Squamous Cell Carcinoma Of The Head And Neck Tissues Research Articles

KDM5B promotes metastasis and epithelial-mesenchymal transition via Wnt/β-catenin pathway in squamous cell carcinoma of the head and neck.

Metastasis determines clinical management decision and restricts the therapeutic efficiency in patients with squamous cell carcinoma of the head and neck (SCCHN). Epigenetic factor KDM5B serves as an oncogene in multiple cancers. However, its role in SCCHN metastasis remains unclear. Our previous study showed that KDM5B is significantly elevated in SCCHN tissue and is positively correlated with metastasis and recurrence. KDM5B overexpression predicted a poor prognosis in both disease-free survival and overall survival, which served as an independent prognostic factor in SCCHN patients. This study further investigates the exact impact of KDM5B in metastasis of SCCHN. We found that KDM5B knockdown significantly inhibits the migration and invasion of SCCHN cells both in vitro and in vivo. On the contrary, forced expression of KDM5B leads to enhanced migration and invasion, accompanied by canonical alterations of epithelial-mesenchymal transition (EMT). Mechanism investigations demonstrated that KDM5B activates Wnt/β-catenin pathway, and inhibition of Wnt/β-catenin pathway via a small molecule inhibitor iCRT-14 partially reverses the enhanced migratory and invasive ability caused by KDM5B in SCCHN cells. Together, our data indicate that KDM5B promotes EMT and metastasis via Wnt/β-catenin pathway in SCCHN, suggesting that KDM5B may be a potential therapeutic target and prognosis biomarker in SCCHN.

Abstract 950: Targeting B7-H3 in squamous cell carcinoma of the head and neck: Preclinical proof-of-concept with the investigational anti-B7-H3 antibody-drug conjugate, MGC018

Abstract Introduction: Squamous cell carcinoma of the head and neck (SCCHN) is the seventh most common cancer worldwide, accounting for ~ 4% of all cancers in the United States. Although PD-1-directed therapy has efficacy in SCCHN, 85-95% of patients progress following initial response. B7-H3 is a member of the B7 family of immunomodulatory molecules, is overexpressed in SCCHN, and correlates with disease severity and poor clinical outcome. Furthermore, consistent with its putative coinhibitory function, B7-H3 expression in SCCHN is inversely correlated with the number of tumor infiltrating CD8+ T-cells. We are developing therapeutics targeting B7-H3, including enoblituzumab, an Fc-engineered anti-B7-H3 monoclonal antibody, and MGC018, a duocarmycin-based B7-H3 ADC, both of which are currently being evaluated in clinical studies. Here the potential of MGC018 was explored in preclinical models as a proof of concept for targeting B7-H3 in SCCHN. Methods: Immunohistochemistry studies were performed to define the expression of B7-H3 in SCCHN tissue microarrays (TMA). Single and repeat-dose in vivo efficacy studies were conducted in CD-1 nude mice with cell line-derived SCCHN human tumor xenografts to explore the relationship between Cmax, exposure and antitumor activity, and to define the minimal efficacious dose in these models. Based on results in these cell-derived xenograft (CDX) studies, in vivo efficacy studies were extended to a panel of SCCHN patient-derived xenograft (PDX) models, which more closely mimic the biological characteristics of the patient tumor and exhibit heterogenous expression of B7-H3. Results: Analysis of B7-H3 expression on a SCCHN TMA confirmed and extended previously reported expression of B7-H3 in SCCHN. Of the SCCHN samples evaluated, 90% (36/40) of the tumor samples were positive for B7-H3: 35% (14/40) had H-scores greater than 200, with the remaining 22 samples equally distributed between the H-score range of 101-200 and 1-100 (~ 28% each). MGC018 demonstrated specific, dose-dependent in vitro cytotoxicity toward SCCHN human tumor cell lines. The in vitro cytotoxicity translated to potent antitumor activity in vivo against SCCHN CDX models, with a single administration of 3 mg/kg resulting in complete responses in 7/7 mice in the FaDu model. In the PDX setting (H-scores 120-283), repeat dose administration every week or two weeks with MGC018 at 3 mg/kg/dose, led to regressions and/or stable disease in 10/18 models, and a delay in tumor growth in 5 additional models. Conclusion: B7-H3 is frequently overexpressed in SCCHN. At clinically relevant dose levels, MGC018 demonstrated potent antitumor activity in vivo toward SCCHN CDX mouse models and the majority of SCCHN PDX mouse models examined. These results support SCCHN as a potential indication that may be responsive to ADC-based treatments directed toward B7-H3. Citation Format: Juniper A. Scribner, Francine Z. Chen, Ying Li, Michael Chiechi, Thomas Son, Jeff Hooley, Scott Koenig, Paul A. Moore, Ezio Bonvini, Chet Bohac, Deryk Loo. Targeting B7-H3 in squamous cell carcinoma of the head and neck: Preclinical proof-of-concept with the investigational anti-B7-H3 antibody-drug conjugate, MGC018 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 950.

