Free-Circulating Methylated DNA in Blood for Diagnosis, Staging, Prognosis, and Monitoring of Head and Neck Squamous Cell Carcinoma Patients: An Observational Prospective Cohort Study.

BACKGROUND: Circulating cell-free DNA methylation testing in blood has recently received regulatory approval for screening of colorectal cancer. Its application in other clinical settings, including staging, prognosis, prediction, and recurrence monitoring is highly promising, and of particular interest in head and neck squamous cell carcinomas (HNSCCs) that represent a heterogeneous group of cancers with unsatisfactory treatment guidelines. METHODS: Short stature homeobox 2 (SHOX2) and septin 9 (SEPT9) DNA methylation in plasma from 649 prospectively enrolled patients (training study: 284 HNSCC/122 control patients; testing study: 141 HNSCC/102 control patients) was quantified before treatment and longitudinally during surveillance. RESULTS: In the training study, 59% of HNSCC patients were methylation-positive at 96% specificity. Methylation levels correlated with tumor and nodal category (P < 0.001). Initially increased methylation levels were associated with a higher risk of death [SEPT9: hazard ratio (HR) = 5.27, P = 0.001; SHOX2: HR = 2.32, P = 0.024]. Disease recurrence/metastases were detected in 47% of patients up to 377 days earlier compared to current clinical practice. The onset of second cancers was detected up to 343 days earlier. In the testing study, sensitivity (52%), specificity (95%), prediction of overall survival (SEPT9: HR = 2.78, P = 0.022; SHOX2: HR = 2.50, P = 0.026), and correlation with tumor and nodal category (P <0.001) were successfully validated. CONCLUSIONS: Methylation testing in plasma is a powerful diagnostic tool for molecular disease staging, risk stratification, and disease monitoring. Patients with initially high biomarker levels might benefit from intensified treatment and posttherapeutic surveillance. The early detection of a recurrent/metastatic disease or a second malignancy could lead to an earlier consecutive treatment, thereby improving patients' outcomes.

Targeting DDR2 in head and neck squamous cell carcinoma with dasatinib.

Squamous cell carcinoma of the head and neck (HNSCC) is the tenth most common tumor entity in men worldwide. Nevertheless therapeutic options are mostly limited to surgery and radio-chemotherapy resulting in 5-year survival rates of around 50%. Therefore new therapeutic options are urgently needed. During the last years, targeting of receptor tyrosine kinases has emerged as a promising strategy that can complement standard therapeutical approaches. Here, we aimed at investigating if the receptor tyrosine kinase DDR2 is a targetable structure in HNSCC. DDR2 expression was assessed on a large HNSCC cohort (554 patients) including primary tumors, lymph node metastases and recurrences and normal mucosa as control. Subsequently, DDR2 was stably overexpressed in two different cell lines (FaDu and HSC-3) using lentiviral technology. Different tumorigenic properties such as proliferation, migration, invasion, adhesion and anchorage independent growth were assessed with and without dasatinib treatment using in-vitro cell models and in-vivo zebrafish xenografts. DDR2 was overexpressed in all tumor tissues when compared to normal mucosa. DDR2 overexpression led to increased migration, invasion, adhesion and anchorage independent growth whereas proliferation remained unaltered. Upon dasatinib treatment migration, invasion and adhesion could be inhibited in-vitro and in-vivo whereas proliferation was unchanged. Our data suggest treatment with dasatinib as a promising new therapeutic option for patients suffering from DDR2 overexpressing HNSCC. Since dasatinib is already FDA-approved we propose to test this drug in clinical trials so that patients could directly benefit from this new treatment option.

