Emerging Role of Decoy Receptor-2 as a Cancer Risk Predictor in Oral Potentially Malignant Disorders.

The aim of this study was to evaluate the expression of the senescence markers, Decoy Receptor 2 (DcR2) and Differentiated Embryo-Chondrocyte expressed gen 1 (DEC1), in oral potentially malignant disorders (OPMDs) to ascertain their possible association with oral cancer risk. The immunohistochemical analysis of DcR2 and DEC1 expression (along with p16 and Ki67 expression) was carried out in 60 patients with clinically diagnosed oral leukoplakia. Fifteen cases (25%) subsequently developed an invasive carcinoma. Correlations between protein marker expression, histological grade and oral cancer risk were assessed. DcR2, DEC1 and Ki67 protein expressions were found to correlate significantly with increased oral cancer risk, and also with an increased grade of dysplasia. Multivariate analysis demonstrated that DcR2 and Ki67 expression are independent predictors of oral cancer development. Our results evidence for the first time the potential of DcR2 as an early biomarker to assess oral cancer risk in patients with oral leukoplakia (HR = 59.7, p = 0.015), showing a superior predictive value to histology (HR = 4.225, p = 0.08). These findings reveal that the increased expression of DcR2 and DEC1 occurred frequently in OPMDs. In addition, DcR2 expression emerges as a powerful biomarker for oral cancer risk assessment in patients with oral leukoplakia.

PD-L1 expression correlates with tumor-infiltrating lymphocytes and better prognosis in patients with HPV-negative head and neck squamous cell carcinomas.

INTRODUCTION: The importance of immune tumor microenvironment in the prognosis of patients with head and neck squamous carcinomas (HNSCC) is increasingly recognized. We analyzed the prognostic relevance of PD-L1 and PD-1 expressions in relation to the infiltration by CD8+ and FOXP3+ tumor-infiltrating lymphocytes (TILs). METHODS: Samples from 372 surgically treated HPV-negative HNSCC patients were evaluated by immunohistochemistry for PD-L1 expression [both tumor proportion score (TPS) and combined proportion score (CPS)], PD-1 expression in immune cells, and density of infiltrating CD8+ and FOXP3+ TILs. PD-L1 expression and CD8+ TIL density were combined to establish the type of tumor microenvironment. RESULTS: 29.5% cases exhibited PD-L1 TPS positivity (≥ 1%), whereas PD-L1 CPS positivity (≥ 1%) was observed in 40% cases. 47.5% cases showed positive PD-1 expression (≥ 1%). PD-L1 and PD-1 positivity correlated with a high density of both CD8+ and FOXP3+ TILs. In univariate analysis, PD-L1 TPS positivity (P = 0.026), PD-L1 CPS positivity (P = 0.004), high density of CD8+ TIL (P = 0.001), and high density of FOXP3+ TIL (P = 0.004) were associated with a better disease-specific survival (DSS). However, in multivariate analysis, only high density of CD8+ TIL was associated with a better DSS (P = 0.002). The type of tumor microenvironment correlated with DSS (P = .008), with the better DSS observed in cases with type I (PD-L1 CPS positivity and high density of CD8+ TIL). CONCLUSIONS: High infiltration by CD8+ TIL is associated with better survival outcomes. Positive PD-L1 expression correlates with a high infiltration by TILs, explaining its association with better prognosis.

The multikinase inhibitor EC-70124 synergistically increased the antitumor activity of doxorubicin in sarcomas.

