Challenges in EGFRvIII Detection in Head and Neck Squamous Cell Carcinoma

ObjectiveHead and neck squamous cell carcinoma (HNSCC) accounts for more than 5% of all cancers worldwide. The mortality rate of HNSCC has remained unchanged (approximately 50%) over the last few decades. Ubiquitous overexpression of wild type EGFR in many solid tumors has led to the development of EGFR targeted therapies. EGFR can be constitutively activated via several mechanisms including the truncated, EGFR variant III isoform (EGFRvIII). EGFRvIII lacks exons 2–7 and has been reported to be present in up to 20–40% of HNSCC. EGFRvIII has been shown to contribute to cetuximab resistance. The mechanisms leading to EGFRvIII expression in HNSCC are unknown. The present investigation was undertaken to determine the etiology of EGFRvIII in HNSCC.ResultsUnlike glioma, EGFRvIII expression in HNSCC did not correlate with EGFR amplification. We found evidence of genomic deletion of the exon 2–7 in 6 of 7 HNSCC cases examined, however, the presence of genomic deletion did not always result in mRNA expression of EGFRvIII. RNA sequencing with automated alignment did not identify EGFRvIII due to microhomology between intron 1 and exon 8. RNA sequencing analyzed by manual alignment methods did not correlate well with RT-PCR and PCR findings.ConclusionThese findings suggest that genomic deletion as well as additional regulatory mechanisms may contribute to EGFRvIII expression in HNSCC. Further, large scale automated alignment of sequencing are unlikely to identify EGFRvIII and an assay specifically designed to detect EGFRvIII may be necessary to detect this altered form of EGFR in HNSCC tumors.     

Decitabine Rescues Cisplatin Resistance in Head and Neck Squamous Cell Carcinoma

Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) reduces survival. In this study we hypothesized that methylation of key genes mediates cisplatin resistance. We determined whether a demethylating drug, decitabine, could augment the anti-proliferative and apoptotic effects of cisplatin on SCC-25/CP, a cisplatin-resistant tongue SCC cell line. We showed that decitabine treatment restored cisplatin sensitivity in SCC-25/CP and significantly reduced the cisplatin dose required to induce apoptosis. We then created a xenograft model with SCC-25/CP and determined that decitabine and cisplatin combination treatment resulted in significantly reduced tumor growth and mechanical allodynia compared to control. To establish a gene classifier we quantified methylation in cancer tissue of cisplatin-sensitive and cisplatin-resistant HNSCC patients. Cisplatin-sensitive and cisplatin-resistant patient tumors had distinct methylation profiles. When we quantified methylation and expression of genes in the classifier in HNSCC cells in vitro, we showed that decitabine treatment of cisplatin-resistant HNSCC cells reversed methylation and gene expression toward a cisplatin-sensitive profile. The study provides direct evidence that decitabine restores cisplatin sensitivity in in vitro and in vivo models of HNSCC. Combination treatment of cisplatin and decitabine significantly reduces HNSCC growth and HNSCC pain. Furthermore, gene methylation could be used as a biomarker of cisplatin-resistance.     

Head and Neck Squamous Cell Carcinoma: Prognosis using molecular approach

Head and neck squamous cell carcinoma (HNSCC) is the fifth most prevalent cancer worldwide. Apart from various known clinicopathogical factors, it is still a major concern as many genetic and epigenetic alterations bring about the possibility of this deadly disease. The aim of this review is to explore the possible role of DNA repair pathways and the polymorphic status of DNA repair genes (XPA, XPC, XPD, XRCC1 and XRCC3) in the onset of HNSCC, along with sequence variations in genes such as Glutathione S-transferases (GSTT1, M1 and P1) that are significantly associated with HNSCC risk. We also focus on the p53 gene mutation induced by various etiological agents and threat factors with its implications towards HNSCC, and emphasise the current therapeutic interventions in treating HNSCC.     