Clinical and molecular implications of MED15 in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) progression depends on various dysregulated pathways. Regulation of diverse pathways is mediated by the mediator complex. The mediator subunit MED15 is essential for transforming growth factor (TGF)-ß signaling and involved in breast and prostate cancers. We investigated the implication of MED15 in HNSCC. IHC for MED15 was performed on 324 tissue samples, and TGF-ß assessed the use of Ki-67 and pSMAD3 as markers. MED15 knockdown followed by proliferation and migration assays, as well as TGF-ß1 treatment followed by MED15 analysis, was also performed. MED15 was overexpressed in 35% of primary tumors, 30% of lymph node metastases, and 70% of recurrences in contrast to no or low expression in benign tumors. MED15 overexpression in primary tumors from patients who developed recurrences was associated with higher mortality rates and occurred at highest frequency in oral cavity or oropharyngeal tumors. Furthermore, MED15 expression correlated between primary tumors and corresponding lymph node metastases. MED15 correlated with proliferation in tissues, and MED15 knockdown reduced proliferation and migration. We observed an association between MED15 and TGF-ß activity in tissues because TGF-ß activation led to increased MED15 expression and reduced pSMAD3 on MED15 knockdown. Taken together, our results implicate MED15 in HNSCC and hint that MED15 overexpression is a clonal event during HNSCC progression. MED15 may serve as a prognostic marker for recurrence and as a therapeutic target.

Implication of the Receptor Tyrosine Kinase AXL in Head and Neck Cancer Progression.

Head and neck squamous cell carcinoma (HNSCC) remains a clinical challenge and identification of novel therapeutic targets is necessary. The receptor tyrosine kinase AXL has been implicated in several tumor entities and a selective AXL small molecule inhibitor (BGB324) is currently being tested in clinical trials for patients suffering from non-small cell lung cancer or acute myeloid leukemia. Our study investigates AXL expression during HNSCC progression and its use as a potential therapeutic target in HNSCC. AXL protein expression was determined in a HNSCC cohort (n = 364) using immunohistochemical staining. For functional validation, AXL was either overexpressed or inhibited with BGB324 in HNSCC cell lines to assess proliferation, migration and invasion. We found AXL protein expression increasing during tumor progression with highest expression levels in recurrent tumors. In HNSCC cell lines in vitro, AXL overexpression increased migration as well as invasion. Both properties could be reduced through treatment with BGB324. In contrast, proliferation was neither affected by AXL overexpression nor by inhibition with BGB324. Our patient-derived data and in vitro results show that, in HNSCC, AXL is important for the progression to more advanced tumor stages. Moreover, they suggest that AXL could be a target for precision medicine approaches in this dismal tumor entity.

Expression and role of the embryonic protein SOX2 in head and neck squamous cell carcinoma.

Recently, SOX2 has been identified as a potential lineage-specific oncogene in lung squamous cell carcinomas. Since head and neck squamous cell carcinomas (HNSCC) are morphologically and clinically highly related to lung squamous cell carcinomas, we hypothesized that SOX2 also plays an oncogenic role in this tumor entity. We assembled a cohort of 496 patients with HNSCC, including 253 metastases and 135 recurrences. SOX2 amplification (FISH) and SOX2 protein expression (immunohistochemistry) were correlated with molecular and clinicopathological parameters. In order to investigate the functional role of SOX2 in human HNSCC, SOX2 knockdown and overexpression in SCC-25 cells were generated by lentiviral constructs and subjected to cell cycle analysis, proliferation and apoptosis assays. Furthermore, SOX2 expression was correlated with the expression of proliferation and apoptosis-related proteins in primary HNSCC samples. SOX2 amplification was detected in 21% of primary HNSCC and mostly observed in a concordant manner between primary tumors and corresponding metastatic tissues. Overall, SOX2 amplification resulted in protein overexpression and was mutually exclusive with human papillomavirus infection. SOX2 protein overexpression was associated with clinicopathological parameters of worse outcome. Functionally, SOX2 induced the expression of the antiapoptotic protein BCL-2 and enhanced resistance to apoptosis-inducing agents including cisplatin, indicating SOX2 as a mediator of therapy resistance in human HNSCC. Targeting SOX2 and related molecular downstream pathways such as BCL-2 may enhance therapy efficacy in SOX2-expressing HNSCC.

Fibroblast growth factor receptor 1 amplification is a common event in squamous cell carcinoma of the head and neck.

