The prognostic value of histopathological grading systems in oral squamous cell carcinomas

Orientadores: Ricardo Della Coletta, Ana Lucia Carrinho Ayroza RangelTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: O carcinoma espinocelular (CEC) representa cerca de 95% de todas as neoplasias malignas que acometem a cavidade oral. Rotineiramente o tratamento e prognóstico desta doença são baseados na localização do tumor e no sistema TNM de classificação dos tumores malignos, no entanto há uma grande variação no comportamento biológico entre tumores no mesmo sítio e dentro do mesmo estadio clínico. Diante deste problema, vários sistemas de gradação histopatológica foram propostos para determinar o prognóstico e o plano do tratamento de pacientes com carcinoma espinocelular (CEC) oral. Este estudo avaliou quatro sistemas de gradação histopatológica ¿ (1) sistema OMS (Organização Mundial de Saúde), (2) sistema MG (gradação de malignidade de margens invasivas profundas), (3) modelo HR (modelo de risco histológico) e (4) escore de risco BD (ninho de células tumorais e profundidade de invasão) ¿ e comparou com dados clínico-patológicos e sobrevida em uma amostra de 113 pacientes com CEC oral primário, excluindo lábios. Os critérios de inclusão foram: pacientes diagnosticados e tratados entre 1998 e 2008, dados clínicos e demográficos completos, tratamento baseado em cirurgia radical com ou sem radioterapia e/ou quimioterapia pós-operatória e disponibilidade de todos os blocos de parafina. Associações significativas com sobrevida na análise univariada foram observadas com todos os sistemas de gradação histopatológica, excetuando o sistema MG. No entanto, aplicando a análise multivariada de COX, apenas o escore de risco BD foi significativamente associado com sobrevida livre de doença como um marcador prognóstico independente. A idade (>56 anos), o tamanho do tumor (estágio T3/T4) e a presença de metástase regional (estágio N+) foram também apontados como marcadores independentes da sobrevida dos pacientes. Nenhuma correlação clara entre os quatro sistemas de gradação foi observada aplicando o teste de correlação de Spearman. Os resultados do presente estudo revelaram uma associação significativa entre o escore de risco BD e a evolução clínica dos pacientes com CEC oral, reforçando a importância deste novo sistema de gradação histopatológica como possível ferramenta prognóstica no pós-operatórioAbstract: Squamous cell carcinoma (SCC) represents almost 95% of all malignant tumors that affect the oral cavity. Routinely the treatment and prognosis of this disease are based on its location and in the TNM classification of malignant tumors, however there is a great variation in the biological behavior among tumors at same location and clinical stage. In the view of those difficulties, several histopathological grading systems were proposed in order to determine the prognostic and the treatment plan of patients with oral squamous cell carcinoma (OSCC). This study evaluated four histopathological grading systems ¿ (1) WHO (World Health Organization) system, (2) MG (malignancy grading of the deep invasive margins) system, (3) HR (histological risk ) model and (4) BD (tumor budding and depth of tumor invasion) risk score ¿ and compared with clinicopathological data and survival in a sample of 113 patients with primary OSCC, excluding lips. The inclusion criteria included who were diagnosed and treated from 1998 to 2008, complete demographic and clinical data, treatment based on radical surgery with or without postoperative radiotherapy and/or chemotherapy, and availability of all paraffin-embedded blocks. Significant associations with survival were observed for all histopathological grading systems, with exception of the MG system. However, when multivariate regression analysis was applied, only BD risk score was significantly associated with disease-free survival as an independent prognostic marker. Age (>56 years), tumor size (T3/T4 stage) and presence of regional metastasis (N+ stage) were also independent markers of reduced survival. No clear correlation between the four grading systems was observed applying the Spearman¿s rank test. The results of the present study revealed a significant association between BD risk score and outcome of OSCC patients, reinforcing the importance of this new histopathological grading system as a possible postoperative prognostic toolDoutoradoPatologiaDoutora em Estomatopatologi

Prognostication for oral carcinomas based on two histological scoring systems (BD and iBD models)

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Comparison of supervised machine learning classification techniques in prediction of locoregional recurrences in early oral tongue cancer