Therapeutic strategies of different HPV status in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the 9^th most common malignant tumor in the world. Based on the etiology, HNSCC has two main subtypes: human papillomavirus (HPV) -related and HPV-unrelated. HPV-positive HNSCC is more sensitive to treatment with favorable survival. Due to the different biological behaviors, individual therapy is necessary and urgently required to deduce the therapeutic intensity of HPV-positive disease and look for a more effective and toxicity-acceptable regimen for HPV-negative disease. EGFR amplification and PI3K/AKT/mTOR pathway aberrant activation are quite common in HPV-positive HNSCC. Besides, HPV infection alters immune cell infiltrating in HNSCC and encompasses a diverse and heterogeneous landscape with more immune infiltration. On the other hand, the chance of HPV-negative cancers harboring mutation on the P53 gene is significantly higher than that of HPV-positive disease. This review focuses on the updated preclinical and clinical data of HPV-positive and HPV-negative HNSCC and discusses the therapeutic strategies of different HPV status in HNSCC.     

头颈部鳞状细胞癌相关肿瘤标志物的研究进展

头颈部鳞状细胞癌(head and neck squamous cell carcinoma,HNSCC)是头颈部恶性肿瘤的主要病理类型,约占所有头颈部肿瘤的90%。而据我们临床所见,大约有70%~80%的患者就诊时已为局部晚期,其治疗效果欠佳,预后差。肿瘤标志物又叫做肿瘤标记物,是指特征性存在于恶性肿瘤细胞,或是由恶性肿瘤细胞异常而产生的物质,或是宿主对于肿瘤的刺激反应而产生的物质,并且能够反映肿瘤发生、发展,以及监测肿瘤对治疗反应的一类物质。作为近年来研究热点的肿瘤标志物,具有简便、经济、快速、无创的特点,更重要的是一些标志物在组织器官发生形态学变化之前就有表达。因此,肿瘤标志物的研究对头颈部鳞状细胞癌的早期诊断以及判断预后都具有十分重要的意义。本文综述近几年来发现的可能与头颈部鳞状细胞癌的发生发展或者预后相关的肿瘤标志物。     

Prognostic Value of Tumor-Infiltrating Lymphocytes for Patients With Head and Neck Squamous Cell Carcinoma

BACKGROUND: The prognostic value of tumor-infiltrating lymphocytes (TILs) in head and neck squamous cell carcinoma (HNSCC) remains controversial. Additionally, there is no standardized approach or cutoff value for evaluating TIL levels. The aim of this study was to establish a feasible method and criterion to assess TIL levels for future clinical practice and research use and to explore the relationship between TIL levels and prognosis. PATIENTS AND METHODS: This retrospective cohort study reviewed the records and pathological sections of 202 patients with HNSCC who were surgically treated at Beijing Stomatological Hospital, Capital Medical University, from January 1998 to January 2011. The predictor variable was the TIL level. The main outcome assessment parameters were disease-free survival (DFS) and disease-specific survival (DSS). RESULT: The T stage (P = .008), smoking history (P = .042), alcohol history (P = .048), need for radiotherapy (P = .012) and microscopic extracapsular spread (ECS) (P = .012) were associated with the TIL level. A cutoff value equal to 70% could be taken as a threshold for TIL assessment, with a TIL level higher than 70% associated with a better prognosis (DFS rate: 51.9%, P = .018; DSS rate: 59.3%, P = .049). The Cox regression model showed that the TIL level was an independent prognostic factor for DFS (hazard ratio (HR): 0.786, 95% CI: 0.618-0.999, P = .049). CONCLUSION: The TIL level is closely related to the prognosis of patients with HNSCC. A threshold value of 70% is appropriate for TIL assessment, as patients with a TIL level higher than 70% show a better prognosis. Thus, the TIL level might serve as an independent predictor for HNSCC recurrence.     

