Negative Oral Squamous Cell Carcinoma Research Articles

HPV and p16 expression association with 5-year survival in oral squamous cell carcinoma patients of north-east India

BackgroundIn our study, we examined the 5-year survival of OSCC patients with HPV positive or negative status along with p16 protein expression. MethodA total of 72 biopsy tissue specimens from histologically confirmed oral squamous cell carcinoma (OSCC) patients were collected. HPV detection and genotyping were performed using HPV E6/E7 and HPV- type-specific multiplex primer for nested-PCR. Immunohistochemistry evaluation of pl6 was conducted. SPSS statistical software (ver 20) was used for data analysis. ResultsHigh risk-HPV (hr-HPV) DNA positivity was found in 27.7% (n = 20) of OSCC patients. Stage III OSCC patients were 7.80 times more likely to survive 5 years than stage IV patients (OR-7.80 CI-95%; P-0.03). Among the hr-HPV positive OSCC patients, we found that the median survival time for the 1st year (95%), 3 years (78.5%), and 5 years (38.5%) was significantly higher than that of the hr-HPV negative [1st year (78.6%), 3 years (45.2%) and 5 years (38.5%)] OSCC patients (P-0.03 The survival of male patients with hr-HPV positive OSCC is 9.75 times greater than the survival of patients with HPV negative OSCC (OR-9.75; CI-95%; P-0.05). The p16 expression level (low to overexpression) group and negative P16 expression group of OSCC patients have not demonstrated a significant association with 5-year survival. ConclusionWe conclude that in OSCC cases of North-East India, the presence of hr-HPV in OSCC cases could be a good predictor of 5-year survival rate. Expression of p16 does not appear to have any significant association with 5-year survival.

Cortactin expression in a Norwegian cohort of human papilloma virus negative oral squamous cell carcinomas of the mobile tongue.

Oral squamous cell carcinoma of the tongue (OTSCC) is the most common malignancy among oral squamous cell carcinomas and is frequently associated with an unfavorable prognosis. Local spread and distant metastasis are important causes of poor prognosis in OTSCC. Cortactin amplification and overexpression, a common molecular alteration in oral squamous cell carcinomas, have been linked to invasion and metastasis of tumor cells. However, the intra-tumor expression pattern and prognostic significance of cortactin in human papillomavirus (HPV) negative OTSCC is not fully investigated. Immunohistochemical analysis using tissue microarray consisting of formalin-fixed and paraffin-embedded HPV negative OTSCC (n=123) specimens showed overexpression of cortactin at tissue cores from invading fronts as compared to the corresponding center cores. High overall cortactin expression was found to be associated with advanced (larger) tumor size and the occurrence of distance metastasis. Kaplan-Meier survival analysis showed that patients with high overall cortactin expression were associated with reduced 5-year survival. Multivariate Cox regression analysis identified high cortactin expression to be an independent prognostic factor in OTSCC. Additionally, siRNA-mediated silencing of cortactin was found to suppress the proliferative and invasive abilities of OTSCC cells in an organotypic co-culture model. Overexpression of cortactin is a promising prognostic marker in HPV-negative OTSCC.

Peri-tumoral infiltrate associates with postoperative prognosis of patients with OSCC: Stronger association in HPV negative patients

PurposeThe current data on the relationship between local inflammatory infiltration and prognosis in oral squamous cell carcinoma (OSCC) are limited and controversial, especially in different HPV status. In this study, we analyzed the relationship between peri-tumoral inflammatory infiltrate (PTI) and HPV status and prognosis of patients with OSCC after surgery. MethodsA retrospective cohort of 99 primary OSCC patients who underwent surgery was constructed. P16 immunohistochemistry was used to determine HPV status. PTI was determined by hematoxylin-eosin staining and quantified into four levels: none (Score 0), weak (Score 1), moderate (Score 2) and strong (Score 3). The associations of PTI with clinico-pathological characteristics, HPV status and survival were examined. ResultsMost OSCC patients had weak to moderate PTI. PTI was significantly associated with lymph node metastasis (P = 0.041), and patients with moderate PTI had significantly better OS (P = 0.009) than those with no PTI. In HPV negative OSCC, patients with moderate PTI also had significantly better OS (P = 0.019) than those with no PTI. However, PTI was not significantly associated with survival in HPV positive OSCC. ConclusionsIn HPV negative OSCC, moderate PTI may suggest a better postoperative prognosis than no PTI.