Background: The proper estimate of the risk of recurrences in early-stage oral tongue squamous cell carcinoma (OTSCC) is mandatory for individual treatment-decision making. However, this remains a challenge even for experienced multidisciplinary centers. Objectives: We compared the performance of four machine learning (ML) algorithms for predicting the risk of locoregional recurrences in patients with OTSCC. These algorithms were Support Vector Machine (SVM), Naive Bayes (NB), Boosted Decision Tree (BDT), and Decision Forest (DF). Materials and methods: The study cohort comprised 311 cases from the five University Hospitals in Finland and A.C. Camargo Cancer Center, Sao Paulo, Brazil. For comparison of the algorithms, we used the harmonic mean of precision and recall called F1 score, specificity, and accuracy values. These algorithms and their corresponding permutation feature importance (PFI) with the input parameters were externally tested on 59 new cases. Furthermore, we compared the performance of the algorithm that showed the highest prediction accuracy with the prognostic significance of depth of invasion (DOI). Results: The results showed that the average specificity of all the algorithms was 71% The SVM showed an accuracy of 68% and F1 score of 0.63, NB an accuracy of 70% and F1 score of 0.64, BDT an accuracy of 81% and F1 score of 0.78, and DF an accuracy of 78% and F1 score of 0.70. Additionally, these algorithms outperformed the DOI-based approach, which gave an accuracy of 63%. With PFI-analysis, there was no significant difference in the overall accuracies of three of the algorithms; PFI-BDT accuracy increased to 83.1%, PFI-DF increased to 80%, PFI-SVM decreased to 64.4%, while PFI-NB accuracy increased significantly to 81.4%. Conclusions: Our findings show that the best classification accuracy was achieved with the boosted decision tree algorithm. Additionally, these algorithms outperformed the DOI-based approach. Furthermore, with few parameters identified in the PFI analysis, ML technique still showed the ability to predict locoregional recurrence. The application of boosted decision tree machine learning algorithm can stratify OTSCC patients and thus aid in their individual treatment planning.Peer reviewe

Histopathological grading systems analysis of oral squamous cell carcinomas of young patients

Background: To analyze the clinicopathological profile of young patients (≤ 40 years) with oral SCC and correlate with a control group (≥ 50 years) by means of histopathological grading systems. Material and Methods: 14 young patients and 14 control patients were selected with similar clinical stage and tumor location. Demographic and clinical data were obtained from patient records and histological sections were evaluated according to four histopathological grading systems. Associations between categories of demographic and clinical data were performed through Chi-square test and Exact Fisher test. The survival analyzes were performed according to the Kaplan-Meier method. Results: The comparison between groups showed a greater association of treatment modalities in younger patients ( p =0.022), they had a higher incidence of local recurrence and regional metastasis ( p =0.018) and lower disease- free survival in 5 years ( p =0.069). There was no difference in 5-year overall survival among the studied groups. There was no difference in histological grading between studied groups according to the four used systems. Conclusions: This study showed that, despite tumors had similar histological grade and more therapeutic modalities were used in the young group, tumors in young patients had a higher incidence of recurrence/metastasis, showing tendency to a more aggressive behavior

Trophoblast cell surface antigen 2 expression predicts outcome in oral squamous cell carcinomas

Background Trophoblast cell surface antigen 2 (TROP2) has unclear clinical role in oral squamous cell carcinomas (OSCC). Here, we investigated the association of TROP2 immunoexpression with clinicopathological parameters and survival of OSCC patients. Subjects and Methods Cancer-specific survival (CSS) and disease-free survival (DFS) were assessed in a cohort composed of 266 OSCC. An independent cohort with 88 OSCC samples matched with the normal oral tissue, as well as 17 metastatic lymph nodes, was used for validation. Results Multivariate analysis showed TROP2 as an independent marker of favorable prognosis for both CSS (HR: 0.60, 95% CI: 0.40-0.90, p = .01) and DFS (HR: 0.57, 95% CI: 0.36-0.89, p = .01). Furthermore, TROP2 protein expression was significantly higher in morphologically normal tissues compared to primary tumors (p <.0001) and lymph node metastases (p = .001), and it was significantly associated with CSS (HR: 0.26, 95% CI: 0.09-0.74, p = .008) in the validation cohort. A pooled mRNA analysis performed on the Oncomine (TM) database confirmed the underexpression in OSCC compared with normal tissues (p = .014). Conclusions In summary, our results point to a favorable prognostic significance of TROP2 overexpression in a large cohort of oral cancer patients, suggesting it as an attractive clinical marker.Peer reviewe