Cytotoxic drugs like doxorubicin remain as the most utilized agents in sarcoma treatment. However, advanced sarcomas are often resistant, thus stressing the need for new therapies aimed to overcome this resistance. Multikinase inhibitors provide an efficient way to target several pro-tumorigenic pathways using a single agent and may constitute a valuable strategy in the treatment of sarcomas, which frequently show an aberrant activation of pro-tumoral kinases. Therefore, we studied the antitumor activity of EC-70124, an indolocarbazole analog that have demonstrated a robust ability to inhibit a wide range of pro-survival kinases. Evaluation of the phospho-kinase profile in cell-of-origin sarcoma models and/or sarcoma primary cell lines evidenced that PI3K/AKT/mTOR, JAK/STAT or SRC were among the most highly activated pathways. In striking contrast with the structurally related drug midostaurin, EC-70124 efficiently prevented the phosphorylation of these targets and robustly inhibited proliferation through a mechanism associated to the induction of DNA damage, cell cycle arrest and apoptosis. In addition, EC-70124 was able to partially reduce tumor growth in vivo. Importantly, this compound inhibited the expression and activity of ABC efflux pumps involved in drug resistance. In line with this ability, we found that the combined treatment of EC-70124 with doxorubicin resulted in a synergistic cytotoxic effect in vitro and an increased antitumor activity of this cytotoxic drug in vivo. Altogether, these results uncover the capability of the novel multikinase inhibitor EC-70124 to counteract drug resistance in sarcoma and highlight its therapeutic potential when combined with current treatments.

Expression of the voltage-gated potassium channel Kv3.4 in oral leucoplakias and oral squamous cell carcinomas.

AIMS: The expression of the voltage-gated potassium channel Kv3.4 was investigated in both oral squamous cell carcinomas (OSCC) and oral leucoplakias to establish its clinical significance during the development and progression of OSCC. MATERIALS AND METHODS: Tissue specimens from 62 patients with oral leucoplakia were collected prospectively and 100 patients with OSCC who underwent surgical treatment were collected retrospectively, and Kv3.4 expression was analysed by immunohistochemistry. RESULTS: Thirty-nine of 100 tumours exhibited Kv3.4-positive expression, and staining was associated with the degree of differentiation (P = 0.05) but showed no impact on patient prognosis. Abnormal Kv3.4 expression was detected in 16% (7 of 43) hyperplastic lesions and at a significantly higher proportion in oral dysplasias (50%, 8 of 16 cases; P = 0.008), whereas expression was negligible in normal adjacent epithelia. Furthermore, patients carrying Kv3.4-positive lesions exhibited a higher progression risk than those with Kv3.4-negative lesions; however, histology but not Kv3.4 expression predicted oral cancer development significantly in this prospective cohort. CONCLUSION: This study provides original evidence to demonstrate the early occurrence and high prevalence of abnormal Kv3.4 expression in oral leucoplakias. Our results support a role for Kv3.4 potassium channel in OSCC tumorigenesis rather than tumour progression and disease outcome.

The TINCR ubiquitin-like microprotein is a tumor suppressor in squamous cell carcinoma.

The TINCR (Terminal differentiation-Induced Non-Coding RNA) gene is selectively expressed in epithelium tissues and is involved in the control of human epidermal differentiation and wound healing. Despite its initial report as a long non-coding RNA, the TINCR locus codes for a highly conserved ubiquitin-like microprotein associated with keratinocyte differentiation. Here we report the identification of TINCR as a tumor suppressor in squamous cell carcinoma (SCC). TINCR is upregulated by UV-induced DNA damage in a TP53-dependent manner in human keratinocytes. Decreased TINCR protein expression is prevalently found in skin and head and neck squamous cell tumors and TINCR expression suppresses the growth of SCC cells in vitro and in vivo. Consistently, Tincr knockout mice show accelerated tumor development following UVB skin carcinogenesis and increased penetrance of invasive SCCs. Finally, genetic analyses identify loss-of-function mutations and deletions encompassing the TINCR gene in SCC clinical samples supporting a tumor suppressor role in human cancer. Altogether, these results demonstrate a role for TINCR as protein coding tumor suppressor gene recurrently lost in squamous cell carcinomas.

Role of HERG1 potassium channel in both malignant transformation and disease progression in head and neck carcinomas.

