The Activation Pattern of Blood Leukocytes in Head and Neck Squamous Cell Carcinoma Is Correlated to Survival

Head and neck squamous cell carcinoma (HNSCC) is known to cause substantial immunosuppression. The present study was designed to characterize blood leukocyte activation in HNSCC and to investigate if the individual activation pattern could be related to tumor progress and survival. The leukocyte activation profile of HNSCC patients and healthy controls was assessed with flow cytometry. HNSCC patients displayed increased numbers of monocytes, neutrophils and total leukocytes as well as an enhanced neutrophil/lymphocyte ratio. In addition, patients had a higher percentage of CD69⁺, CD71⁺ and CD98⁺ T cell subsets and NK cells, and a reduced expression of L-selectin in CD14^highCD16⁺ monocytes and neutrophils, when compared to controls. These changes could be correlated to both tumor burden and spread to lymph nodes. Among the cancer patients an increased neutrophil/lymphocyte ratio, a low neutrophil and CD14^high CD16⁺ monocyte activation state and an elevated CD4/CD8 ratio were related to poor survival. In contrast, a high percentage of CD98⁺ Th cells appeared to be associated with a better outcome. Taken together, the present data indicate that HNSCC causes activation of blood leukocytes and that the individual activation pattern can be linked to prognosis.     

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     

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.     

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.     

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.     

Slug Is a Predictor of Poor Prognosis in Esophageal Squamous Cell Carcinoma Patients

Background

Slug, a regulator of epithelial mesenchymal transition, was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. This study aimed to determine the clinical significance of Slug overexpression in ESCC and determine its correlation with clinicopathological parameters and disease prognosis for ESCC patients.

Methods

Immunohistochemical analysis of Slug expression was carried out in archived tissue sections from 91 ESCCs, 61 dysplastic and 47 histologically normal esophageal tissues. Slug immunopositivity in epithelial cells was correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients.

Results

Increased expression of Slug was observed in esophageal dysplasia [cytoplasmic, 24/61 (39.3%) cases, p = 0.001, odd’s ratio (OR) = 4.7; nuclear, 11/61 (18%) cases, p < 0.001, OR = 1.36] in comparison with normal esophageal tissues. The Slug expression was further increased in ESCCs [cytoplasmic, 64/91 (70.3%) p < 0.001, OR = 10.0; nuclear, 27/91 (29.7%) p < 0.001, OR = 1.42]. Kaplan Meier survival analysis showed significant association of nuclear Slug accumulation with reduced disease free survival of ESCC patients (median disease free survival (DFS) = 6 months, as compared to those that did not show overexpression, DFS = 18 months; p = 0.006). In multivariate Cox regression analysis nuclear Slug expression [p= 0.005, Hazard’s ratio (HR) = 2.269, 95% CI = 1.289 - 3.996] emerged as the most significant independent predictor of poor prognosis for ESCC patients.

Conclusions

Alterations in Slug expression occur in early stages of development of ESCC and are sustained during disease progression. Slug may serve as a diagnostic biomarker and as a predictor of poor disease prognosis to identify ESCC patients that are likely to show recurrence of the disease.     

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。     

Markers of Epithelial to Mesenchymal Transition in Association with Survival in Head and Neck Squamous Cell Carcinoma (HNSCC)

Background

Elucidating the molecular phenotype of cancers with high metastatic potential will facilitate the development of novel therapeutic approaches to the disease. Gene expression profiles link epithelial to mesenchymal transition (EMT) phenotype with high-risk HNSCC. We sought to determine the role of protein biomarkers of EMT in head and neck squamous carcinoma (HNSC) prognosis.

Methods

Protein expression analysis of EGFR, β-catenin and E-cadherin was performed on a cohort of 102 patients with HNSCC recruited between 1992 and 2005 using automated quantitative protein analysis (AQUA). We evaluated associations with clinicopathological parameters and prognosis.

