014 at The First Affiliated Hospital of Guangzhou Medical University. The included patients were 51 males, 0 female, without distant metastasis. Clinical characteristics were summarized in Table 1. All patients included might take additional postoperative treatment according to the guideline of NCCN, such as neoadjuvant therapy. And the patients with any adjuvant therapy before surgery were excluded. Tumor stage was categorized on the basis of TNM system by AJCC [2010], and the tumor pathological grade was according to WHO Classification of Head and Neck Tumors [2017]. The research was approved by the ethical committee of The First Affiliated Hospital of Guangzhou Medical University.

Histologic evaluation
Histologic evaluation was performed on 4 μm hematoxylin and eosin (H&E)-stained slides (1,7). Histologic evaluation was completed by three pathologists who were without any knowledge of the clinical data or the other pathologists' results. At a later stage, all three pathologists contributed to a consensus assessment for all the variables. Reproducibility was measured using Gwet's agreement coefficients.
For TSR assessment, the most tumor areas were selected with 4× objective, then, TSR scoring was evaluated using 10× objective. Only fields where stroma and tumor cells are both present were eligible. Stromal cells ratio ≤50% was defined as low stroma ratio (high TSR) and >50% were high stroma (low TSR) ( Figure 1A).
Tumor budding was defined as small tumor nests composed of <5 tumor cells. For evaluating tumor budding, tumor slides were scanned at 10× objective. Subsequently, tumor budding was counted at the most invasion area in 10 fields at ×200 magnification. The tumor budding was analyzed by two ways: the total numbers of tumor budding under 10 HPFs and the maximum numbers per field among 10 HPFs ( Figure 1B). >10 tumor budding/10 HPFs or >10 tumor budding/HPF was defined as high total tumor budding or high maximum tumor budding, respectively. <10 tumor budding/10 HPFs or <10 tumor budding/HPF was defined as low total tumor budding or low maximum tumor budding, respectively. Single cell invasion was considered present if any was observed after examination of 50 HPFs at the most invasive area with the maximal number of the smallest tumor nests ( Figure 1C).
Nuclear atypia was identified using 40× objective in the area with the highest abnormal nuclear phenotype after scanning the whole slide at 10× objective. For nuclear diameter, we screened 10 HPFs with the largest nuclei and then examined the average nuclear diameter of 100 tumor cells. Small lymphocytes (≈4.0 μm) served as the reference. The nuclear diameter >4 small lymphocytes was classified as large nuclei, while, ≤4 small lymphocytes was classified as small nuclei ( Figure 1D). Mitosis evaluation was performed in 50 HPFs areas that contained the highest mitotic activity. Mitotic numbers ≥15/10 HPFs were classified as high mitotic     As for the multivariate analysis (Table 3), after adjusting for clinical stage, total tumor budding was an independent predictor of the LSCC patients' prognosis except for T status. Also, the histologic subtype was independently correlated with 5-year RFS (HR =3.381) and the nest size was independently correlated with 5-year OS (HR =1.843). And we found that high-grade tumor budding was associated with higher T stage (P=0.000), smaller nest size (including single-cell) (P=0.005), larger nuclear diameter (P=0.040), advanced clinical stage (P=0.004), worse poorly pathological differentiation (P=0.048) and higher TSR (P=0.005) (shown in Table 4).

Discussion
In previous literatures, unfavorable prognostic factors for laryngeal squamous cell carcinoma are listed as advanced tumor stage, subglottic localization, high microscopic grade, increased number and size of metastasizing lymph nodes, presence of extracapsular extension, epidermal growth factor receptor expression, and tumor budding, etc. (14)(15)(16).
In the present study, we evaluated the prognostic value of histologic factors on the basis of H&E analysis.
Stromal cells take a main role in the cancer invasion and metastasis (17). The TSR has been documented as  B E F a significantly independent prognostic factor in several epithelial cancers (18). Also, TSR scoring can be completed based on H&E-stained slides without additional costs and taken less than a minute. TSR evaluation is highly reproducible due to its simplicity and reliability. Therefore, TSR scoring can be as a part of the routine histopathologic report. The patients with higher TSR showed a worse    Non-keratinization 6 5 prognosis. But it was not as we expected that TSR was an independent predictor for the LSCC patients' outcome in Cox-regression analysis. The limited sample may attribute to the result. Tumor budding has been documented as a morphologic phenomenon of tumor invasion and to be a poor prognostic predictor in several cancers (19)(20)(21)(22). In our research, we counted the total numbers of tumor buds using 10 HPFs and the maximum numbers in 1 HPF. Only total budding number is independently associated with 5-year RFS and OS. It has been uncovered that tumor budding is related with epithelial mesenchymal transition (EMT) to enhance tumor cell migration and invasion (23)(24)(25)(26). Tumor budding maximum number does not show independent effect, which may be due to being difficult to agree between different pathologists when counting the maximal number of tumor budding in 1 HPF. Budding seems to be associated with the nuclear location of b-catenin, which is related to E-cadherin aberrations, along with loss of the expression of epithelial cell adhesion molecule (Ep-CAM). These changes are caused by the loss of intercellular adhesions (27). In the process of EMT, cells lose epithelial features and gain mesenchymal characteristics. Dysregulation of EMT can enhance cancer invasion and metastasis formation. However, the detailed molecular mechanism by which tumor budding enhances tumor invasion and metastasis is unclear.
The degree of keratinization was found to be independently related to the occurrence and survival of OSCC patients (28,29). Our results supported this conclusion, so as a possible risk factor for recurrence, keratinization should be considered in decisions regarding adjuvant therapy. In several kinds of squamous cell carcinoma, tumor cell nest size was highly prognostic on these malignancies (7), and tumor nest size showed independent prognosis on the LSCC patients' 5-year OS. Further studies are necessary.
In conclusion, similar with previous researches about other malignancies, tumor budding based on the H&Estained specimens, which can be carried out easily, can be a part of routine histopathologic reporting for LSCC. We think tumor budding will be better stratify patients with LSCC and provide better prognostic outcome. As it is a limited sample research, it needs an enlarged and multicenter LSCC samples to certify the conclusion.