A validated predictive model for risk of nodal metastasis in node negative oral squamous cell carcinoma of the buccal mucosa and tongue.

Lymph node metastasis in oral squamous cell carcinoma (OSCC) is influenced by clinical and histopathological variables. The aim of this study was to develop a simple model to predict nodal metastasis of OSCC in clinically negative necks (cN0). Data from patients who underwent surgery for treatment of OSCC of the tongue or buccal mucosa with neck dissection were used for model development and validation. Nodal metastasis was significantly associated with gender, age, tumor size, site, pattern of invasion and depth of invasion on univariate analysis. All the five variables except age were retained at the variable selection step of the model development and were used in the final model because it was not significant at 0.10 significance level after adjusting for other variables. Regression coefficients of the model were used to estimate risks of nodal metastases for each combination of clinicopathological characteristics. A 10-fold cross-validation was used to assess the model. The average of the resultant 10 AUCs (along with its 95% confidence interval estimated using bootstrap) was used as the overall validated measure of the model. A risk chart was produced using probability of nodal metastasis predicted by the model for each combination of five characteristics. The model's ability to identify patients with nodal metastases as assessed by the area under the ROC curve (AUC) was 0.752. The model based on established clinicopathological variables has been internally validated on a large cohort of patients and offers practicability for use in OSCCs of the tongue and buccal mucosa.

Pathogenic TP53 mutations influence chemotherapy response and survival rate of HPV-negative oral carcinomas

Background/Aim. Oral squamous cell carcinoma (OSCC) is the most common tumor type of head and neck carcinomas, characterized by a high recurrence rate and patients? poor survival. Further elucidation of the function and regulation of the TP53, a pivotal tumor suppressor gene, would provide advances in predicting the clinical behavior, prognosis, and chemotherapy response of OSCC patients. Thus, we investigated the association of TP53 gene mutations with survival and response to cisplatin chemotherapy in human papilloma virus (HPV)-negative OSCC patients. Methods. The potential clinical relevance of TP53 mutations was analyzed in 82 patients with HPV-negative OSCC. All patients underwent radiotherapy, and 25 patients received cisplatin chemotherapy. A negative HPV status was deter-mined by type-specific polymerase chain reaction (PCR) for high-risk HPV 16, 18, 31, and 33. Targeted sequencing of TP53 exons 4?8 was assessed by Sanger sequencing. Results. Of 82 HPV-negative OSCC patients, 49 (59.79%) had TP53 mutations, and 26 patients (31.7%) carried pathogenic TP53 mutations. Patients with pathogenic TP53 mutations had significantly reduced overall survival (p = 0.009). Recurrence status, but not TP53 mutations, was an independent marker of poor survival in our cohort [hazard ratio (HR) = 4.733, 95% confidence interval (95% CI): 2.027?11.053, p = 0.0001]. In the sub-cohort of patients who underwent cisplatin-based chemotherapy, pathogenic TP53 mutations were predictors of poor response to chemotherapy (p = 0.026). Conclusion. Our findings indicate that pathogenic TP53 mutations in HPV-negative OSCC tumors could be a prognostic marker of patients? reduced overall survival. In addition, pathogenic TP53 mutations in HPV-negative OSCC could be a marker of poor chemotherapy response of OSCC patients.

The Prognostic Value of Human Papilloma Virus Infection in Oral Cavity Squamous Cell Carcinoma: A Meta-Analysis.

Human papilloma virus (HPV) infection has been confirmed as a favorable prognostic factor in oropharyngeal cancer. However, the prognostic value of HPV in oral squamous cell carcinoma (OSCC) is still unclear. Therefore, a systematic review and meta-analysis was performed to evaluate the prognostic value of HPV infection in OSCC patients. Systematic literature review with meta-analysis. A systematic literature review was conducted in accordance with PRISMA guidelines in PubMed, EMBASE, and MEDLINE databases. The primary outcomes were overall survival (OS), disease-free survival (DFS), disease-specific survival (DSS), and secondary outcomes were local control (LC), regional control (RC), and distant control (DC). A total of 22 articles with 3065 OSCC patients were included in this study. Meta-analysis demonstrated that compared to HPV-negative OSCC patients, HPV-positive OSCC patients had a significantly shorter OS (hazard ratio [HR]=1.45, 95% confidence intervals [CI], 1.10-1.93) and lower DC (HR=2.16, 95% CI, 1.54-3.04). There was no significant difference in DFS (HR=1.20, 95% CI, 0.63-2.26), DSS (HR=1.20, 95% CI, 0.63-2.26), LC (HR=1.44, 95% CI, 0.97-2.14), and RC (HR=1.50, 95% CI, 0.98-2.30) between HPV-positive and negative OSCC patients. Sensitivity analysis confirmed the above results. Our systematic review and meta-analysis reveal that HPV-positive is associated with significantly decreased OS and DC, suggesting HPV infection is an adverse prognostic factor in OSCC. Laryngoscope, 132:1760-1770, 2022.