Recently, we characterized fibroblast growth factor receptor 1 amplification as a target for a rational therapy in lung squamous cell carcinoma. Patients harboring this genetic event are currently eligible for treatment with antifibroblast growth factor receptor small-molecule inhibitors in phase I clinical trials. This has the potential to significantly improve standard therapy for lung squamous cell carcinoma patients. The aim of this study was to elucidate whether fibroblast growth factor receptor 1 amplification is also a common genetic event in head and neck squamous cell carcinoma. For this purpose, we assembled a cohort of 555 patients, including 264 with metastatic disease and 147 with recurrent disease. Formalin-fixed, paraffin-embedded material of primary tumors, metastases and recurrences were assessed for fibroblast growth factor receptor 1 copy number status using fluorescence in situ hybridization. Human papilloma virus status was detected by p16 immunohistochemistry staining and PCR-ELISA. Molecular parameters were correlated with each other and with clinicopathological data. We found 15% of primary head and neck squamous cell carcinoma to display a fibroblast growth factor receptor 1 amplification. In nearly all cases, metastatic and recurrent tumor samples shared the same amplification status as the corresponding primary tumor. Fibroblast growth factor receptor 1 amplification was associated with nicotine and alcohol consumption, but was mutually exclusive with human papilloma virus infection. Amplification of the gene was associated with parameters of worse outcome. Our data identify fibroblast growth factor receptor 1 amplification as a frequent event in primary and metastatic head and neck squamous cell carcinoma and represents a potential biomarker for more aggressive disease. Fibroblast growth factor receptor 1-amplified tumors could potentially comprise a subset of head and neck squamous cell carcinoma against which targeted small-molecule inhibitors hold therapeutic efficacy.

Transarterial endovascular treatment in the management of life-threatening intra- and postoperative haemorrhages after otorhinolaryngological surgery.

Management of life-threatening postsurgical bleeding is complex. If conservative or surgical therapy is demanding, an endovascular treatment can be considered. The goal of this study was to evaluate the outcome of endovascular approaches in the diagnosis and therapy of otherwise intractable postoperative haemorrhages with a study design of outcomes research. Charts of all patients with postsurgical bleedings receiving endovascular treatment were reviewed for clinical outcome, complications, and demographic data. 15 patients were identified. They had rhinosurgery (12/15), tonsillectomy (2/15) or transoral tumour debulking (1/15) prior to the endovascular procedure. In more than 70%, the source of bleeding was directly located angiographically and subsequently superselectively embolized. The remaining patients suffered from post-rhinosurgical epistaxis and underwent a bilateral embolization of the sphenopalatine artery. All bleedings were successfully controlled and no procedure-related complication was noted. In conclusion, endovascular treatment of life-threatening postsurgical haemorrhages should be considered if the source of bleeding is unknown or if surgery is difficult and may result in devastating postoperative complications.

Recurrent HNSCC Harbor an Immunosuppressive Tumor Immune Microenvironment Suggesting Successful Tumor Immune Evasion.

PURPOSE: Recurrent tumors (RT) of head and neck squamous cell carcinoma (HNSCC) occur in up to 60%, with poor therapeutic response and detrimental prognosis. We hypothesized that HNSCC RTs successfully evade antitumor immune response and aimed to reveal tumor immune microenvironment (TIME) changes of primary tumors (PT) and corresponding RTs. EXPERIMENTAL DESIGN: Tumor-infiltrating leukocytes (TIL) of 300 PTs and 108 RTs from two large independent and clinically well-characterized HNSCC cohorts [discovery cohort (DC), validation cohort (VD)] were compared by IHC. mRNA expression analysis of 730 immune-related genes was performed for 18 PTs and RTs after adjuvant chemoradiotherapy (CRT). The effect of chemotherapy and radiation resistance was assessed with an in vitro spheroid/immunocyte coculture model. RESULTS: TIME analysis revealed overall decrease of TILs with significant loss of CD8+ T cells (DC P = 0.045/VC P < 0.0001) and B lymphocytes (DC P = 0.036/VC P < 0.0001) in RTs compared with PTs in both cohorts. Decrease predominantly occurred in RTs after CRT. Gene expression analysis confirmed loss of TILs (P = 0.0004) and B lymphocytes (P < 0.0001) and showed relative increase of neutrophils (P = 0.018), macrophages (P < 0.0001), dendritic cells (P = 0.0002), and mast cells (P = 0.0057) as well as lower overall expression of immune-related genes (P = 0.018) in RTs after CRT. Genes involved in B-lymphocyte functions and number of tertiary lymphoid structures showed the strongest decrease. SPP1 and MAPK1 were upregulated in vivo and in vitro, indicating their potential suitability as therapeutic targets in CRT resistance. CONCLUSIONS: HNSCC RTs have an immunosuppressive TIME, which is particularly apparent after adjuvant CRT and might substantially contribute to poor therapeutic response and prognosis.