Nicotine Promotes Acquisition of Stem Cell and Epithelial-to-Mesenchymal Properties in Head and Neck Squamous Cell Carcinoma

The ability of nicotine to enhance the malignancy of cancer cells is known; however, the possibility that nicotine could regulate a cancer stem cell phenotype remains to be well-established. In this study we sought to determine whether long-term exposure to nicotine could promote cancer stem cell-like properties in two head and neck squamous cell carcinoma cell lines, UMSCC-10B and HN-1. Nicotine treatment induced epithelial-to-mesenchymal transition (EMT) in both cell lines by repressing E-cadherin expression, and led to the induction of stem cell markers Oct-4, Nanog, CD44 and BMI-1, which was reversed upon ectopic re-expression of E-cadherin. Nicotine-treated cells formed spheres at a higher efficiency than non-treated cells, formed larger tumors when injected into mice, and formed tumors with 4-fold greater efficiency compared to control cells when injected at limiting doses. Consistent with previous literature, nicotine-treated cells demonstrated a greater capacity for survival and also a higher tendency to invade. Comparison of microRNA profiles between nicotine and control cells revealed the upregulation of miR-9, a repressor of E-cadherin, and the downregulation of miR-101, a repressor of EZH2. Taken together, these results suggest that nicotine may play a critical role in the development of tobacco-induced cancers by regulating cancer stem cell characteristics, and that these effects are likely mediated through EMT-promoting, microRNA-mediated pathways. Further characterization of such pathways remains a promising avenue for the understanding and treatment of tobacco-related cancers.     

Upregulation of SQLE Contributes to Poor Survival in Head and Neck Squamous Cell Carcinoma

Recently, increasing attention has been paid to the role of Squalene epoxidase (SQLE) in several types of cancers. However, its functional role in tumor progression of head and neck squamous cell carcinoma (HNSCC) is still unclear. We performed bioinformatic analyses and relative experiments to assess the potential mechanism of SQLE-mediated HNSCC malignancy. And the results showed that SQLE was significantly upregulated in tumor samples compared with peritumor samples. Mechanistically, miR-584-5p downregulation may lead to the upregulation of SQLE in HNSCC. Moreover, high SQLE expression in HNSCC was associated with TNM stage, distant metastasis, and poor survival, indicating that SQLE be involved in the progression of HNSCC. Furtherly, SQLE boosted proliferation, migration, invasion of HNSCC cells in vitro and in vivo. Bioinformatic studies showed that PI3K/Akt signaling participated in HNSCC progression mediated by SQLE overexpression, which is confirmed by in vitro and in vivo analysis. Particularly, treatment with terbinafine, an inhibitor of SQLE widely used in the treatment of fungal infections, showed a therapeutic influence on HNSCC. Our findings demonstrate that SQLE plays a vital role in HNSCC progression, providing research evidence for SQLE as a prospective HNSCC therapeutic target and for terbinafine as a candidate drug of HNSCC treatment in the future     

Correlations Between DCE MRI and Histopathological Parameters in Head and Neck Squamous Cell Carcinoma