Wnt3a promotes radioresistance via autophagy in squamous cell carcinoma of the head and neck.

The canonical Wnt/β‐catenin signalling pathway and autophagy play critical roles in cancer progression. However, the role of Wnt‐mediated autophagy in cancer radioresistance remains unclear. In this study, we found that irradiation activated the Wnt/β‐catenin and autophagic signalling pathways in squamous cell carcinoma of the head and neck (SCCHN). Wnt3a is a classical ligand that activated the Wnt/β‐catenin signalling pathway, induced autophagy and decreased the sensitivity of SCCHN to irradiation both in vitro and in vivo. Further mechanistic analysis revealed that Wnt3a promoted SCCHN radioresistance via protective autophagy. Finally, expression of the Wnt3a protein was elevated in both SCCHN tissues and patients' serum. Patients showing high expression of Wnt3a displayed a worse prognosis. Taken together, our study indicates that both the canonical Wnt and autophagic signalling pathways are valuable targets for sensitizing SCCHN to irradiation.

KDM5B overexpression predicts a poor prognosis in patients with squamous cell carcinoma of the head and neck.

Purpose: Lysine demethylase (KDM) 5B, as a member of the histone lysine demethylase family, is overexpressed and functions abnormally in various human cancers. However, its expression in the squamous cell carcinoma of the head and neck (SCCHN) remains unclear.Methods: KDM5B expression was analyzed by immunohistochemistry and correlated with clinicopathological parameters in 103 archival SCCHN tissue samples and 24 adjacent noncancerous epithelial tissues.Results: We found that KDM5B expression was higher in SCCHN than that in adjacent noncancerous tissues. This was closely associated with lymph node metastasis and tumor recurrence. In addition, Kaplan-Meier analysis revealed that patients with high KDM5B expression had shorter disease-free and overall survival times than those with low KDM5B expression. Importantly, both univariate and multivariate analysis demonstrated that KDM5B level was an independent prognostic factor in SCCHN patients.Conclusions: These results indicate that KDM5B is a valuable biomarker that can be used to predict SCCHN patient outcome.

Protein methyltransferases and demethylases dictate CD8+ T-cell exclusion in squamous cell carcinoma of the head and neck.

A subset of patients with recurrent/metastatic squamous cell carcinoma of the head and neck (SCCHN) benefit from pembrolizumab and nivolumab, but the majority of patients do not probably due to lack of activated cytotoxic CD8+ T-cells in their tumor tissues. Herein, we aim to investigate whether specific protein methyltransferases (PMTs) and demethylases (PDMTs) could play any roles in the CD8+ T-cell exclusion process in HPV-negative SCCHN. RNA sequencing data from the TCGA database were interrogated for HPV-negative SCCHN patients using a 10-gene chemokine signature that classifies SCCHN tissues into CD8+ T-cell inflamed and non-CD8+ T-cell inflamed phenotypes. Among 53 PMT/PDMT genes examined in the TCGA HPV-negative SCCHN database, expression levels of 15 PMT/PDMT genes were significantly negatively correlated with the chemokine signature score and CD8 mRNA expression levels. The expression level of each of these 15 PMT/PDMT genes showed significantly negative correlations with immune-active chemokines, as well as HLA class I and APM molecules. siRNA-mediated knockdown of a candidate PMT, SMYD3, led to upregulation of CXCL9, CXCL10, CXCL11 and TAP1 at mRNA and protein levels in HPV-negative SCCHN cell lines. These findings demonstrate that overexpression of some PMTs and PDMTs seems to be related with the non-CD8+ T-cell inflamed phenotype and may drive CD8+ T-cell exclusion in HPV-negative SCCHN. This study suggests that chromatin modifiers contribute to CD8+ T-cell exclusion and antigen presentation capacity of HPV-negative SCCHN, supporting that targeting of specific PMTs and/or PDMTs could enhance CD8+ T-cell infiltration and increase the proportion of patients that may benefit from immunotherapy.