PD-L1 (CD274) and PD-L2 (PDCD1LG2) promoter methylation is associated with HPV infection and transcriptional repression in head and neck squamous cell carcinomas.

BACKGROUND: DNA methylation of the immune checkpoint gene PD-L1 has recently been shown to be associated with PD-L1 mRNA expression in various malignancies. This study aimed to investigate the association of PD-L1 and PD-L2 methylation with mRNA expression, immune cell infitration, protein expression and human papilloma virus (HPV) infection in head and neck squamous cell carcinoma (HNSCC) patients. RESULTS: DNA methylation of PD-L1 and PD-L2 correlates inversely with mRNA expression (PD-L1: p ≤ 0.002; PD-L2: p ≤ 0.014). Methylation of specific CpG-sites of both PD-L1 and PD-L2 were further significantly associated with HPV infection in the TCGA cohort. Immune cell infiltrates correlated significantly with PD-L1 and PD-L2 methylation. In the validation cohort, PD-L1 protein expression was associated with PD-L1 hypomethylation (p = 0.012). CONCLUSIONS: DNA methylation of PD-L1 and PD-L2 is associated with transcriptional silencing and HPV infection in HNSCCs. Additional studies are warranted to test PD-L1 and PD-L2 methylation as predictive biomarkers for response to immunotherapies (e.g. pembrolizumab and nivolumab) that target the PD-L1/PD-L2/PD-1 immune checkpoint axis. MATERIALS AND METHODS: PD-L1 and PD-L2 promoter methylation and its mRNA expression were analyzed based on Infinium HumanMethylation450 BeadChip and RNA-Seq (both Illumina, Inc.) data in a representative HNSCC patient cohort (n = 528) enrolled by The Cancer Genome Atlas (TCGA) Research Network. A validation cohort consisting of 168 HNSCC patients treated at the University Hospital Bonn was analyzed regarding PD-L1 and PD-L2 promoter methylation by means of methylation-specific quantitative real-time PCR. PD-L1 protein expression in the validation cohort was quantified via immunohistochemistry (PD-L1 antibody clone 22C3, Dako/Agilent Technologies, Inc.).

Comparison of quantification algorithms for circulating cell-free DNA methylation biomarkers in blood plasma from cancer patients.

BACKGROUND: SHOX2 and SEPT9 methylation in circulating cell-free DNA (ccfDNA) in blood are established powerful and clinically valuable biomarkers for diagnosis, staging, prognosis, and monitoring of cancer patients. The aim of the present study was to evaluate different quantification algorithms (relative quantification, absolute quantification, quasi-digital PCR) with regard to their clinical performance. METHODS: Methylation analyses were performed in a training cohort (141 patients with head and neck squamous cell carcinoma [HNSCC], 170 control cases) and a testing cohort (137 HNSCC cases, 102 controls). DNA was extracted from plasma samples, bisulfite-converted, and analyzed via quantitative real-time PCR. SHOX2 and SEPT9 methylations were assessed separately and as panel [mean SEPT9/SHOX2 ] using the ΔCT method for absolute quantification and the ΔΔCT-method for relative quantification. Quasi-digital PCR was defined as the number of amplification-positive PCR replicates. The diagnostic (sensitivity, specificity, area under the curve (AUC) of the receiver operating characteristic (ROC)) and prognostic accuracy (hazard ratio (HR) from Cox regression) were evaluated. RESULTS: Sporadic methylation in control samples necessitated the introduction of cutoffs resulting in 61-63% sensitivity/90-92% specificity (SEPT9/training), 53-57% sensitivity/87-90% specificity (SHOX2/training), and 64-65% sensitivity/90-91% specificity (mean SEPT9/SHOX2 /training). Results were confirmed in a testing cohort with 54-56% sensitivity/88-90% specificity (SEPT9/testing), 43-48% sensitivity/93-95% specificity (SHOX2/testing), and 49-58% sensitivity/88-94% specificity (mean SEPT9/SHOX2 /testing). All algorithms showed comparable cutoff-independent diagnostic accuracy with largely overlapping 95% confidence intervals (SEPT9: AUCtraining = 0.79-0.80; AUCtesting = 0.74-0.75; SHOX2: AUCtraining = 0.78-0.81, AUCtesting = 0.77-0.79; mean SEPT9/SHOX2 : AUCtraining = 0.81-0.84, AUCtesting = 0.80). The accurate prediction of overall survival was possible with all three algorithms (training cohort: HR SEPT9 = 1.23-1.90, HR SHOX2 = 1.14-1.85, HRmeanSEPT9/SHOX2 =1.19-1.89 ; testing cohort: HR SEPT9 =1.22-1.67, HR SHOX2 = 1.15-1.71, HRmeanSEPT9/SHOX2 = 1.12-1.77). CONCLUSION: The concordant clinical performance based on different quantification algorithms allows for the application of various diagnostic platforms for the analysis of ccfDNA methylation biomarkers.