A Plausible Proposition of CCL20-Related Mechanism in Fusobacterium nucleatum-Associated Oral Carcinogenesis.

Objective: The objective of this prospective observational case–control study is to evaluate the prevalence of Fusobacterium nucleatum in the tissues of oral squamous cell carcinoma (OSCC). Reconnoitering the CCL20-related mechanism of carcinogenesis in Fusobacterium nucleatum-positive OSCC is another objective. Methodology: Tissues from 50 OSCC patients and 30 healthy oral tissues were collected. The prevalence of Fusobacterium nucleatum was evaluated in both tumour and healthy tissue by polymerase chain reaction. The immunohistochemistry of OSCC tissues was conducted to evaluate the difference in the expression of CCL20 between Fusobacterium nucleatum-positive and -negative OSCC tissues. Results: Fusobacterium nucleatum was significantly (p < 0.001) prevalent in OSCC tissues (74%), compared to healthy tissues (26%). No association of Fusobacterium nucleatum or CCL20 immuno-expression with any clinical or histopathological features of OSCC was observed. While the intensity of CCL20 immuno-expression did not differ (p = 0.053), the CCL20-positive cell population was significantly different (p = 0.034) between Fusobacterium nucleatum-positive and -negative OSCC. Conclusion: Fusobacterium nucleatum is possibly prevalent in oral cancer tissues in the Indian population. By using immunohistochemistry, this is the first study to propose that the carcinogenesis in Fusobacterium nucleatum-positive OSCC may be CCL20-related. The findings enrich the knowledge of mechanisms involved in Fusobacterium nucleatum-mediated oral carcinogenesis.

CD68+ Macrophage Infiltration Associates With Poor Outcome of HPV Negative Oral Squamous Carcinoma Patients Receiving Radiation: Poly(I:C) Enhances Radiosensitivity of CAL-27 Cells but Promotes Macrophage Recruitment Through HMGB1.

Patients with human papillomavirus (HPV) negative oral squamous cell carcinoma (OSCC) generally have poor clinical outcomes and worse responses to radiotherapy. It is urgent to explore the underlining mechanisms of the distinct prognoses between HPV negative and HPV positive OSCC and to develop effective therapy strategy to increase the survival rate of HPV negative OSCC patients. We conducted a retrospective cohort of 99 resected OSCC patients to evaluate the prognosis of HPV negative and HPV positive OSCC patients receiving radiation or not. We further addressed the association of CD68+ macrophage infiltration with HPV status and the effects on survival of OSCC patients. We also used the TCGA-OSCC cohort for further verification. Based on the cohort study, we applied a synthetic dsRNA polymer, polyriboinosinic-polyribocytidylic acid (poly(I:C)), on CAL-27 (HPV negative OSCC cells). We co-cultured its condition medium with THP-1 derived macrophage and examined the cytokines and macrophage migration. We found that high CD68+ macrophage infiltration associated with poor overall survival in HPV negative OSCC patients receiving radiation. In vitro, poly(I:C) could induce apoptosis and enhance the radiosensitivity, but increase macrophage recruitment. Targeting HMGB1 could inhibit IL-6 induction and macrophage recruitment. Our findings indicated that CD68+ macrophage might play an important role in the outcomes of HPV negative OSCC patients receiving radiation. Our findings also suggested that radiation combined poly(I:C) might be a potential therapy strategy to increase the radiation response and prognosis of HPV negative OSCC. Notably, HMGB1 should be targeted to inhibit macrophage recruitment and enhance overall therapy effects.