NLRP3 inflammasome activation promotes inflammation-induced carcinogenesis in head and neck squamous cell carcinoma

BackgroundNLRP3 inflammasome acts as a danger signal sensor that triggers and coordinates the inflammatory response. However, the roles of NLRP3 inflammasome in the tumorigenesis and development of cancer stem cells (CSCs) of squamous cell carcinoma of the head and neck (SCCHN) remain ambiguous.MethodsIn our study, tissue microarrays, ELISA, sphere-forming assay, colony formation assay and Western blot analysis were performed to evaluate the effect of NLRP3 inflammasome on the development of CSCs in human SCCHN tissue specimen, cell lines, and transgenic mouse SCCHN model.ResultsThe components of NLRP3 inflammasome, namely, NLRP3, ASC, Caspase-1, and IL-18 were correlated with CSCs markers BMI1, ALDH1 and CD44 in human SCCHN specimens. Moreover, NLRP3, Caspase-1, IL-1β, and IL-18 were highly expressed in SCCHN cell lines. NLRP3 inflammasome activated by LPS and ATP promoted sphere-forming and colony formation capacities along with an upregulation of BMI1, ALDH1 and CD44. In addition, NLRP3 inflammasome blockade by NLRP3 inhibitor MCC950 reduced sphere and colony number, also decreased the expression of BMI1, ALDH1 and CD44 in SCCHN cell lines. Expression of NLRP3, ASC, Caspase-1, IL-1β, IL-18, BMI1, ALDH1 and CD44 was upregulated in Tgfbr1/Pten 2cKO mouse SCCHN model, and NLRP3 inflammasome expression was closely related to those CSCs makers in mice SCCHN. However, MCC950 treatment reduced the expression of NLRP3 inflammasome, CSCs markers BMI1, ALDH1 and CD44 in Tgfbr1/Pten 2cKO mice SCCHN. In addition, blockade of NLRP3 inflammasome can also delayed the tumor-burdened speed in SCCHN mice.ConclusionsOur study demonstrates that NLRP3 inflammasome was upregulated and associated with the carcinogenesis and CSCs self-renewal activation in SCCHN. NLRP3 inflammasome can be a potential target in the development of novel approaches for head and neck squamous cell carcinoma therapy.

Jak3 is involved in CCR7-dependent migration and invasion in metastatic squamous cell carcinoma of the head and neck.

Patients with cervical lymph node metastasis in squamous cell carcinoma of the head and neck (SCCHN) exhibit a poor prognosis and low 5-year survival rate. It has been proven that chemokine receptor 7 (CCR7) promotes cellular migration and invasion in metastatic SCCHN. In the present study, the metastatic SCCHN PCI-37B cell line was utilized to explore the role of Janus activated kinase-3 (Jak3) in the CCR7-mediated signaling pathway in metastatic SCCHN cells. It was observed that phospho-Jak3 was expressed in SCCHN tissues. In addition, when the PCI-37B cells were analyzed in response to chemokine ligand 19 (CCL19), the ligand of CCR7, at the indicated time points, the results of the present study demonstrated that CCR7 induced Jak3 activation, and inhibition of Jak3 activity using a specific inhibitor, ZM39923, significantly attenuated CCR7-induced Jak3 phosphorylation. Migration and invasion assays and immunofluorescence staining experiments demonstrated that CCL19 promoted cell migration, invasion and F-actin rearrangment in CCR7-expressing SCCHN cells partially due to the activation of the Jak3 signaling pathway. These results demonstrate that the Jak3 signaling pathway is important for the CCR7-induced malignant biological behavior of SCCHN cells.

Elevated expression of Derlin-1 associates with unfavorable survival time of squamous cell carcinoma of the head and neck and promotes its malignance

Derlin-1 is over-expressed to function as an oncoprotein in breast, lung and colon cancers. However, the implications of Derlin-1 involved in squamous cell carcinoma of the head and neck (SCCHN) remain unknown. This study aims to investigate the effects of Derlin-1 expression on SCCHN tissues and cells. The potential mechanism of Derlin-1 regulating SCCHN cell proliferation, apoptosis and metastasis was also indicated in this work. Western blot and immunohistochemistry (IHC) assays showed that Derlin-1 was over-expressed in 114 SCCHN samples and five SCCHN cell lines. We found that the expression of Derlin-1 was positively associated with lymph node metastasis, clinical stage and recurrence in our SCCHN patients' samples. Survival analysis indicated that high expression of Derlin-1 was significantly associated with shorter overall survival (OS) and disease-free survival (DFS). Knock down expression of Derlin-1 in SCCHN cell lines was found to inhibit cell proliferation, metastasis and promote cell apoptosis. Further experiments showed that signals of PI3K/Akt, p53 and Smad2/3 may involve in these processes. In all, Derlin-1 might be a novel prognostic marker of SCCHN patients and plays an oncogenic role in SCCHN cell progression.