PD-L1: a novel prognostic biomarker in head and neck squamous cell carcinoma.

BACKGROUND: The PD-1 receptor and its ligands PD-L1 and PD-L2 are known to be significantly involved in T-cell regulation. Recent studies suggest that PD-L1 expression in malignant tumors contributes to an immunosuppressive microenvironment and disruption of antitumoral immune response. Drugs targeting this pathway are already tested in clinical trials against several tumor entities with promising results. However, until now comprehensive data with regard to PD-L1 and PD-L2 expression in head and neck squamous cell carcinoma (HNSCC) is still lacking. PATIENTS AND METHODS: We assessed PD-L1 and PD-L2 expression via immunohistochemistry in two independent cohorts of 293 HNSCC patients. RESULTS: A significant subset of HNSCC showed high expression levels of PD-L1. Most remarkable, we detected a strong correlation between PD-L1 expression and overall survival time in both HNSCC cohorts. Further, in multivariate cox proportional hazard models, PD-L1 dominates as the strongest prognostic factor of patient's outcome in HNSCC, leaving even tumor stage and distant metastasis behind. Moreover, strong PD-L1 expression was associated with the presence of distant metastases in a subset of cases. CONCLUSIONS: In summary, while the significance of PD-L2 in HNSCC seems to minor, we show that PD-L1 expression is common in HNSCC and, more importantly, a both robust and strong prognostic biomarker. In this respect, our results provide hints on further application of therapies targeting the PD-1/PD-L1 pathway in HNSCC. Investigation of response and outcome of patients receiving anti-PD-1/PD-L1 containing therapies in correlation with PD-L1 expression analysis should be an important task for the future. STATEMENT OF TRANSLATIONAL RELEVANCE: In spite of improved treatment options and increasing knowledge of molecular alterations in HNSCC, the survival rate has not been dramatically changed in the past decades. Pies are missing in HNSCC. One promising candidate in cancer immune therapy is PD-L1. Drugs targeting PD-L1 or its receptor PD-1 are subject of several clinical studies in different cancer entities. However, comprehensive data about PD-L1 expression in HNSCC and therefore a rational basis for anti PD-L1/PD-1 therapy in HNSCC is lacking. Here, we provide wide-ranging data about PD-L1 expression in HNSCC of all major localizations. We observed a strong correlation between expression of PD-L1 and reduced overall survival time. Furthermore, high PD-L1 expression was identified as a strong prognostic factor of patient's outcome when verified together with recognized prognostic factors.

PITX3 DNA methylation is an independent predictor of overall survival in patients with head and neck squamous cell carcinoma.