BACKGROUND: Dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) can characterize perfusion and vascularization of tissues. DCE MRI parameters can differentiate between malignant and benign lesions and predict tumor grading. The purpose of this study was to correlate DCE MRI findings and various histopathological parameters in head and neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: Sixteen patients with histologically proven HNSCC (11 cases primary tumors and in 5 patients with local tumor recurrence) were included in the study. DCE imaging was performed in all cases and the following parameters were estimated: K^trans, V_e, K_ep, and iAUC. The tumor proliferation index was estimated on Ki 67 antigen stained specimens. Microvessel density parameters (stained vessel area, total vessel area, number of vessels, and mean vessel diameter) were estimated on CD31 antigen stained specimens. Spearman''s non-parametric rank sum correlation coefficients were calculated between DCE and different histopathological parameters. RESULTS: The mean values of DCE perfusion parameters were as follows: K^trans 0.189 ± 0.056 min⁻¹, K_ep 0.390 ± 0.160 min⁻¹, V_e 0.548 ± 0.119%, and iAUC 22.40 ± 12.57. Significant correlations were observed between K_ep and stained vessel areas (r = 0.51, P = .041) and total vessel areas (r = 0.5118, P = .043); between V_e and mean vessel diameter (r = −0.59, P = .017). Cell count had a tendency to correlate with V_e (r = −0.48, P = .058). In an analysis of the primary HNSCC only, a significant inverse correlation between K^trans and KI 67 was identified (r = −0.62, P = .041). Our analysis showed significant correlations between DCE parameters and histopathological findings in HNSCC.Dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) has been reported as a technique which is able to characterize perfusion and vascularization of tissues [1], [2]. It has been shown that DCE MRI can be helpful to differentiate between malignant and benign lesions [1]. For example, Yuan et al. reported that lung cancer had a larger volume transfer constant (K^trans) and a lower volume of the extravascular extracellular leakage space (V_e) in comparison to benign lesions [3]. Similar results were reported by Li et al. for breast lesions [4]. Furthermore, according to Cho et al., DCE MRI parameters can be used to distinguish prostatic cancer from benign changes [5]. Moreover, DCE MRI parameters can also predict tumor grading. As reported previously, K^trans correlated well with Gleason score in prostatic cancer [5], [6]. According to other reports, K^trans and V_e correlated with glioma grade [7], [8].DCE MRI parameters were also associated with prognosis in several malignancies [9], [10]. Koo et al. showed that breast cancers with higher K^trans or lower V_e had poor prognostic factors and were often of the triple-negative subtype [10].According to the literature, DCE MRI parameters can predict response to therapy in different tumors. For instance, some authors mentioned that low pretreatment K^trans in regional lymph node metastases in head and neck cancer was associated with a poor response to concurrent chemoradiation therapy [11].Furthermore, Andersen et al. showed that DCE MR parameters obtained prior to chemoradiotherapy predicted survival of patients with cervical cancer [12].Presumably, DCE MRI parameters may be based on tissue composition, such as cellularity and vascular density. However, in this regard there are contradictory data in the literature. While some studies identified significant correlations between DCE MRI and histopathological parameters, others did not [13], [14], [15], [16].The purpose of this study was to correlate DCE MRI findings and various histopathological parameters in head and neck squamous cell carcinoma (HNSCC).     

mTOR信号通路及其在头颈肿瘤研究中的进展

哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)在肿瘤的发生、侵袭、转移,甚至肿瘤的局部复发都起着重要的作用。头颈肿瘤作为全球最常见的癌症死亡原因之一,有多元的逐步恶化的过程。近年来研究表明m TOR信号通路的异常表达是头颈肿瘤最常见的病理改变之一,且m TOR信号通路的多种组成元件与头颈肿瘤患者的预后有明显的相关性,这不仅可为头颈肿瘤的特异性靶向治疗提供依据,而且也可为预测头颈肿瘤患者的预后提供参考。本文就m TOR信号通路及其在头颈肿瘤中的作用做一综述,旨在对m TOR信号通路和头颈肿瘤及其预后的关系有更进一步的认识。     

Clinical and genomic characterization of neutral tumor evolution in Head and Neck Squamous Cell Carcinoma

Recent studies suggest that a significant proportion of cancers undergo neutral tumor evolution. We applied neutral evolution model in HNSCC patients from The Cancer Genome Atlas (TCGA). To ensure the accuracy of classification results, a sample with the purity of tumor <0.7 was excluded. A tumor sample was considered to evolve neutrally if R² ≥ 0.98. We found that about 16% of HNSCC patients undergo neutral tumor evolution. We showed that neutral evolution HNSCC patients have better prognosis and higher activities of immune response pathways, and the numbers of co-occurring mutation events and significantly positive selection mutations are significantly less than non-neutral evolution HNSCC patients. In conclusion, we described a comprehensive clinical and genomic characteristics of neutral tumor evolution in Head and Neck Squamous Cell Carcinoma (HNSCC), and provided evidence that the evolution history of HNSCC has both clinical and biological implications.     