Metadherin regulation of vascular endothelial growth factor expression is dependent upon the PI3K/Akt pathway in squamous cell carcinoma of the head and neck.

Our previous study indicated overexpression of metadherin (MTDH) is an adverse prognostic factor in squamous cell carcinoma of the head and neck (SCCHN) and promotes SCCHN cell proliferation and invasion. However, its mechanism remains unclear. Recent studies have indicated that MTDH is a cancer-metastasis-associated molecule that participates in the process of angiogenesis. Therefore, the study is aimed to investigate that whether vascular endothelial growth factor (VEGF), as one of the most potent proangiogenic cytokines, is regulated by MTDH and the role of the phosphatidylinositide 3-kinases/Protein Kinase B (PI3K/Akt) pathway in this process of regulation and the clinical significance of both MTDH and VEGF in SCCHN.Immunohistochemistry was used to assay the expression of MTDH and VEGF in a cohort of 189 SCCHN patients with intact follow-up information. The expression of MTDH was then upregulated or inhibited by lentivirus-mediated MTDH Complementary deoxyribonucleic acid or MTDH short hairpin ribonucleic acid (shRNA) to observe the resulting alterations in VEGF expression and the PI3K/Akt signaling pathway in SCCHN cell lines. In addition, the PI3K/Akt pathway was modulated to observe the resulting changes in the MTDH-mediated expression of VEGF.The immunohistochemistry data showed that MTDH expression is positively correlated with VEGF expression in SCCHN tissues. Moreover, the overexpression of MTDH in SCCHN Tu686 and 5-8F cells led to increases in the expression of VEGF, and this effect was accompanied by activation of the PI3K/Akt pathway. Conversely, shRNA-mediated knockdown of MTDH led to decreased VEGF expression. In addition, inhibition of the Akt signaling pathway reversed the upregulation of VEGF resulting from MTDH overexpression. Moreover, the survival analysis revealed that VEGF is an independent prognostic factor, and a combined survival analysis based on both MTDH and VEGF showed synergistic effects in the prognosis evaluation of SCCHN patients.The findings of the present study demonstrate that MTDH regulates the expression of VEGF via the PI3K/Akt signaling pathway, indicating the potential role of the MTDH-mediated activation of VEGF signaling pathway in SCCHN angiogenesis and metastasis.

Elevated expression of T-lymphoma invasion and metastasis inducing factor 1 in squamous-cell carcinoma of the head and neck and its clinical significance

PurposeT-lymphoma invasion and metastasis inducing factor 1 (Tiam1) overexpression has been reported in a variety of human cancers. However, the investigation of Tiam1 in squamous-cell carcinoma of the head and neck (SCCHN) is extremely rare. The aim of the present study is to assess Tiam1 expression and to explore its role in SCCHN. MethodsTiam1 expression in 119 primary SCCHN tissue specimens was analysed by immunohistochemistry and correlated with clinicopathological parameters and patients’ survival. Additionally, 12 paired SCCHN tissues were evaluated for Tiam1 expression by Western blotting. ResultsWestern blotting indicated that Tiam1 expression levels in SCCHN carcinomas were significantly higher than those in the corresponding paraneoplastic tissues (P<0.001). Immmunohistochemistry staining revealed that Tiam1 was detected in all primary tumour samples, moreover, Tiam1 overexpression was significantly correlated with lymph node metastasis (P<0.001), clinical stage (P<0.001), histological grade (P=0.001) and recurrence (P<0.001). Survival analysis demonstrated that high Tiam1 expression was significantly correlated with shorter disease-free survival and overall survival (both P<0.001). When combining the Tiam1 expression and lymph node status, Kaplan–Meier survival showed that patients with Tiam1 overexpression/lymph node metastasis (+) had both shorter disease-free and overall survival than others (both P<0.001). Multivariate Cox proportional hazards model analysis confirmed that lymph node metastasis, histological grade and Tiam1 overexpression were statistically significant, independent predictor of prognosis for patients with SCCHN. ConclusionTiam1 may contribute to SCCHN progression, and represent as a novel prognostic indicator as well as a potential therapeutic target for SCCHN.