Results

There were 67 patients with primary squamous cell carcinoma of the head and neck in this cohort who met inclusion criteria and for whom we had complete E-cadherin, beta-catenin and EGFR expression data. High E-cadherin expressers had longer 5-year progression-free survival (PFS) compared to those with low E-cadherin (59.7% versus 40.6%, p = 0.04) and overall survival (OS) (69.6% versus 44.3%, p  = 0.05). Kaplan-Meier analysis showed that patients with low beta-catenin-expressing tumors trended toward worse 5-year PFS (p = 0.057). High EGFR expressers had inferior OS compared to low EGFR expressers (27.7% vs. 54%, p = 0.029). In the multivariable analysis context, E-cadherin remained an independent predictor of improved OS (HR = 0.204, 95% CI 0.043 to 0.972, p = 0.046) while EGFR trended towards significance for OS.

Conclusions

The putative markers of EMT defined within a panel of HNSCC using AQUA are associated with tumors of poor prognosis.     

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.     

Human Papillomavirus Infection in 674 Chinese Patients with Laryngeal Squamous Cell Carcinoma

Objectives

Previous reports suggest a strong association between human papillomavirus (HPV) and the etiology of laryngeal squamous cell carcinoma (LSCC). However, clinical data regarding the HPV infection rate among LSCC patients remain largely inconsistent.

Methods

In total, 674 LSCC patients from three major hospitals in Shanghai were enrolled in this study. We determined the patients'' HPV infection status using immunohistochemistry and the GenoArray HPV genotyping assay and calculated their long-term survival rate using the Kaplan-Meier method.

Results

The total P16-positive rate according to immunostaining results was 7.57% (51/674). None of the P16-negative patients were HPV-positive according to the HPV genotyping test. The rate of HPV infection among patients with LSCC was 4.9% (33/674). HPV infection was more common among nonsmokers (P<0.05), nondrinkers (P<0.05), and patients with supraglottic LSCC (P<0.05). Of the 33 HPV-positive patients, 28 (84.8%) were infected with HPV-16, 2 with HPV-18, 1 with HPV-31, 1 with HPV-33 and 1 with HPV-45. The 3-year overall survival rate and progression-free survival rate were higher in HPV-positive than HPV-negative patients, but the difference was not statistically significant (76.3% vs. 70.7%, P = 0.30 and 65.1% vs. 58.3%, P = 0.37, respectively).

Conclusion

HPV was not a main causal factor in LSCC carcinogenesis in this Chinese population. HPV infection did not alter patients'' overall survival or progression-free survival rates in this study.     

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。     

High SEPT9_v1 Expression Is Associated with Poor Clinical Outcomes in Head and Neck Squamous Cell Carcinoma

The purpose of this work was to determine SEPT9_v1 expression levels in head and neck squamous cell carcinoma (HNSCC) and to analyze whether SEPT9_v1 expression is relevant to clinical outcomes. Recently, the SEPT9 isoform SEPT9_v1 has been implicated in oncogenesis, and methylation of the SEPT9 promoter region was reported in HNSCC. These findings led us to hypothesize that SEPT9_v1 could be differently expressed in HNSCC. To determine whether SEPT9_v1 is expressed in HNSCC, tissue microarray immunohistochemical analysis was performed using a SEPT9_v1-specific antibody. Tissue microarrays stained with a polyclonal SEPT9_v1-specific antibody was used to determine protein expression levels in HNSCC tissue samples, some with known clinical outcomes. This analysis showed that SEPT9_v1 is in fact highly expressed in HNSCC compared with normal epithelium, and high expression levels directly correlated with poor clinical outcomes. Specifically, a high SEPT9_v1 expression was associated with decreased disease-specific survival (P = .012), time to indication of surgery at primary site (P = .008), response to induction chemotherapy (P = .0002), and response to chemotherapy (P = .02), as well as advanced tumor stage (P = .012) and N stage (P = .0014). The expression of SEPT9_v1 was also strongly correlated with smoking status (P = .00094). SEPT9_v1 is highly expressed in HNSCC, and a high expression of SEPT9_v1 is associated with poor clinical outcomes. These data indicate that SEPT9_v1 warrants additional investigation as a potential biomarker for HNSCC.