BACKGROUND: Molecular biomarkers assisting risk-group assignment and subsequent treatment stratification are urgently needed for patients with squamous cell cancer of the head and neck region (HNSCC). Aberrant methylation is a frequent event in cancer and, therefore, a promising source for potential biomarkers. Here, the methylation status of the paired-like homeodomain transcription factor 3 (PITX3) was evaluated in HNSCC. METHODS: Using a quantitative real-time PCR, PITX3 methylation was assessed in a cohort of 326 HNSCC patients treated for localized or locally advanced disease (training cohort). The results were validated with Infinium HumanMethylation450 BeadChip data from a 528 HNSCC patient cohort (validation cohort) generated by The Cancer Genome Atlas (TCGA) Research Network. RESULTS: PITX3 methylation was significantly higher methylated in tumor compared to normal adjacent tissue (NAT; training cohort: median methylation NAT 32.3%, tumor 71.8%, p < 0.001; validation cohort: median methylation NAT 16.9%, tumor 35.9%, p < 0.001). PITX3 methylation was also significantly correlated with lymph node status both in the training (p = 0.006) and validation (p < 0.001) cohort. PITX3 methylation was significantly higher in HPV-associated (p16-positive) tumors compared to p16-negative tumors (training cohort: 73.7 vs. 66.2%, p = 0.013; validation cohort: 40.0 vs. 33.1%, p = 0.015). Hypermethylation was significantly associated with the risk of death (training cohort: hazard ratio (HR) = 1.80, [95% confidence interval (CI) 1.20-2.69], p = 0.005; validation cohort: HR = 1.43, [95% CI 1.05-1.95], p = 0.022). In multivariate Cox analyses, PITX3 added independent prognostic information. Messenger RNA (mRNA) expression analysis revealed an inverse correlation with PITX3 methylation in the TCGA cohort. CONCLUSIONS: PITX3 DNA methylation is an independent prognostic biomarker for overall survival in patients with HNSCC and might aid in the process of risk stratification for individualized treatment.

MAGE expression in head and neck squamous cell carcinoma primary tumors, lymph node metastases and respective recurrences-implications for immunotherapy.

Melanoma associated antigens (MAGE) are potential targets for immunotherapy and have been associated with poor overall survival (OS) in head and neck squamous cell carcinoma (HNSCC). However, little is known about MAGE in lymph node metastases (LNM) and recurrent disease (RD) of HNSCC.To assess whether MAGE expression increases with metastasis or recurrence, a tissue microarray (TMA) of 552 primary tumors (PT), 219 LNM and 75 RD was evaluated by immunohistochemistry for MAGE antigens using three monoclonal antibodies to multiple MAGE family members. Mean expression intensity (MEI) was obtained from triplicates of each tumor specimen.The median MEI compared between PT, LNM and RD was significantly higher in LNM and RD. In paired samples, MEI was comparable in PT to respective LNM, but significantly different from RD. Up to 25% of patients were negative for pan-MAGE or MAGE-A3/A4 in PT, but positive in RD. The prognostic impact of MAGE expression was validated in the TMA cohort and also in TCGA data (mRNA). OS was significantly lower for patients expressing pan-MAGE or MAGE-A3/A4 in both independent cohorts.MAGE expression was confirmed as a prognostic marker in HNSCC and may be important for immunotherapeutic strategies as a shared antigen.

PITX2 and PANCR DNA methylation predicts overall survival in patients with head and neck squamous cell carcinoma.

BACKGROUND: Squamous cell carcinoma of the head and neck region (HNSCC) is a common malignant disease accompanied by a high risk of local or distant recurrence after curative-intent treatment. Biomarkers that allow for the prediction of disease outcome can guide clinicians with respect to treatment and surveillance strategies. Here, the methylation status of PITX2 and an adjacent lncRNA (PANCR) were evaluated for their ability to predict overall survival in HNSCC patients. RESULTS: PITX2 hypermethylation was associated with a better overall survival (hazard ratio, HR = 0.51, 95%CI: 0.35-0.74, p<0.001), while PANCR hypermethylation was significantly associated with an increased risk of death (HR = 1.64, 95%CI: 1.12-2.39, p=0.010). METHODS: Quantitative, methylation-specific real-time PCR assays for PITX2 and PANCR were employed to measure bisulfite-converted DNA from formalin-fixed, paraffin-embedded (FFPE) tissues in a cohort of 399 patients with localized or locally advanced HNSCC who received curative-intent treatment (surgery with optional adjuvant radiochemotherapy or definite radiochemotherapy). CONCLUSIONS: PITX2 and PANCR methylation status were shown to be independent predictors for overall survival in HNSCC patients. Tissue-based methylation testing could therefore potentially be employed to identify patients with a high risk for death who might benefit from a more radical or alternative treatment.

Evaluation of FGFR3 as a Therapeutic Target in Head and Neck Squamous Cell Carcinoma.