Metadherin regulates metastasis of squamous cell carcinoma of the head and neck via AKT signalling pathway-mediated epithelial–mesenchymal transition

Our recent study suggested that metadherin (MTDH) is overexpressed in laryngeal squamous cell carcinoma. Here, we further investigated its role in promoting metastasis of squamous cell carcinoma of the head and neck (SCCHN). An immunohistochemistry analysis demonstrated that MTDH is elevated and positively correlated with metastasis in 189 primary SCCHN tissues. In vitro experiments demonstrated that MTDH overexpression enhanced the migratory and invasive ability of SCCHN cells. Moreover, MTDH induced epithelial–mesenchymal transition (EMT) by both regulating morphological changes and mediating the expression of the biomolecular makers E-cadherin and vimentin. In addition, MTDH mediated AKT activation, and all of the above effects were nearly completely blocked by the inhibition of AKT. Our results suggested that MTDH might promote the metastasis of SCCHN through AKT signalling pathway mediated-EMT.

Abstract LB-323: Subclassification of squamous cell carcinomas of the head and neck based on HER/ErbB and c-MET receptor protein expression and activation profiles

Abstract The response rates of HER1 antibody monotherapy (∼13%) are comparable to the added response rates of HER1 antibody therapy when used in combination with chemotherapy (∼16%) in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN). Measures of HER1/ErbB1/EGFR protein levels by immunohistochemistry (IHC) or gene copy number by fluorescent in-situ hybridization (FISH) do not correlate with response, therefore, alternative gene or protein expression signatures are needed to identify patients most likely to respond to HER1 targeted therapies. Previously, quantitative VeraTag proximity immunoassays performed on formalin-fixed, paraffin imbedded SCCHN tumor tissue identified an activated HER1 signature (HER1-HER1 homodimers; HER1pY1173) that was significantly more pronounced in SCCHN compared to other carcinoma of the head and neck. In this study, we analyzed the expression of HER1, 2, 3 and c-MET along with the activation of HER1 and HER3 in 56 SCCHN tumors. Our results identified two distinct protein expression profiles: an activated HER1 profile, and a second profile characterized by the different expression levels of HER2, HER3, phosphorylated HER3, and c-MET relative to activated HER1. Our working hypothesis is that the first profile correlates with sensitivity to HER1 targeted therapies while the second profile correlates with either sensitivity or resistance to HER1 targeted therapies. HER1 protein levels measured by VeraTag assay varied over a ∼20-fold dynamic range, and significantly correlated with HER1 IHC staining and mRNA levels determined by qRT-PCR. HER2 and HER3 protein expression spanned a 15- to 20-fold range, and correlated with mRNA levels. Several of the highest HER1 expressing tumors have increased gene copy number; however, similar high values were measured in tumors without amplification, indicating that additional mechanisms of high HER1 expression exist in SCCHN. Although high HER1 activation measured by HER1-HER1 dimer and HER1pY1173 correlates with HER1 levels (r=∼0.6, p&amp;lt;0.05), and are associated with the highest levels of HER1 expression measured by multiple methods, not all high HER1 expressing tumors displayed highly activated HER1. These observations may explain why total HER1 expression in SCCHN tumors, determined by IHC or FISH, does not correlate with response to targeted therapies. On the other hand, approximately 16% of the SCCHN tumors studied here (9/56) exhibit a highly activated HER1 profile; similar in magnitude to the response rate seen with HER1 targeted antibody therapy. Future studies are designed to further validate the HER1 activation signature as a predictor of response to HER1 targeted therapies and/or the attenuation of response due to HER2, HER3 or c-MET expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-323. doi:10.1158/1538-7445.AM2011-LB-323

Bone Morphogenetic Protein-4-induced Epithelial-Mesenchymal Transition and Invasiveness Through Smad1-mediated Signal Pathway in Squamous Cell Carcinoma of the Head and Neck