Inverse Immunological Responses Induced by Allergic Rhinitis and Head and Neck Squamous Cell Carcinoma

Several epidemiological studies have investigated the relation between allergy and cancer with contradicting conclusions, and reports on immunological differences are scarce. By focusing on inflammation, the present study was designed to compare the immune response induced by allergic rhinitis (AR) and head and neck squamous cell carcinoma (HNSCC). Blood and serum was obtained from patients with symptomatic seasonal AR, and newly detected HNSCC, as well as healthy controls. Peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMN) were isolated and cultured with or without the toll-like receptor ligands, Pam₃CSK₄, LPS, R837, and CpG. Cellular activation and cytokine release were assessed with ELISA, Luminex Multiplex Immunoassay, flow cytometry, and real-time RT-PCR. Sera from HNSCC patients showed elevated levels of innate immune cytokines, and exhibited a response profile consistent with an increased innate immune reaction. In contrast, sera and stimulated PBMC from AR patients displayed increased concentrations of T cell related cytokines, consistent with an adaptive immune response. The presented data demonstrate that AR and HNSCC induce two distinct immunological processes, indicating an inverse association between the immunological responses seen in patients with allergy and cancer of the upper airway.     

Deguelin Induces Both Apoptosis and Autophagy in Cultured Head and Neck Squamous Cell Carcinoma Cells

Head and neck squamous cell carcinoma (HNSCC) represents more than 5% of all cancers diagnosed annually in United States and around the world. Despite advances in the management of patients with this disease, the survival has not been significantly improved, and the search for potential alternative therapies is encouraging. Here we demonstrate that deguelin administration causes a significant HNSCC cell death. Deguelin induces both cell apoptosis and autophagy by modulating multiple signaling pathways in cultured HNSCC cells. Deguelin inhibits Akt signaling, and down-regulates survivin and cyclin-dependent kinase 4 (Cdk4) expressions, by disrupting their association with heat shock protein-90 (Hsp-90). Deguelin induces ceramide production through de novo synthase pathway to promote HNSCC cell death. Importantly, increased ceramide level activates AMP-activated protein kinase (AMPK), which then directly phosphorylates Ulk1 and eventually leads to cell autophagy. We found that a low dose of deguelin sensitized HNSCC cells to 5-FU. Finally, using a nude mice Hep-2 xenograft model, we also showed a significant anti-tumor ability of deguelin in vivo. Together, we suggest that deguelin may represent a novel and effective chemo-agent against HNSCC.     

Inactivation of p16INK4a in Primary Tumors and Cell Lines of Head and Neck Squamous Cell Carcinoma

Inactivation of the p16^INK4a gene by mutation and deletion is common in head and neck squamous cell carcinoma (HNSCC). The present study demonstrates that hypermethylation of the 5 CpG islands can serve as an alternative mechanism for the inactivation of the p16^INK4a gene in this tumor. We studied 11 HNSCC cell lines and 17 oral squamous cell carcinoma (OSCC) primary tumors for p16^INK4a gene status by protein/mRNA and DNA genetic/epigenetic analyses to determine the incidence of its inactivation. Our study indicates that: (1) inactivation of p16 protein is frequent in HNSCC cell lines (6/11, 54.5%) and OSCC primary tumors (15/17, 88.2%), (2) inactivation of p16^INK4a protein is commonly associated with the presence of gene alteration such as mutation, homozygous deletion and especially aberrant methylation, and (3) genomic sequencing of bisulfite-modified DNA shows that the carcinoma develops a heterogeneous pattern of hypermethylation.     

Epigenetic and Genetic Alterations Affect the WWOX Gene in Head and Neck Squamous Cell Carcinoma

Different types of genetic and epigenetic changes are associated with HNSCC. The molecular mechanisms of HNSCC carcinogenesis are still undergoing intensive investigation. WWOX gene expression is altered in many cancers and in a recent work reduced WWOX expression has been associated with miR-134 expression in HNSCC. In this study we investigated the WWOX messenger RNA expression levels in association with the promoter methylation of the WWOX gene and miR-134 expression levels in 80 HNSCC tumor and non-cancerous tissue samples. Our results show that WWOX expression is down-regulated especially in advanced-stage tumor samples or in tumors with SCC. This down-regulation was associated with methylation of the WWOX promoter region but not with miR-134 expression. There was an inverse correlation between the expression level and promoter methylation. We also analyzed whole exons and exon/intron boundries of the WWOX gene by direct sequencing. In our study group we observed 10 different alterations in the coding sequences and 18 different alterations in the non-coding sequences of the WWOX gene in HNSCC tumor samples. These results indicate that the WWOX gene can be functionally inactivated by promoter methylation, epigenetically or by mutations affecting the sequences coding for the enzymatic domain of the gene, functionally. We conclude that inactivation of WWOX gene contributes to the progression of HNSCC.     