Evidence indicates that human ether à-go-go-related gene 1 (HERG1) voltage-gated potassium channels could represent new valuable membrane therapeutic targets and diagnostic/prognostic biomarkers in various cancers. This study is the first to investigate the expression pattern of HERG1 potassium channel subunit in both primary tumors and precancerous lesions to establish its clinical and biological role during the development and progression of head and neck squamous cell carcinomas. HERG1 protein expression was evaluated by immunohistochemistry in paraffin-embedded tissue specimens from 133 patients with laryngeal/hypopharyngeal squamous cell carcinomas and 75 patients with laryngeal dysplasia, and correlated with clinical data. Our findings demonstrate that HERG1 is frequently aberrantly expressed in a high percentage of primary tumors (87%), whereas expression was negligible in both stromal cells and normal-adjacent epithelia. HERG1 expression increased during head and neck squamous cell carcinoma progression and was significantly associated with lymph node metastasis (P=0.04), advanced disease stages (P<0.001), regional tumor recurrence (P=0.004), distant metastasis (P=0.03) and reduced disease-specific survival (P=0.012, log-rank test). HERG1-positive expression was also detected in 31 (41%) of 75 laryngeal dysplasias. Interestingly, HERG1 expression increased with the grade of dysplasia; however, HERG1 expression but not histology correlated significantly with increased laryngeal cancer risk (P=0.007). In addition, functional studies in head and neck squamous cell carcinoma-derived cell lines further revealed that HERG1 expression promotes anchorage-dependent and -independent cell growth and invasive capability, although independently of its ion-conducting function. Our data demonstrate that HERG1 expression is a biologically and clinically relevant feature in head and neck squamous cell carcinoma progression and also during malignant transformation, and a promising candidate as cancer risk marker and therapeutic target for head and neck squamous cell carcinoma prevention and treatment.

Expression and clinical significance of the Kv3.4 potassium channel subunit in the development and progression of head and neck squamous cell carcinomas.

The concept of ion channels as membrane therapeutic targets and diagnostic/prognostic biomarkers has attracted growing attention. We therefore investigated the expression pattern and clinical significance of the Kv3.4 potassium channel subunit during the development and progression of head and neck squamous cell carcinomas (HNSCCs). KCNC4 mRNA levels were determined by real-time RT-PCR in both HNSCC tissue specimens and derived cell lines. Kv3.4 protein expression was evaluated by immunohistochemistry in paraffin-embedded tissue specimens from 84 patients with laryngeal/pharyngeal squamous cell carcinomas and 67 patients with laryngeal dysplasias. Molecular alterations were correlated with clinicopathological parameters and patient outcome. Increased KCNC4 mRNA levels were found in 15 (54%) of 28 tumours, compared to the corresponding normal epithelia and varied mRNA levels were detected in 12 HNSCC-derived cell lines analysed. Increased Kv3.4 protein expression was observed in 34 (40%) of 84 carcinomas and also at early stages of HNSCC tumourigenesis. Thus, 35 (52%) of 67 laryngeal lesions displayed Kv3.4-positive staining in the dysplastic areas, whereas both stromal cells and normal adjacent epithelia exhibited negligible expression. No significant correlations were found between Kv3.4-positive expression in HNSCC and clinical data; however, Kv3.4 expression tended to diminish in advanced-stage tumours. Interestingly, patients carrying Kv3.4-positive dysplasias experienced a significantly higher laryngeal cancer incidence than did those with negative lesions (p = 0.0209). In addition, functional studies using HNSCC cells revealed that inhibition of Kv3.4 expression by siRNA leads to the inhibition of cell proliferation via selective cell cycle arrest at the G2/M phase without affecting apoptosis. Collectively, these data demonstrate for the first time that Kv3.4 expression is frequently increased during HNSCC tumourigenesis and correlated significantly with a higher cancer risk. Our findings support a role for Kv3.4 in malignant transformation and provide original evidence for the potential clinical utility of Kv3.4 expression as a biomarker for cancer risk assessment.

SDCBP Modulates Stemness and Chemoresistance in Head and Neck Squamous Cell Carcinoma through Src Activation.