Effector T cell responses unleashed by regulatory T cell ablation exacerbate oral squamous cell carcinoma

SummaryImmune suppression by CD4+FOXP3+ regulatory T (Treg) cells and tumor infiltration by CD8+ effector T cells represent two major factors impacting response to cancer immunotherapy. Using deconvolution-based transcriptional profiling of human papilloma virus (HPV)-negative oral squamous cell carcinomas (OSCCs) and other solid cancers, we demonstrate that the density of Treg cells does not correlate with that of CD8+ T cells in many tumors, revealing polarized clusters enriched for either CD8+ T cells or CD4+ Treg and conventional T cells. In a mouse model of carcinogen-induced OSCC characterized by CD4+ T cell enrichment, late-stage Treg cell ablation triggers increased densities of both CD4+ and CD8+ effector T cells within oral lesions. Notably, this intervention does not induce tumor regression but instead induces rapid emergence of invasive OSCCs via an effector T cell-dependent process. Thus, induction of a T cell-inflamed phenotype via therapeutic manipulation of Treg cells may trigger unexpected tumor-promoting effects in OSCC.

Systematic screening identifies a TEAD4-S100A13 axis modulating cisplatin sensitivity of oral squamous cell carcinoma cells.

This study aimed to screen prognosis-related S100 protein family members in human paxpillomaviruses (HPV)-negative oral squamous cell carcinoma (OSCC) and their molecular regulations. Bioinformatic screening was conducted based on single-cell RNA-seq data from Puram 2017 dataset and bulk-seq data from the Cancer Genome Atlas (TCGA). HPV-negative OSCC cell lines CAL-27 and SCC-4 were used as in vitro cell models. Among 21 S100 protein family member genes, S100A13 upregulation was associated with unfavorable progression-free survival and disease-specific survival of OSCC patients. Gene Set Enrichment Analysis showed that the higher S100A13 expression group had elevated genes enriched in DNA repair and oxidative phosphorylation. S100A13knockdown increased cisplatin sensitivity, while its overexpression decreased the sensitivity of CAL-27 and SCC-4 cells. S100A13gene had complex alternative transcription patterns. ENST00000440685 is one of the major protein-coding transcripts and was the only transcript elevated in the tumor group. TEAD4 could bind to the promoter of ENST00000440685 and increase its transcription. TEAD4 overexpression alleviated the tumor-suppressive effect of cisplatin in terms of colony formation, the expression of apoptotic proteins, and DNA damage. However, S100A13knockdown partly abrogated the protective effects of TEAD4 overexpression. This study revealed a novel TEAD4-S100A13 axis that might modulate cisplatin sensitivity of OSCC tumor cells.

Combined In Situ Hybridization and Immunohistochemistry on Archival Tissues Reveals Stromal microRNA-204 as Prognostic Biomarker for Oral Squamous Cell Carcinoma.

Simple SummaryIn addition to the transformation of epithelial cells, dysfunction of stroma is crucial in carcinogenesis; cancer-associated stroma can regulate the phenotype of cancer cells and thereby influence the clinical outcome. Our study aimed to investigate the correlation of stromal miR-204 with progression of oral squamous cell carcinoma (OSCC) and assert its clinical utility. We first established a chromogen-based method that combined immunohistochemistry and in situ hybridization for exact delimitation of stroma from the tumor islands and concomitant visualization of miRs, and have developed a guide to digital miR quantification using the publicly available tool ImageJ and the licensed software Aperio ImageScope. We have then applied the method for investigating stromal miR-204 as a putative prognostic biomarker on an OSCC cohort and identified expression of miR204 in the stroma at tumor front as an independent prognostic biomarker for this disease.Micro-RNAs (miRs) are emerging as important players in carcinogenesis. Their stromal expression has been less investigated in part due to lack of methods to accurately differentiate between tumor compartments. This study aimed to establish a robust method for dual visualization of miR and protein (pan-cytokeratin) by combining chromogen-based in situ hybridization (ISH) and immunohistochemistry (IHC), and to apply it to investigate stromal expression of miR204 as a putative prognostic biomarker in oral squamous cell carcinoma (OSCC). Four different combinations of methods were tested and ImageJ and Aperio ImageScope were used to quantify miR expression. All four dual ISH-IHC methods tested were comparable to single ISH in terms of positive pixel area percentage or integrated optical density of miRs staining. Based on technical simplicity, one of the methods was chosen for further investigation of miR204 on a cohort of human papilloma virus (HPV)-negative primary OSCC (n = 169). MiR204 stromal expression at tumor front predicted recurrence-free survival (p = 0.032) and overall survival (p = 0.036). Multivariate Cox regression further confirmed it as an independent prognostic biomarker in OSCC. This study provides a methodological platform for integrative biomarker studies based on simultaneous detection and quantification of miRs and/or protein and reveals stromal miR204 as a prognostic biomarker in OSCC.