Bitter Melon Reduces Head and Neck Squamous Cell Carcinoma Growth by Targeting c-Met Signaling

Head and neck squamous cell carcinoma (HNSCC) remains difficult to treat, and despite of advances in treatment, the overall survival rate has only modestly improved over the past several years. Thus, there is an urgent need for additional therapeutic modalities. We hypothesized that treatment of HNSCC cells with a dietary product such as bitter melon extract (BME) modulates multiple signaling pathways and regresses HNSCC tumor growth in a preclinical model. We observed a reduced cell proliferation in HNSCC cell lines. The mechanistic studies reveal that treatment of BME in HNSCC cells inhibited c-Met signaling pathway. We also observed that BME treatment in HNSCC reduced phosphoStat3, c-myc and Mcl-1 expression, downstream signaling molecules of c-Met. Furthermore, BME treatment in HNSCC cells modulated the expression of key cell cycle progression molecules leading to halted cell growth. Finally, BME feeding in mice bearing HNSCC xenograft tumor resulted in an inhibition of tumor growth and c-Met expression. Together, our results suggested that BME treatment in HNSCC cells modulates multiple signaling pathways and may have therapeutic potential for treating HNSCC.     

Decreased Plakophilin-1 Expression Promotes Increased Motility in Head and Neck Squamous Cell Carcinoma Cells

Desmosomes are prominent cell-cell adhesive junctions found in a variety of epithelial tissues, including the oral epithelium. The transmembrane core of the desmosome is composed of the desmosomal cadherins that interact extracellularly to mediate cell-cell adhesion. The cytoplasmic domain of desmosomal cadherins interact with plaque proteins that in turn interact with the keratin intermediate filament cytoskeleton. Plakophilin 1 is a major desmosomal plaque component that functions to recruit intermediate filaments to sites of cell-cell contact via interactions with desmoplakin. Decreased assembly of desmosomes has been reported in several epithelial cancers. We examined plakophilin-1 expression in an esophageal squamous cell carcinoma tissue microarray and found that plakophilin-1 expression inversely correlates with tumor grade. In addition, we sought to investigate the effect of plakophilin-1 expression on desmosome assembly and cell motility in oral squamous cell carcinoma cell lines. Cell lines expressing altered levels of plakophilin-1 were generated and the ability of these cells to recruit desmoplakin to sites of cell-cell contact was examined. Our results show that decreased expression of plakophilin-1 results in decreased desmosome assembly and increased cell motility and invasion. These data lead us to propose that loss of plakophilin-1 expression during head and neck squamous cell carcinoma (HNSCC) progression may contribute to an invasive phenotype.     

Investigation of 6-[18F]-Fluoromaltose as a Novel PET Tracer for Imaging Bacterial Infection

Despite advances in the field of nuclear medicine, the imaging of bacterial infections has remained a challenge. The existing reagents suffer from poor sensitivity and specificity. In this study we investigate the potential of a novel PET (positron emission tomography) tracer that overcomes these limitations.

Methods

6-[¹⁸F]-fluoromaltose was synthesized. Its behavior in vitro was evaluated in bacterial and mammalian cultures. Detailed pharmacokinetic and biodistribution profiles for the tracer were obtained from a murine model.

Results

6-[¹⁸F]-fluoromaltose is taken up by multiple strains of pathogenic bacteria. It is not taken up by mammalian cancer cell lines. 6-[¹⁸F]-fluoromaltose is retained in infected muscles in a murine model of bacterial myositis. It does not accumulate in inflamed tissue.

Conclusion

We have shown that 6-[¹⁸F]-fluoromaltose can be used to image bacterial infection in vivo with high specificity. We believe that this class of agents will have a significant impact on the clinical management of patients.