To characterize the mechanisms that govern chemoresistance, we performed a comparative proteomic study analyzing head and neck squamous cell carcinoma (HNSCC) cells: CCL-138 (parental), CCL-138-R (cisplatin-resistant), and cancer stem cells (CSCs). Syntenin-1 (SDCBP) was upregulated in CCL-138-R cells and CSCs over parental cells. SDCBP depletion sensitized biopsy-derived and established HNSCC cell lines to cisplatin (CDDP) and reduced CSC markers, Src activation being the main SDCBP downstream target. In mice, SDCBP-depleted cells formed tumors with decreased mitosis, Ki-67 positivity, and metastasis over controls. Moreover, the fusocellular pattern of CCL-138-R cell-derived tumors reverted to a more epithelial morphology upon SDCBP silencing. Importantly, SDCBP expression was associated with Src activation, poor differentiated tumor grade, advanced tumor stage, and shorter survival rates in a series of 382 HNSCC patients. Our results reveal that SDCBP might be a promising therapeutic target for effectively eliminating CSCs and CDDP resistance.

Impact of notch signaling on the prognosis of patients with head and neck squamous cell carcinoma.

OBJECTIVES: The function of NOTCH signaling (oncogenic or oncosuppressive) remains controversial in head and neck squamous cell carcinomas (HNSCC). The purpose of this work is to investigate the role of NOTCH pathway in HNSCC prognosis. METHODS: Immunohistochemical NOTCH1 and HES1 expression was jointly evaluated and correlated with other NOTCH1 targets, p21 (WAF1/Cip1) and Cyclin D1, using an unbiased cohort of 372 surgically treated HPV-negative HNSCC patients. RESULTS: Membranous NOTCH1 expression was detected in 197 (61%) out of 324 evaluable tumor samples, and nuclear NOTCH1 expression in 91 samples (28%). Nuclear HES1 expression was found in 224 (67%) cases. Membranous and nuclear NOTCH1 expression were consistently and significantly correlated with nuclear HES1 (P < 0.001) and p21 (P = 0.03) expression, but not with Cyclin D1. NOTCH1 expression was significantly associated to early stages (I-II), non-recurrent disease, and better disease-specific (DSS) and overall survival (OS) rates (P < 0.001). Moreover, triple-positive cases (NOTCH1+/HES1+/p21+) exhibited significantly improved DSS (P < 0.001) and OS (P = 0.004), thus reinforcing the association of NOTCH pathway activation with a better prognosis in HNSCC. Multivariate analysis further revealed membranous NOTCH1 expression as a robust independent predictor of better DSS (HR = 0.554; 95% IC 0.412-0.745; P < 0.001) and better OS (HR = 0.640; 95% CI 0.491-0.835; P = 0.001). CONCLUSION: These findings show the association of NOTCH pathway activation with a better prognosis in HNSCC patients, also revealing membranous NOTCH1 expression as a robust independent predictor of improved survival. Accordingly, these results suggest a tumor suppressive rather than an oncogenic role for NOTCH pathway in HNSCC.

TSPAN1: A Novel Protein Involved in Head and Neck Squamous Cell Carcinoma Chemoresistance.

Sensitization of resistant cells and cancer stem cells (CSCs) represents a major challenge in cancer therapy. A proteomic study revealed tetraspanin-1 (TSPAN1) as a protein involved in acquisition of cisplatin (CDDP) resistance (Data are available via ProteomeXchange with identifier PXD020159). TSPAN1 was found to increase in CDDP-resistant cells, CSCs and biopsies from head and neck squamous cell carcinoma (HNSCC) patients. TSPAN1 depletion in parental and CDDP-resistant HNSCC cells reduced cell proliferation, induced apoptosis, decreased autophagy, sensitized to chemotherapeutic agents and inhibited several signaling cascades, with phospho-SRC inhibition being a major common target. Moreover, TSPAN1 depletion in vivo decreased the size and proliferation of parental and CDDP-resistant tumors and reduced metastatic spreading. Notably, CDDP-resistant tumors showed epithelial-mesenchymal transition (EMT) features that disappeared upon TSPAN1 inhibition, suggesting a link of TSPAN1 with EMT and metastasis. Immunohistochemical analysis of HNSCC specimens further revealed that TSPAN1 expression was correlated with phospho-SRC (pSRC), and inversely with E-cadherin, thus reinforcing TSPAN1 association with EMT. Overall, TSPAN1 emerges as a novel oncogenic protein and a promising target for HNSCC therapy.