Tumor Microenvironment, HLA Class I and APM Expression in HPV-Negative Oral Squamous Cell Carcinoma.

Simple SummaryOral squamous cell carcinoma has developed different strategies to escape from T-cell-mediated immune surveillance, which is mediated by changes in the composition of cellular and soluble components of the tumor microenvironment as well as an impaired expression of molecules of the antigen processing machinery leading to a downregulation of HLA class I surface antigens. In depth characterization of these escape mechanisms might help to develop strategies to overcome this tolerance. In this study, human papilloma virus negative oral squamous cell carcinoma lesions were analyzed regarding the protein expression of major components of the HLA class I antigen processing/presentation pathway in correlation to the intra-tumoral immune cell composition, IFN-γ signaling and clinical parameters, which was further confirmed by bioinformatics analyses of datasets obtained from The Cancer Genome Atlas. This novel knowledge could be used for optimizing the design of immunotherapeutic approaches of this disease.Progression of oral squamous cell carcinoma (OSCC) has been associated with an escape of tumor cells from the host immune surveillance due to an increased knowledge of its underlying molecular mechanisms and its modulation by the tumor microenvironment and immune cell repertoire. In this study, the expression of HLA class I (HLA-I) antigens and of components of the antigen processing machinery (APM) was analyzed in 160 pathologically classified human papilloma virus (HPV)-negative OSCC lesions and correlated to the intra-tumoral immune cell response, IFN-γ signaling and to the patient’s outcome. A heterogeneous but predominantly lower constitutive protein expression of HLA-I APM components was found in OSCC sections when compared to non-neoplastic cells. Tumoral HLA-I APM component expression was further categorized into the three major phenotypes HLA-Ihigh/APMhigh, HLA-Ilow/APMlow and HLA-Idiscordant high/low/APMhigh. In the HLA-Ihigh/APMhigh group, the highest frequency of intra-tumoral CD8+ T cells and lowest number of CD8+ T cells close to FoxP3+ cells were found. Patients within this group presented the most unfavorable survival, which was significantly evident in stage T2 tumors. Despite a correlation with the number of intra-tumoral CD8+ T cells, tumoral JAK1 expression as a surrogate marker for IFN-γ signaling was not associated with HLA-I/APM expression. Thus, the presented findings strongly indicate the presence of additional factors involved in the immunomodulatory process of HPV-negative OSCC with a possible tumor-burden-dependent complex network of immune escape mechanisms beyond HLA-I/APM components and T cell infiltration in this tumor entity.

Cumulative suppressive index as a predictor of relapse free survival and overall survival in Human Papilloma Virus-negative oral squamous cell carcinomas with negative resection margins.

This study aimed to analyze margin status and the impact of the immune elements on recurrence in patients with oral squamous cell carcinoma (OSCC), employing a prognostic biomarker, cumulative suppressive index (CSI), which reflects FoxP3+, PD-L1+, and CD8+ cell spatial relationships in the tumor microenvironment. Cox proportional hazards regression was used to evaluate the interactive effect of the margin by CSI discrepancy (high, 3-4 vs low, 0-2) on recurrence free survival (RFS) and overall survival (OS) in 119 patients with stage I to IVA OSCC. In cases with negative margins, multivariable analysis showed high CSI was significantly associated with worse RFS (HR = 2.59, 95% CI [1.03, 6.49], P = .04) and OS (HR = 5.49, 95% CI [1.48, 20.35], P = .01) compared to low CSI. However, high CSI was not significantly associated with recurrence in cases with positive margins. Immune architecture analysis can augment our current histopathological risk assessment of margin status.

Usefulness of Sentinel Lymph Node Biopsy for Oral Cancer: A Systematic Review and Meta-Analysis.