BACKGROUND: Although head and neck squamous cell carcinoma (HNSCC) is the sixth most common tumour entity worldwide, it remains a clinical challenge. Large-scale explorative genomic projects have identified several genes as potential targets for therapy, including fibroblast growth factor receptor 3 (FGFR3). AIMS: The aim of this study was to investigate the biological significance of wild-type and mutated FGFR3 to evaluate its potential as a novel therapeutic target in HNSCC. METHODS: FGFR3 protein expression was analysed in a large HNSCC tissue cohort (n = 536) and FGFR3 mRNA expression from The Cancer Genome Atlas (TCGA; n = 520). Moreover, FGFR3 wild-type and mutant versions were overexpressed in vitro, and both proliferation and migration was assessed with and without BGJ398 (a specific FGFR1-3 inhibitor) treatment. RESULTS: Although FGFR3 expression for both cohorts decreased during tumour progression, high FGFR3 expression levels were observed in a small subset of patients. In vitro, FGFR3 overexpression led to increased proliferation, whereas migration was not altered. Moreover, FGFR3-overexpressing cells were more sensitive to BGJ398. Cells overexpressing FGFR3 mutant versions showed increased proliferation compared to wild-type FGFR3 under serum-reduced conditions and were largely as sensitive as the wild-type protein to BGJ398. CONCLUSIONS: Taken together, the results of this study demonstrate that although FGFR3 expression decreases during HNSSC progression, it plays an important role in tumour cell proliferation and thus may be a potential target for therapy in selected patients suffering from this dismal tumour entity.

MERTK as a novel therapeutic target in head and neck cancer.

Although head and neck cancer (HNSCC) is the sixth most common tumor entity worldwide therapy options remain limited leading to 5-year survival rates of only 50 %. MERTK is a promising therapeutic target in several tumor entities, however, its role in HNSCC has not been described yet. The aim of our study was to investigate the biological significance of MERTK and to evaluate its potential as a novel therapeutic target in this dismal tumor entity. In two large HNSCC cohorts (n=537 and n=520) we found that MERTK is overexpressed in one third of patients. In-vitro, MERTK overexpression led to increased proliferation, migration and invasion whereas MERTK inhibition with the small molecule inhibitor UNC1062 or MERTK knockdown reduced cell motility via the small GTPase RhoA.Taken together, we are the first to show that MERTK is frequently overexpressed in HNSCC and plays an important role in tumor cell motility. It might therefore be a potential target for selected patients suffering from this dismal tumor entity.

Ectopic Myoglobin Expression Is Associated with a Favourable Outcome in Head and Neck Squamous Cell Carcinoma Patients.

BACKGROUND/AIM: Ectopic myoglobin (MB) expression, mediated by alternative and hypoxia-inducible transcription, has recently been demonstrated in several epithelial tumours. This study aimed to examine the expression of MB in hormone-independent head and neck squamous cell carcinomas (HNSCCs). PATIENTS AND METHODS: Using imunohistochemistry, ectopic MB expression was analyzed on tissue microarrays (TMAs) of 524 patients with localized and locally advanced primary and recurrent HNSCC who had undergone surgical treatment with curative intent. Associations of MB expression with survival and clinicopathological parameters were analyzed. RESULTS: MB expression was found in 45.8% of HNSCC patients being significantly lower in normal adjacent tissue (NAT) compared to primary and recurrent tumours (p<0.001) and significantly associated with a favourable overall survival (OS) in HNSCC [p=0.037, hazard ratio (HR)=0.72, 95% confidence interval (CI)=0.53-0.98]. Furthermore, MB expression negatively correlated with human papillomavirus (HPV) status (p=0.013). CONCLUSION: MB is differentially expressed in HNSCC and correlates with a better OS.

A new bright-field dual-colour chromogenic and silver in situ hybridization method for the detection of FGFR1 gene copy number status.