Lectin-Like Transcript 1 (LLT1) Checkpoint: A Novel Independent Prognostic Factor in HPV-Negative Oropharyngeal Squamous Cell Carcinoma.

Lectin-like transcript 1 (LLT1) expression by tumor cells contributes to immune evasion, thereby emerging as a natural killer (NK) cell-mediated immunotherapeutic target. This study is the first to investigate LLT1 expression (encoded by CLEC2D gene) in head and neck cancers to ascertain its impact on patient prognosis. LLT1 expression was analyzed by immunohistochemistry in a homogeneous cohort of human papillomavirus (HPV)-negative oropharyngeal squamous cell carcinomas (OPSCC), and correlated with clinical data. Results were further validated using transcriptomic data from the TCGA database. Tumoral LLT1 expression was detected in 190/221 (86%) OPSCC specimens, whereas normal pharyngeal epithelium was negative. Patients harboring LLT1-positive tumors showed significantly lower disease-specific (DSS) and overall survival (OS) (p = 0.049 and p = 0.036, respectively, log-rank test). High density of LLT1-positive tumor-infiltrating lymphocytes (TIL) was also frequently detected in 160 (73%) OPSCC samples, and significantly associated with better DSS and OS (p < 0.001 and p = 0.007, respectively). Multivariate Cox analysis further revealed that tumoral LLT1 expression and infiltration of LLT1-positive TIL were independent prognostic factors for DSS and OS. CLEC2D mRNA levels are also significantly increased in primary tumors compared to normal tissue. Strikingly, the prognostic impact of CLEC2D mRNA levels varied depending on HPV status in OPSCC, and among distinct cancer types. CLEC2D expression was significantly correlated with NK cell infiltration using the MCP-counter model. These findings uncover LLT1/CLEC2D as an independent prognostic factor in HPV-negative OPSCC, and a potential novel target for immunotherapy.

Expression of E-cadherin and ß-catenin in Laryngeal and Hypopharyngeal Squamous Cell Carcinomas. / Expresión de E-cadherina y ß-catenina en carcinomas de células escamosas de laringe e hipofaringe.

INTRODUCTION AND OBJECTIVES: Dysfunction of the E-cadherin/catenin complex is directly related to carcinogenesis and metastases development. The aim of this paper is to investigate the prognostic significance of E-cadherin and ß-catenin expression in surgically treated laryngeal and hypopharyngeal squamous cell carcinomas. MATERIAL AND METHODS: Tumour tissue samples were obtained from 133 consecutive patients with squamous cell carcinomas of the head and neck: 68 of the larynx and 65 hypopharyngeal carcinomas, who underwent surgical treatment in our hospital between 2000 and 2005. E-cadherin and ß-catenin expression was analysed by immunohistochemistry, quantifying the percentage of stained cells and the intensity of staining. RESULTS: E-cadherin and ß-catenin expression was evaluable in 59 laryngeal carcinomas and in 58 cases of hypopharyngeal carcinomas. In the laryngeal tumours, a significant association was found between the low expression of membrane ß-catenin with T4 tumours and tumour recurrence. In the hypopharynx there was a significant association between positive expression of nuclear ß-catenin and poor histological differentiation (P=.02). In the multivariate analysis, only the presence of lymph node metastases was an independent predictive factor of decreased disease-specific survival in laryngeal squamous cell carcinomas. CONCLUSIONS: The expression of E-cadherin and ß-catenin does not show prognostic significance in laryngeal and hypopharyngeal squamous cell carcinomas over the TNM classification.

Prognostic Significance of the Pluripotency Factors NANOG, SOX2, and OCT4 in Head and Neck Squamous Cell Carcinomas.