We assessed the diagnostic accuracy of sentinel lymph node biopsy (SLNB) for detecting neck nodal metastasis in early oral squamous cell carcinoma (OSCC) as an alternative to elective neck dissection. A systematic search for relevant literature was conducted in the PubMed, SCOPUS, Embase, Web of Science, and Cochrane databases. Two reviewers individually searched the five databases up to November 2019. For studies that met inclusion criteria, data on patient diagnoses were pooled, including true positives, true negatives, false positives, and false negatives. Methodological quality was checked with the Quality Assessment of Diagnostic Accuracy Studies (version 2) tool. In total, 98 observational or retrospective studies were included. The diagnostic odds ratio of SLNB was 326.165 (95% confidence interval [CI]: 231.477-459.587; I2 = 0%). The area under the summary receiver operating characteristic curve was 0.982. Sensitivity was 0.827 (95% CI: 0.804-0.848), and specificity was 0.981 (95% CI: 0.975-0.986). The correlation between sensitivity and the false positive rate was -0.076, which indicates that heterogeneity did not exist. Subgroup analyses were performed with the subgroups reference test type, publication year, and study type. No significant difference was found within the reference test type subgroup. However, differences within the publication year and study type subgroups were significant, where the retrospective study subgroup was significantly more sensitive and specific than the prospective study subgroup. Results of this meta-analysis imply that the high specificity of SLNB supports its role as a diagnostic tool for patients with clinical tumor stage (CT)1-2 clinically negative (N0) OSCC. More studies should be done to further verify the results of this study. 2a Laryngoscope, 131:E459-E465, 2021.

Nuclear Magnetic Resonance Metabolomics Biomarkers for Identifying High Risk Patients with Extranodal Extension in Oral Squamous Cell Carcinoma.

Extranodal extension (ENE) is an independent adverse prognostic factor in oral squamous cell carcinoma (OSCC), and is difficult to identify preoperatively. We aimed to discover biomarkers for high risk patients with ENE. Tandem tissue, plasma, and urine samples of 110 patients with OSCC were investigated through 600-MHz nuclear magnetic resonance (NMR) metabolomics analysis. We found that the levels of creatine, creatine phosphate, glycine, and tyramine in plasma significantly decreased in stage IV ENE positive OSCC compared with stage IV ENE negative OSCC. To understand the underlying mechanism behind the alteration of plasma metabolites, our tissue analysis revealed that the carnitine level significantly increased in tumors but significantly decreased in the adjacent normal tissue in advanced stage OSCC, in addition to decreased levels of alanine and pyruvate in tumor tissues. The global metabolomics analysis on tumor tissues also showed that stage IV tumors with an ENE positive status demonstrated higher levels of aspartate, butyrate, carnitine, glutamate, glutathione, glycine, glycolate, guanosine, and sucrose but lower levels of alanine, choline, glucose, isoleucine, lactate, leucine, myo-inositol, O-acetylcholine, oxypurinol, phenylalanine, pyruvate, succinate, tyrosine, valine, and xanthine than tumors with an ENE negative status. We concluded that metabolomics alterations in tumor tissues correspond to an increase in the tumor stage and are detectable in plasma samples. Metabolomic alterations of OSCC can serve as potential diagnostic markers and predictors of ENE in patients with stage IV OSCC.

Salivary proteins from dysplastic leukoplakia and oral squamous cell carcinoma and their potential for early detection

Dysplastic leukoplakia (LP) of the oral cavity is a potentially malignant condition for oral squamous cell carcinoma (OSCC), early detection of which remains an unmet clinical need. In an effort to develop non-invasive biomarker based method for early detection of the disease, differential proteomic profiling was carried out with the saliva from patients with risk habits and diagnosed with LP and those with lymph node negative and positive OSCC in comparison to healthy controls with risk habits. Ninety three proteins were observed at elevated level (≥1.5 fold), and 30 were prioritized based on a scoring system comprising of confidence of identification, presence in the various specimen groups, functional relevance, and their secretory potential. Verification was carried out in independent patient cohorts for 8 selected, representative, upregulated proteins using ELISA. Three of them CD44, S100A7, and S100P were significantly altered in patients with LP as well as OSCC and can be regarded as a panel of biomarker candidates for early detection of the malignancy. Other members may also be investigated in a targeted manner to expand the portfolio of biomarkers for early detection. The mass spectrometry data are available via ProteomeXchange with identifier PXD015722. SignificanceThere is an unmet clinical need for non-invasive, biomarker based methods for the improved early detection and the subsequent management of oral cancer. The study represents differential proteome profiling of the saliva of patients with oral dysplastic leukoplakia (LP) – a potentially malignant lesion, patients diagnosed with oral squamous cell carcinoma (OSCC), and healthy controls to identify potential markers for the purpose of early detection of malignancy. From among the matched and prioritized proteins with elevated levels in the saliva of patients with LP and those with OSCC, eight were verified. Three of them – CD44, S100A7 and S100P appeared promising candidates as biomarkers for early detection of the neoplastic predisposition and may form the basis of clinical assays for this purpose.