Bone morphogenetic proteins (BMPs) have recently been shown to be involved in the genesis and progression of a wide variety of carcinomas. The present study was undertaken to estimate the effect of BMP-4 on squamous cell carcinoma of the head and neck (SCCHN) in tissue and cell levels. In this study, immunohistochemistry, Western blotting and RT-PCR were utilized to detect the expression of BMP-4, Smad1 and phosphorylated Smad1 in SCCHN tissues or SCCHN cell lines. Those three proteins in tissues were further correlated with prognosis of SCCHN by Kaplan-Meier analysis. The epithelial-mesenchymal transition (EMT)-associated changes in SCCHN cells were detected after stimulation by human BMP-4 recombinant protein and knockdown of Smad1 gene. Meanwhile, the effect on invasiveness and migration was evaluated by invasion and scratch assays, respectively. BMP-4 and p-Smad1 protein were overexpressed in SCCHN tissues with cervical lymph node metastasis, which was significantly higher than those without metastasis. The expression of BMP-4 and p-Smad1 protein was negatively correlated with the prognosis of SCCHN. BMP-4 promoted the invasiveness and migration through EMT, which was demonstrated by morphological alterations, loss of E-cadherin, increase of vimentin and activation of the Smad1 signal pathway. Knockdown of Smad1 expression suppressed BMP-4 induced EMT in both cell lines and weakened the invasiveness and migration of Tu686 and Tu212 in vitro. Our results demonstrate that BMP-4 protein may contribute to the malignant metastasis of SCCHN, which presents as a novel prognostic marker and a potential therapeutic target for patients with SCCHN.

Elevated expression of HMGB1 in squamous-cell carcinoma of the head and neck and its clinical significance

PurposeHMGB1 overexpression has been reported in a variety of human cancers. However, the role of HMGB1 in squamous-cell carcinoma of the head and neck (SCCHN) remains unclear. The aim of the present investigation was to analyse HMGB1 protein expression in both SCCHN tissue and cell levels and to assess its prognostic significance in SCCHN. MethodsHMGB1 protein expression in 103 primary SCCHN tissue specimens was analysed by immunohistochemistry and correlated with clinicopathological parameters and patient outcome. Additionally, HMGB1 protein expression was evaluated in cell level by Western blotting. ResultsBy Western blotting analysis, all the 5 SCCHN cell lines overexpressed HMGB1 protein, whereas the non-transformed immortalised cell line NP-69 had relatively weak HMGB1 protein expression. Immunohistochemical staining revealed that HMGB1 protein was detected in 91 (91/103, 88.3%) primary tumour samples, but only in 7 (7/16, 43.75%) adjacent non-carcinoma samples (p<0.001); moreover, HMGB1 overexpression was significantly associated with T classification (p=0.001), clinical stage (p<0.001), recurrence (p<0.001) and lymph node metastasis (p<0.001). Survival analysis demonstrated that high HMGB1 expression was significantly associated with shorter disease-free and overall survival (both p<0.001), especially in late patients with SCCHN. When HMGB1 expression and lymph node status were combined, patients with HMGB1 overexpression/lymph node (+) had both poorer disease-free and overall survival than others (both p<0.001). Multivariate analysis further demonstrated that HMGB1 was an independent prognostic factor for patients with SCCHN. ConclusionsHMGB1 protein may contribute to the malignant progression of SCCHN, and present as a novel prognostic marker and a potential therapeutic target for patients with SCCHN.

Clinical significance of EphA2 expression in squamous-cell carcinoma of the head and neck

EphA2 receptor tyrosine kinase is frequently overexpressed and functionally altered in a variety of human cancers. The study aimed to assess EphA2 expression and to explore its roles in squamous-cell carcinoma of the head and neck (SCCHN). EphA2 expression in 98 primary SCCHN tissue specimens was analyzed by immunohistochemistry and correlated with clinicopathological parameters. Additionally, 13 paired SCCHN tissues and 6 SCCHN cell lines were evaluated for EphA2 expression by RT-PCR and immunoblotting. EphA2 overexpressed in SCCHN tissues and SCCHN cell lines. More importantly, high EphA2 expression was significantly associated with tumor site, T classification, clinical stage, recurrence, and lymph node metastasis, respectively. Patients with high EphA2 expression had both poorer disease-free survival and overall survival than patients with low EphA2 expression. Multivariate Cox regression analysis revealed that EphA2 overexpression was an independent prognostic factor for patients with SCCHN. These findings suggested that EphA2 may contribute to SCCHN progression and represent a novel prognostic indicator for patients with SCCHN.