Cancer stem cells (CSCs) play major roles in tumor initiation, progression, and resistance to cancer therapy. Several CSC markers have been studied in head and neck squamous cell carcinomas (HNSCC), including the pluripotency factors NANOG, SOX2, and OCT4; however, their clinical significance is still unclear. NANOG, SOX2, and OCT4 expression was evaluated by immunochemistry in 348 surgically-treated HNSCC, and correlated with clinicopathological parameters and patient outcomes. mRNA expression was further analyzed in 530 The Cancer Genome Atlas (TCGA) HNSCC. NANOG protein expression was detected in 250 (72%) cases, more frequently in patients with lymph node metastasis (p = 0.003), and was an independent predictor of better survival in multivariate analysis. While OCT4 expression was undetectable, SOX2 expression was observed in 105 (30%) cases, and strongly correlated with NANOG expression. Combined expression of both proteins showed the highest survival rates, and double-negative cases the worst survival. Strikingly, the impact of NANOG and SOX2 on outcome varied depending on tumor site and lymph node infiltration, specifically showing prognostic significance in pharyngeal tumors. Correlation between NANOG and SOX2 at mRNA and protein was specifically observed in node positive (N+) patients, and consistently correlated with better survival rates. According to our findings, NANOG protein expression is frequent in HNSCC, thereby emerging as an independent predictor of better prognosis in pharyngeal tumors. Moreover, this study uncovers a differential impact of NANOG and SOX2 expression on HNSCC prognosis, depending on tumor site and lymph node infiltration, which could facilitate high-risk patient stratification.

SOX2 Expression and Transcriptional Activity Identifies a Subpopulation of Cancer Stem Cells in Sarcoma with Prognostic Implications.

Stemness in sarcomas is coordinated by the expression of pluripotency factors, like SOX2, in cancer stem cells (CSC). The role of SOX2 in tumor initiation and progression has been well characterized in osteosarcoma. However, the pro-tumorigenic features of SOX2 have been scarcely investigated in other sarcoma subtypes. Here, we show that SOX2 depletion dramatically reduced the ability of undifferentiated pleomorphic sarcoma (UPS) cells to form tumorspheres and to initiate tumor growth. Conversely, SOX2 overexpression resulted in increased in vivo tumorigenicity. Moreover, using a reporter system (SORE6) which allows to monitor viable cells expressing SOX2 and/or OCT4, we found that SORE6+ cells were significantly more tumorigenic than the SORE6- subpopulation. In agreement with this findings, SOX2 expression in sarcoma patients was associated to tumor grade, differentiation, invasive potential and lower patient survival. Finally, we studied the effect of a panel of anti-tumor drugs on the SORE6+ cells of the UPS model and patient-derived chondrosarcoma lines. We found that the mithramycin analogue EC-8042 was the most efficient in reducing SORE6+ cells in vitro and in vivo. Overall, this study demonstrates that SOX2 is a pro-tumorigenic factor with prognostic potential in sarcoma. Moreover, SORE6 transcriptional activity is a bona fide CSC marker in sarcoma and constitutes an excellent biomarker for evaluating the efficacy of anti-tumor treatments on CSC subpopulations.

GARP promotes the proliferation and therapeutic resistance of bone sarcoma cancer cells through the activation of TGF-ß.

Sarcomas are mesenchymal cancers with poor prognosis, representing about 20% of all solid malignancies in children, adolescents, and young adults. Radio- and chemoresistance are common features of sarcomas warranting the search for novel prognostic and predictive markers. GARP/LRRC32 is a TGF-ß-activating protein that promotes immune escape and dissemination in various cancers. However, if GARP affects the tumorigenicity and treatment resistance of sarcomas is not known. We show that GARP is expressed by human osteo-, chondro-, and undifferentiated pleomorphic sarcomas and is associated with a significantly worse clinical prognosis. Silencing of GARP in bone sarcoma cell lines blocked their proliferation and induced apoptosis. In contrast, overexpression of GARP promoted their growth in vitro and in vivo and increased their resistance to DNA damage and cell death induced by etoposide, doxorubicin, and irradiation. Our data suggest that GARP could serve as a marker with therapeutic, prognostic, and predictive value in sarcoma. We propose that targeting GARP in bone sarcomas could reduce tumour burden while simultaneously improving the efficacy of chemo- and radiotherapy.