Comprehensive Data of P53 R282 Gene Mutation with Human Papillomaviruses (HPV)-Associated Oral Squamous Cell Carcinoma (OSCC).

Alterations of the P53 gene and human papillomavirus (HPV) infection are associated with development of oral squamous cell carcinoma (OSCC). We aimed to identify mutation of P53 exon 8 codon 282 in OSCC and correlate these with HPV infection as well as histopathological grade of OSCC. Samples of known HPV infection status were studied including oral lesion cells, formalin-fixed paraffin embedded (FFPE) tissues from OSCC and exfoliated oral cells of matched age-sex controls. P53 exon 8 mutation was detected using the polymerase chain reaction (PCR). Mutation of codon 282 was identified by allele-specific oligonucleotide typing (ASO) using EvaGreen real-time PCR. The PCR products were analyzed by gel electrophoresis and melting curve analysis. Mutation of P53 exon 8 was seen in 81.7% and 69.6% of FFPE OSCC tissues and oral lesion cells, respectively. This was significantly higher than in controls (16.7%). Frequency of mutation did not differ between HPV-positive samples (62.5% and 81.8% in oral lesion cells and FFPE tissue samples, respectively) and HPV-negative samples (73.3% and 81.5% in oral lesion cells and FFPE tissue samples, respectively). This finding is similar to P53 codon 282 mutation that was found only in FFPE tissues (35.0%) and oral lesion cells (32.6%) from both HPV-positive and negative OSCC. Interestingly, frequency of mutation was higher in well-differentiated OSCC with HPV-infection (28.1%) than without HPV (14.8%). This result demonstrated a mutation hot spot in P53 associated with oral carcinogenesis and might be useful to guide chemotherapeutic modality for HPV-associated OSCC in northeast Thailand.

Expression of Kif5b protein is significantly associated with the progression, recurrence and prognosis of oral squamous cell carcinoma.

Kinesin family member 5b (Kif5b), a conventional kinesin, mainly participates in lysosome and mitochondria transportation. Some studies have indicated that Kif5b may be associated with the development of a variety of tumors. However, the role Kif5b plays in oral squamous cell carcinoma (OSCC) has yet to be determined. Our study aimed at investigating the expression level of Kif5b in primary OSCC and discussing its clinical significance in patients' outcomes. We measured Kif5b expression in 82 OSCC tissue samples with immunohistochemistry. The associations between the expression level of Kif5b and clinicopathological characteristics as well as patients' survival were statistically assessed. Kif5b level was significantly associated with tumor size (p=0.034), histological differentiation (p=0.028), disease recurrence (p=0.018), surrounding tissue invasion (p=0.045), recurrence time (p=0.036) and survival status (p=0.030). Kaplan-Meier cumulative survival analyses indicated that high expression of Kif5b was linked to worse overall survival (p=0.0112) and disease-free survival (p=0.0085). The univariate and multivariate Cox proportional hazard analysis further identified the expression status of Kif5b as an independent variable that correlated with patients' survival and recurrence. Furthermore, in 54 early-stage, clinically node negative OSCC patients, Kif5b expression were correlated with histological differentiation (p=0.034), disease recurrence (p=0.038) and surrounding tissue invasion (p=0.029). Univariate and multivariable logistic regression results showed that only Kif5b expression level could influence the probability of recurrence. Our results reveal that Kif5b expression is associated with poor clinical outcome in OSCC and even in early-stage, clinically node negative OSCC and may be a potential target for OSCC treatment.

Abstract 38: Development of a DC-targeted microvesicle vaccine to intercept the progression of oral preneoplasia to cancer