Tenascin-C and fibronectin expression divide early stage tongue cancer into low- and high-risk groups

Background:Oral tongue squamous cell carcinoma (OTSCC) metastasises early, especially to regional lymph nodes. There is an ongoing debate on which early stage (T1-T2N0) patients should be treated with elective neck dissection. We need prognosticators for early stage tongue cancer. Methods: Mice immunisation with human mesenchymal stromal cells resulted in production of antibodies against tenascin-C (TNC) and fibronectin (FN), which were used to stain 178 (98 early stage), oral tongue squamous cell carcinoma samples. TenascinC and FN expression in the stroma (negative, moderate or abundant) and tumour cells (negative or positive) were assessed. Similar staining was obtained using corresponding commercial antibodies. Results: Expression of TNC and FN in the stroma, but not in the tumour cells, proved to be excellent prognosticators both in all stages and in early stage cases. Among early stages, when stromal TNC was negative, the 5-year survival rate was 88%. Correspondingly, when FN was negative, no cancer deaths were observed. Five-year survival rates for abundant expression of TNC and FN were 43% and 25%, respectively. Conclusions: Stromal TNC and, especially, FN expressions differentiate patients into low-and high-risk groups. Surgery alone of early stage primary tumours might be adequate when stromal FN is negative. Aggressive treatments should be considered when both TNC and FN are abundant.Peer reviewe

Machine learning application for prediction of locoregional recurrences in early oral tongue cancer: a Web-based prognostic tool

Estimation of risk of recurrence in early-stage oral tongue squamous cell carcinoma (OTSCC) remains a challenge in the field of head and neck oncology. We examined the use of artificial neural networks (ANNs) to predict recurrences in early-stage OTSCC. A Web-based tool available for public use was also developed. A feedforward neural network was trained for prediction of locoregional recurrences in early OTSCC. The trained network was used to evaluate several prognostic parameters (age, gender, T stage, WHO histologic grade, depth of invasion, tumor budding, worst pattern of invasion, perineural invasion, and lymphocytic host response). Our neural network model identified tumor budding and depth of invasion as the most important prognosticators to predict locoregional recurrence. The accuracy of the neural network was 92.7%, which was higher than that of the logistic regression model (86.5%). Our online tool provided 88.2% accuracy, 71.2% sensitivity, and 98.9% specificity. In conclusion, ANN seems to offer a unique decision-making support predicting recurrences and thus adding value for the management of early OTSCC. To the best of our knowledge, this is the first study that applied ANN for prediction of recurrence in early OTSCC and provided a Web-based tool.Peer reviewe

Secretome profiling of oral squamous cell carcinoma-associated fibroblasts reveals organization and disassembly of extracellular matrix and collagen metabolic process signatures

An important role has been attributed to cancer-associated fibroblasts (CAFs) in the tumorigenesis of oral squamous cell carcinoma (OSCC), the most common tumor of the oral cavity. Previous studies demonstrated that CAF-secreted molecules promote the proliferation and invasion of OSCC cells, inducing a more aggressive phenotype. In this study, we searched for differences in the secretome of CAFs and normal oral fibroblasts (NOF) using mass spectrometry-based proteomics and biological network analysis. Comparison of the secretome profiles revealed that upregulated proteins involved mainly in extracellular matrix organization and disassembly and collagen metabolism. Among the upregulated proteins were fibronectin type III domain-containing 1 (FNDC1), serpin peptidase inhibitor type 1 (SERPINE1), and stanniocalcin 2 (STC2), the upregulation of which was validated by quantitative PCR and ELISA in an independent set of CAF cell lines. The transition of transforming growth factor beta 1 (TGF-beta 1)-mediating NOFs into CAFs was accompanied by significant upregulation of FNDC1, SERPINE1, and STC2, confirming the participation of these proteins in the CAF-derived secretome. Type I collagen, the main constituent of the connective tissue, was also associated with several upregulated biological processes. The immunoexpression of type I collagen N-terminal propeptide (PINP) was significantly correlated in vivo with CAFs in the tumor front and was associated with significantly shortened survival of OSCC patients. Presence of CAFs in the tumor stroma was also an independent prognostic factor for OSCC disease-free survival. These results demonstrate the value of secretome profiling for evaluating the role of CAFs in the tumor microenvironment and identify potential novel therapeutic targets such as FNDC1, SERPINE1, and STC2. Furthermore, type I collagen expression by CAFs, represented by PINP levels, may be a prognostic marker of OSCC outcome.Peer reviewe