PD-L1 Expression in Tumor Cells Is an Independent Unfavorable Prognostic Factor in Oral Squamous Cell Carcinoma.

BACKGROUND: The immune checkpoint PD-1 and its ligand PD-L1 are involved in the induction of immunological tolerance of solid tumors including oral squamous cell carcinoma (OSCC). The aim of the study was to establish the clinical and prognostic significance of PD-L1 in OSCC. METHODS: Tissue microarrays of 125 resected OSCC were stained with two different commercially available PD-L1 antibodies (clones E1L3N and 22C3), alongside PD-1 immunostaining. RESULTS: PD-L1 expression in more than 10% of tumor cells was associated with poorer survival, and established as a clinically relevant cut-off point. This relevant PD-L1 expression was detected in 10% to 15% OSCC specimens depending on the anti-PD-L1 antibody, and showed an inverse correlation with tobacco and alcohol consumption. We consistently found that PD-L1 expression was associated with tumor recurrence and lower disease-specific survival. Multivariate analysis further revealed that neck node metastasis (HR 2.304; P = 0.009) and tumor PD-L1 expression (HR 2.571; P = 0.01) were significant independent factors for poor prognosis. CONCLUSIONS: PD-L1 expression in more than 10% of tumor cells was a significant and independent factor of poor prognosis in OSCC. IMPACT: PD-L1 expression in more than 10% of tumor cells was consistently established as a clinically relevant cut-off point by using two different antibodies. Remarkably, PD-L1 expression emerges as an independent poor prognosis marker in patients with OSCC.

SOX2 Expression Is an Independent Predictor of Oral Cancer Progression.

Potentially malignant oral lesions, mainly leukoplakia, are common. Malignant transformation varies widely, even in the absence of histological features such as dysplasia. Hence, there is a need for novel biomarker-based systems to more accurately predict the risk of cancer progression. The pluripotency transcription factor SOX2 is frequently overexpressed in cancers, including oral squamous cell carcinoma (OSCC), thereby providing a link between malignancy and stemness. This study investigates the clinical relevance of SOX2 protein expression in early stages of oral carcinogenesis as a cancer risk biomarker, and also its impact on prognosis and disease outcome at late stages of OSCC progression. SOX2 expression was evaluated by immunohistochemistry in 55 patients with oral epithelial dysplasia, and in 125 patients with OSCC, and correlated with clinicopathological data and outcomes. Nuclear SOX2 expression was detected in four (7%) cases of oral epithelial dysplasia, using a cut-off of 10% stained nuclei, and in 16 (29%) cases when any positive nuclei was evaluated. Univariate analysis showed that SOX2 expression and histopathological grading were significantly associated with oral cancer risk; and both were found to be significant independent predictors in the multivariate analysis. Nuclear SOX2 expression was also found in 49 (39%) OSCC cases, was more frequent in early tumor stages and N0 cases, and was associated with a better survival. In conclusion, SOX2 expression emerges as an independent predictor of oral cancer risk in patients with oral leukoplakia. These findings underscore the relevant role of SOX2 in early oral tumorigenesis rather than in tumor progression.

The Emerging Role of NANOG as an Early Cancer Risk Biomarker in Patients with Oral Potentially Malignant Disorders.