Abstract Background: HPV negative oral squamous cell carcinoma (OSCC) is diagnosed in more than 300,000 people each year worldwide and more than half of these will die within five years despite standard treatment. These cancers are often preceded by the appearance of a preneoplastic lesion, which offers a unique opportunity to identify patients at high risk of developing OSCC and offer them a vaccine that may prevent development of this non-viral cancer. Cancer cell line-derived proteasome-blocked autophagosomes are DC-targeted microvesicles containing tumor-derived short-lived proteins (SLiPs) and defective ribosomal products (DRiPs). SLiPs and DRiPs are short-lived because they are targeted to and degraded by the proteasome, explaining why they are thought to make up the dominant epitopes presented on the surface of cancer cells. This may explain their ability to induce broad anticancer immunity in preclinical models and in patients receiving the vaccine. The human vaccine studied here, DPV-001, was derived from two cancer cell lines. Characterization of the vaccine by mass spectroscopy identified that the vaccine contains more than 100 antigens whose genes are over-expressed by most cancers. Many of these antigens have single amino acid variants that may serve as immunogenic mimetopes, or altered peptide ligands. Further, DPV-001 contains at least 13 antigens from the NCI priority list and multiple toll-like receptor (TLR) agonists packaged into stable double membrane microvesicles that are targeted to CLEC9A+ antigen presenting cells (APCs). We recently reported that every patient receiving vaccine in an NCI-supported phase I/II trial developed or augmented immunity against a large number of putative cancer antigens. Methods: Recently, gene sets developed from a preclinical 4-NQ0 mouse model of oral cancer were shown to enrich for patients with oral preneoplasia that progressed to squamous cell carcinoma (p=0.0495, Foy J-P, 2016). The progressive gene set (PGS) contained 162 genes and we compared this list to results of mass spectroscopy analysis of the DPV-001 vaccine. Results: 94 of the 162 genes contained in the PGS, which enriched for patients with poor oral cancer-free survival, were identified in the DPV-001 vaccine. Conclusions: DPV-001 contains more than 90 proteins for genes (PGS) whose over-expression is associated with progression of oral preneoplasia to OSCC. Based on significant preclinical data documenting induction of protective or therapeutic immunity in animals receiving a DRibble vaccine, coupled with safety data from a phase I/II clinical trial, and evidence that vaccination induces broad immunity against putative cancer antigens in every patient studied to date, we are proposing a pilot clinical trial of DPV-001 in patients with severe oral dysplasia. Citation Format: R. Bryan Bell, Rom Leidner, Carlo B. Bifulco, Christopher Dubay, Tarsem Moudgil, Sam Bookhardt, Glenna McDonnell, Christopher Paustian, Hong-Ming Hu, Walter J. Urba, Traci L. Hilton, Bernard Fox. Development of a DC-targeted microvesicle vaccine to intercept the progression of oral preneoplasia to cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 38.

Plasticity of PD-L1 expression between nodal metastases and primary tumors in p16 negative squamous cell carcinoma of the oral cavity

Background: the recent achievements of immunotherapy in many different areas (melanoma, kidney, lung and bladder carcinoma) stimulate the investigation about the action of check points inhibitors in other tumors, particularly in case of tumors with an high mutational load, that are potentially responsive to immunotherapy approaches, like p16 negative oral squamous cell carcinoma. Phase III studies are ongoing, and there are also preliminary data about the PD-L1 tissue expression, in correlation with tumor grade and stage. However, investigations concerning the possible up/down regulation of this ligand in primary vs nodal metastasis are still largely inadequate. Material and methods: 16 oral squamous cell carcinoma T2-T4, node positive (N1), p16 negative, moderately differentiated (G2), underwent immunostaining with anti PD-L1 rabbit MoAb SP 142, using a VENTANA BenchMark Ultra platform with OptiView detection kit. Both primary tumors and nodal metastases were immunostained. Results were evaluated according to Roche PD-L1 (SP142) scoring system. In particular, neoplastic cell immunoreactivity (TC) and immune cells (lymphocytes, macrophages, dendritic cells and granulocytes) intratumoral and peritumoral immunoreactivity (IC) were evaluated. CD3 immunostaing of both primary tumors and nodal metastases were also performed, in order to facilitate the interpretation of the results. Results: the entire group of 16 cases showed PD-L1 expression in perineoplastic IC, ranging from 1% to 50%, according to the scoring. No significant variations of expression between primary and secondary nodal sites were observed in all cases but one. Conversely, 10 out of 16 showed no expression of PD-L1 on TC (0%) both in primary tumor and nodal metastasis. The other six cases showed: case 4: T 0%, N 60%; case 5: T 80%, N 5%; case 6: T 70%, N 5%, case 11: T 50%, N 0%; case 13: T 30%, N 10%, case 16: T 40%, N 1%. Conclusions: in the light of the relevance of PD-L1 expression in term of immunotherapy efficacy in other areas (lung and bladder carcinoma), this documented plasticity (37.5% of cases) might be an interesting information for future trials.