Abstract A126: Analysis of excision repair cross complementing group 1 (ERCC1) expression in squamous cell carcinoma of the head and neck (SCCHN)

Abstract Introduction: ERCC1 is a key enzyme in the nucleotide excision repair pathway and increased expression of ERCC1 is associated with cisplatin resistance in some cancers. Given the widespread use of platinum therapy for the treatment of SCCHN, we characterized the range of ERCC1 expression in SCCHN specimens using an immunofluorescence-based assay (automated quantitative analysis-tissue microarray [AQUA-TMA]). There is currently inadequate data regarding the range of ERCC1 expression in SCCHN, and how ERCC1 levels correlates with treatment outcomes, ERCC1 has been studied in SCCHN by standard immunohistochemistry, however, this method is limited by variability of techniques and subjectivity of interpretation, Therefore, there is a need for a highly quantitative technique which can be accomplished on formalin fixed paraffin embedded tissue and small samples, Our goal is to develop a reproducible assay that can eventually be incorporated into standard patient care practice. Methods: Tissue microarrays containing head and neck squamous cell carcinoma samples obtained from patients treated at Fox Chase Cancer Center from 1999–2006 were constructed. Slides were stained by a modified indirect immunofluorescence method. Sections were incubated with ERCC1 antibody (8F1, Lab Vision) and wide-spectrum screening rabbit cytokeratin antibody (Dako Z0622). Prolong Gold mounting medium (P36931; Molecular Probes) containing 4,6-Diamidino-2-phenylindole (DAPI) was used to define tissue nuclei. A binary image (tumor mask) was created from the cytokeratin image of each histospot. ERCC1 levels were measured using fluorescent immunohistochemistry on the HistoRx PM-2000 image analysis platform and the data analyzed using AQUA algorithms. The in situ biomarker profiling system generated quantitative measurements of patient protein levels based on AQUA scores derived from fluorescent output of labeled markers. Both nuclear and non-nuclear (i.e., non-membrane/cytoplasmic) ERCC1 expression was measured Results: 41 SCCHN specimens (24 oral cavity, 7 oropharynx, 5 glottic tumors, 3 unknown primary, 2 other) were analyzed. There were 25 males and 16 females and age at diagnosis ranged from 25 to 80 years (mean 64). The majority of patients were treated primarily by surgery. The range of AQUA scores was 33.66 to 1696.39 (mean 501.78, stnd deviation 284). The 25th percentile was 337.35, and the 75th percentile was 639.37. ERCC1 was predominantly nuclear (i.e., non-membrane/cytoplasmic) in the SCCHN tissue samples. Conclusions: 1) It is feasible to determine a quantitative value of ERCC1 expression with AQUA. 2) There is a wide range of ERCC1 expression in SCCHN. 3) ERCC1 is predominantly localized to the nuclear compartment. 4) Ongoing work is being done to correlate ERCC1 levels with clinical features, sensitivity to platinum therapy, and survival. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A126.

Examining expression of folate receptor in squamous cell carcinoma of the head and neck as a target for a novel nanotherapeutic drug.

Nanotechnology-based drug delivery approaches may help increase therapeutic efficacy and decrease side effects of chemotherapeutics. We investigated expression levels of folate receptor in squamous cell carcinoma of the head and neck (SCCHN) to evaluate folate receptor as a target for nanotherapy. Folate receptor expression levels in archival SCCHN tissues were analyzed by immunohistochemistry and correlated with clinical parameters. Folate receptor was detected in 45% of primary tumors and 40% of corresponding lymph node metastases. Folate receptor expression in primary tumors of the metastatic group strongly correlated with the corresponding lymph node metastases (p = .0002). Folate receptor expression was inversely correlated with disease-free survival in nonmetastatic (p = .0048), metastatic (p = .0127), and lymph node metastases (p <.001) groups, and with overall survival in the lymph node metastases group (p <.0001). Folate receptor is expressed in a significant proportion of primary SCCHN and corresponding lymph node metastases tissues, and correlates with worse clinical outcome. These findings provide support for folate receptor-mediated nanotherapeutics in SCCHN.