NANOG, a key regulator of pluripotency and self-renewal in embryonic and adult stem cells, is frequently overexpressed in multiple cancers, including oral squamous cell carcinoma (OSCC). It has been frequently associated with poor outcomes in epithelial cancers, and recently implicated in laryngeal tumorigenesis. On this basis, we investigated the role of NANOG protein expression as an early cancer risk biomarker in oral potentially malignant disorders (OPMD), and the impact on prognosis and disease outcomes in OSCC patients. NANOG expression was evaluated by immunohistochemistry in 55 patients with oral epithelial dysplasia, and 125 OSCC patients. Correlations with clinical and follow-up data were assessed. Nuclear NANOG expression was detected in 2 (3.6%) and cytoplasmic NANOG expression in 9 (16.4%) oral dysplasias. NANOG expression increased with the grade of dysplasia. Cytoplasmic NANOG expression and the histopathological grading were significantly correlated with oral cancer risk, although dysplasia grading was the only significant independent predictor of oral cancer development in multivariate analyses. Cytoplasmic NANOG expression was also detected in 39 (31%) OSCC samples. Positive NANOG expression was significantly associated with tobacco and alcohol consumption, and was more frequent in pN0 tumors, early I-II stages. These data unveil the clinical relevance of NANOG in early stages of OSCC tumorigenesis rather than in advanced neoplastic disease. NANOG expression emerges as an early predictor of oral cancer risk in patients with OPMD.

Frequent Alteration of Annexin A9 and A10 in HPV-Negative Head and Neck Squamous Cell Carcinomas: Correlation with the Histopathological Differentiation Grade.

The annexin protein superfamily has been implicated in multiple physiological and pathological processes, including carcinogenesis. Altered expression of various annexins has frequently been observed and linked to the development and progression of various human malignancies. However, information is lacking on the expression and clinical significance of annexin A9 (ANXA9) and A10 (ANXA10) in head and neck squamous cell carcinomas (HNSCC). ANXA9 and ANXA10 expression was evaluated in a large cohort of 372 surgically treated HPV-negative HNSCC patients and correlated with the clinicopathologic parameters and disease outcomes. Down-regulation of ANXA9 expression was found in 42% of HNSCC tissue samples, compared to normal epithelia. ANXA9 expression in tumors was significantly associated with oropharyngeal location and histological differentiation grade (P < 0.001). In marked contrast, ANXA10 expression was absent in normal epithelium, but variably detected in the cytoplasm of cancer cells. Positive ANXA10 expression was found in 64% of tumors, and was significantly associated with differentiation grade (P < 0.001), being also more frequent in oropharyngeal tumors (P = 0.019). These results reveal that the expression of both ANXA9 and ANXA10 is frequently altered in HNSCC and associated to the tumor differentiation grade, suggesting that they could be implicated in the pathogenesis of these cancers.

The Differential Impact of SRC Expression on the Prognosis of Patients with Head and Neck Squamous Cell Carcinoma.

Aberrant SRC expression and activation is frequently detected in multiple cancers, and hence, targeting SRC has emerged as a promising therapeutic strategy. Different SRC inhibitors have demonstrated potent anti-tumor activity in preclinical models, although they largely lack clinical efficacy as monotherapy in late-stage solid tumors, including head and neck squamous cell carcinomas (HNSCC). Adequate selection and stratification of patients who may respond to and benefit from anti-SRC therapies is therefore needed to guide clinical trials and treatment efficacy. This study investigates the prognostic significance of active SRC expression in a homogeneous cohort of 122 human papillomavirus (HPV)-negative, surgically treated HNSCC patients. Immunohistochemical evaluation of the active form of SRC by means of anti-SRC Clone 28 monoclonal antibody was specifically performed and subsequently correlated with clinical data. The expression of p-SRC (Tyr419), total SRC, and downstream SRC effectors was also analyzed. Our results uncovered striking differences in the prognostic relevance of SRC expression in HNSCC patients depending on the tumor site. Active SRC expression was found to significantly associate with advanced disease stages, presence of lymph node metastasis, and tumor recurrences in patients with laryngeal tumors, but not in the pharyngeal subgroup. Multivariate Cox analysis further revealed active SRC expression as an independent predictor of cancer-specific mortality in patients with laryngeal carcinomas. Concordantly, expression of p-SRC (Tyr419) and the SRC substrates focal adhesion kinase (FAK) and the Arf GTPase-activating protein ASAP1 also showed specific associations with poor prognosis in the larynx. These findings could have important implications in ongoing Src family kinase (SFK)-based clinical trials, as these new criteria could help to improve patient selection and develop biomarker-stratified trials.