Stanniocalcin 2 contributes to aggressiveness and is a prognostic marker for oral squamous cell carcinoma

Stanniocalcin 2 (STC2), a glycoprotein that regulates calcium and phosphate homeostasis during mineral metabolism, appears to display multiple roles in tumorigenesis and cancer progression. This study aimed to access the prognostic value of STC2 in oral squamous cell carcinoma (OSCC) and its implications in oral tumorigenesis. STC2 expression was examined in 2 independent cohorts of OSCC tissues by immunohistochemistry. A loss-of-function strategy using shRNA targeting STC2 was employed to investigate STC2 in vitro effects on proliferation, apoptosis, migration, invasion, epithelial-mesenchymal transition (EMT) and possible activation of signaling pathways. Moreover, STC2 effects were assessed in vivo in a xenograft mouse cancer model. High expression of STC2 was significantly associated with poor disease-specific survival (HR: 2.67, 95% CI: 1.37-5.21, p = 0.001) and high rate of recurrence with a hazard ratio of 2.80 (95% CI: 1.07-5.71, p = 0.03). In vitro downregulation of STC2 expression in OSCC cells attenuated proliferation, migration and invasiveness while increased apoptotic rates. In addition, the STC2 downregulation controlled EMT phenotype of OSCC cells, with regulation on E-cadherin, vimentin, Snaill, Twist and Zeb2. The reactivation of STC2 was observed in the STC2 knockdown cells in the in vivo xenograft model, and no influence on tumor growth was observed. Modulation of STC2 expression levels did not alter consistently the phosphorylation status of CREB, ERK, JNK, p38, p70 S6K, STAT3, STAT5A/B and AKT. Our findings suggest that STC2 overexpression is an independent marker of OSCC outcome and may contribute to tumor progression via regulation of proliferation, survival and invasiveness of OSCC cells.Peer reviewe

Fascin promotes migration and invasion and is a prognostic marker for oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) prognosis is related to clinical stage and histological grade. However, this stratification needs to be refined. We conducted a comparative proteome study in microdissected samples from normal oral mucosa and OSCC to identify biomarkers for malignancy. Fascin and plectin were identified as differently expressed and both are implicated in several malignancies, but the clinical impacts of aberrant fascin and plectin expression in OSCCs remains largely unknown. Immunohistochemistry and real-time quantitative PCR were carried out in ex vivo OSCC samples and cell lines. A loss-of-function strategy using shRNA targeting fascin was employed to investigate in vitro and in vivo the fascin role on oral tumorigenesis. Transfections of microRNA mimics were performed to determine whether the fascin overexpression is regulated by miR-138 and miR-145. We found that fascin and plectin are frequently upregulated in OSCC samples and cell lines, but only fascin overexpression is an independent unfavorable prognostic indicator of disease-specific survival. In combination with advanced T stage, high fascin level is also an independent factor of disease-free survival. Knockdown of fascin in OSCC cells promoted cell adhesion and inhibited migration, invasion and EMT, and forced expression of miR-138 in OSCC cells significantly decreased the expression of fascin. In addition, fascin downregulation leads to reduced filopodia formation and decrease on paxillin expression. The subcutaneous xenograft model showed that tumors formed in the presence of low levels of fascin were significantly smaller compared to those formed with high fascin levels. Collectively, our findings suggest that fascin expression correlates with disease progression and may serve as a prognostic marker and therapeutic target for patients with OSCC.Peer reviewe