Down‐regulation of S100A2 in lymph node metastases of head and neck cancer

Our cDNA microarray analysis of squamous cell carcinoma of the head and neck (SCCHN) previously identified that S100A2 was down-regulated in highly metastatic 686LN-M3s cell lines established through in vivo selection using a metastatic xenograft mouse model. S100A2, a putative tumor suppressor, has been found to be down-regulated in several types of primary tumor as compared with the normal tissue. Only a few reports have explored its expression status and function in metastasis. To further confirm down-regulation of S100A2 in human metastasis, we examined S100A2 expression using immunohistochemical analysis of paraffin-embedded SCCHN tissues. The samples included primary SCCHN tumors (Tu-1) and involved lymph nodes (Met-1) from the same patients, and primary tumors in node-negative patients (Tu-2). Most of these tumors expressed S100A2 but lymph node metastases showed a pattern of reduced staining for S100A2 compared with primary tumors. A similar expression pattern of S100A2 was also observed in several SCCHN cell lines by reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting. Particularly, S100A2 expression was lower in 686LN than Tu686 and hardly detectable in the metastatic derivatives 686LN-M3s. Further study of S100A2 promoter showed higher methylation intensity in these metastatic derivatives than in Tu686 and 686LN. S100A2 was down-regulated in lymph node metastasis of SCCHN, suggesting that instead of being a putative tumor suppressor, S100A2 may play a role in the metastasis of SCCHN.

Identification of differentially expressed genes in HPV-positive and HPV-negative oropharyngeal squamous cell carcinomas

Human papillomaviruses (HPVs) have been implicated in the pathogenesis of a subset of squamous cell carcinoma of the head and neck (SCCHN). The goal of this study was to compare the cellular gene expression profiles of HPV-positive and HPV-negative oropharyngeal carcinomas with those of the normal oral epithelium. Using Affymetrix Human U133A GeneChip, our results showed that 397 genes were differentially expressed in HPV-positive SCCHN compared to the normal oral epithelium. The upregulated genes included those involved in cell cycle regulation ( CDKN2A), cell differentiation ( SFRP4) and DNA repair ( RAD51AP1), while the downregulated genes included those involved in proteolysis ( PRSS3). We also found 162 differentially expressed genes in HPV-negative SCCHN compared to the normal oral mucosa. The upregulated genes included those involved in cell proliferation ( AKR1C3) and transcription regulation ( SNAPC1), while downregulated genes included those involved in apoptosis ( CLU) and RNA processing ( RBM3). Our studies also identified a subgroup of 59 differentially expressed genes in HPV-positive SCCHN as compared to both HPV-negative SCCHN and normal oral tissues. Such upregulated genes included those involved in nuclear structure and meiosis ( SYCP2), DNA repair ( RFC5), and transcription regulation ( ZNF238). Genes involved in proteolysis ( KLK8) and signal transduction ( CRABP2) were found to be downregulated in HPV-positive SCCHN. The results of GeneChip experiments were validated by quantitative real-time RT-PCR analysis of a few representative genes. Our results reveal specific gene expression patterns in HPV-positive and HPV-negative oropharyngeal squamous carcinomas that may serve as potential biomarkers for the development of SCCHN.

Associations betweenXPC expression, genotype, and the risk of head and neck cancer

Squamous cell carcinoma of the head and neck (SCCHN) is a relatively rare cancer with a poor prognosis. In the present study, we investigated susceptibility biomarkers for SCCHN in 155 Koreans (73 SCCHN cases and 82 controls). Expression of the xeroderma pigmentosum group C (XPC) DNA-repair gene was measured by TaqMan fluorogenic real-time RT-PCR using RNA isolated from peripheral blood samples. In addition, the XPC-PAT genotype [an intronic and biallelic poly(AT) insertion/deletion polymorphism] was determined by PCR of peripheral blood DNA. Among known environmental and physical risk factors, age, gender, tobacco smoking, and alcohol consumption were associated with the SCCHN risk (P < 0.01). Lower XPC expression levels were found for the SCCHN cases, particularly in the larynx, than for the controls (P < 0.05); the geometric means (standard deviations) of XPC expression normalized by 18S rRNA in the cases and controls were 5.89 (8.13) and 15.14 (9.77), respectively. However, the distributions of the XPC-PAT genotypes were similar in the cases and controls. In addition, there were no associations between the expression of XPC and the PAT genetic polymorphism. XPC expression was not affected by age, gender, tobacco smoking, or alcohol consumption. After adjusting for SCCHN-associated lifestyle factors, the effective strength of XPC expression on SCCHN risk was weaker (0.05 < P < 0.1) than those of tobacco smoking and age (P < 0.05). There was a positive association between XPC mRNA expression in blood and SCCHN tissues (n = 9; P < 0.01); therefore, peripheral blood appears to be a reasonable surrogate tissue for XPC expression in tumor tissue. In conclusion, the XPC-PAT polymorphism had no effect on XPC expression or SCCHN risk. However, XPC expression may influence SCCHN risk.