Recently, the fibroblast growth factor receptor 1 (FGFR1) has been identified as the first actionable target in squamous cell lung cancer. Clinical trials testing specific FGFR inhibitors are in progress, and patients are selected based on their FGFR1 gene copy number status. Fluorescent in situ hybridization is the most commonly used method for detecting FGFR1 amplifications, but it has its limitations. In this paper, we describe a new non-fading and easy to assess assay for detecting FGFR1 amplification using a combination of chromogenic and silver in situ hybridization. We assessed 394 patients diagnosed with head and neck squamous cell carcinoma with the new assay and compared the results with those obtained by FGFR1 fluorescent in situ hybridization. We could assess copy number by the fluorescent in situ hybridization in 86.8 % (342/394) of cases, whereas with chromogenic and silver in situ hybridization, this was 79.4 % (313/394). By fluorescent in situ hybridization, a FGFR1 amplification was detected in 12.6 % (43/342) of cases, a low-level amplification (LLA) in 7.6 % (26/342) and a high-level amplification (HLA) in 5.0 % (17/342). By chromogenic and silver in situ hybridization, a FGFR1 amplification was found in 10.2 % (32/313) (5.7 % LLA, 4.5 % HLA). The two techniques showed highly concordant results (Pearson's correlation coefficient = 0.971, p < 0.01).

Fibroblast growth factor receptor-1 as a potential therapeutic target in sinonasal cancer.

BACKGROUND: Despite multimodal treatment, sinonasal malignancies have an unfavorable prognosis. The purpose of this study was to elucidate if these tumors harbor amplifications of the fibroblast growth factor receptor 1 (FGFR1) gene, which has recently been identified as a potential therapeutic target in squamous cell lung cancer. METHODS: One hundred twelve primary tumors (including squamous cell carcinoma [SCC], carcinoma associated with an inverted papilloma, sinonasal undifferentiated carcinoma [SNUC], adenocarcinoma, adenoid cystic carcinoma [ACC], esthesioneuroblastoma, and 9 corresponding lymph node metastases) were assessed by fluorescence in situ hybridization (FISH) for FGFR1 copy number status. Human papillomavirus (HPV) status was assessed by p16 immunohistochemical as a surrogate marker. RESULTS: FGFR1 amplification was found in subsets of sinonasal SCCs (20%), carcinomas associated with an inverted papilloma (33%), and SNUCs (5%). In all cases, metastatic tumor samples shared the same FGFR1 amplification status as the corresponding primary tumor tissue. None of the FGFR1-amplified tumors expressed p16. CONCLUSION: FGFR1 amplification represents a potential molecular target in a subset of patients with sinonasal cancer. © 2014 Wiley Periodicals, Inc. Head Neck 36: 1253-1257, 2014.

Sex determining region Y-box 2 (SOX2) amplification is an independent indicator of disease recurrence in sinonasal cancer.

OBJECTIVES: The transcription factor SOX2 (3q26.3-q27) is an embryonic stem cell factor contributing to the induction of pluripotency in terminally differentiated somatic cells. Recently, amplification of the SOX2 gene locus has been described in squamous cell carcinoma (SCC) of different organ sites. Aim of this study was to investigate amplification and expression status of SOX2 in sinonasal carcinomas and to correlate the results with clinico-pathological data. MATERIALS AND METHODS: A total of 119 primary tumor samples from the sinonasal region were assessed by fluorescence in-situ hybridization and immunohistochemistry for SOX2 gene amplification and protein expression, respectively. Of these, 59 were SSCs, 18 sinonasal undifferentiated carcinomas (SNUC), 10 carcinomas associated with an inverted papilloma (INVC), 19 adenocarcinomas (AD) and 13 adenoid cystic carcinomas (ACC). RESULTS: SOX2 amplifications were found in subsets of SCCs (37.5%), SNUCs (35.3%), INVCs (37.5%) and ADs (8.3%) but not in ACCs. SOX2 amplification resulted in increased protein expression. Patients with SOX2-amplified sinonasal carcinomas showed a significantly higher rate of tumor recurrences than SOX2 non-amplified tumors. CONCLUSION: This is the first study assessing SOX2 amplification and expression in a large cohort of sinonasal carcinomas. As opposed to AD and ACC, SOX2 amplifications were detected in more than 1/3 of all SCCs, SNUCs and INVCs. We therefore suggest that SNUCs are molecularly closely related to SCCs and INVCs and that these entities represent a subgroup of sinonasal carcinomas relying on SOX2 acquisition during oncogenesis. SOX2 amplification appears to identify sinonasal carcinomas that are more likely to relapse after primary therapy, suggesting that these patients might benefit from a more aggressive therapy regime.