HPV & head and neck cancer: a descriptive update

The incidence of head and neck squamous cell carcinoma (HNSCC) has been gradually increasing over the last three decades. Recent data have now attributed a viral aetiology to a subset of head and neck cancers. Several studies indicate that oral human papillomavirus (HPV) infection is likely to be sexually acquired. The dominance of HPV 16 in HPV+ HNSCC is even greater than that seen in cervical carcinoma of total worldwide cases. Strong evidence suggests that HPV+ status is an important prognostic factor associated with a favourable outcome in head and neck cancers. Approximately 30 to 40% of HNSCC patients with present with early stage I/II disease. These patients are treated with curative intent using single modality treatments either radiation or surgery alone. A non-operative approach is favored for patients in which surgery followed by either radiation alone or radiochemotherapy may lead to severe functional impairment. Cetuximab, a humanized mouse anti-EGFR IgG1 monoclonal antibody, improved locoregional control and overall survival in combination with radiotherapy in locally advanced tumours but at the cost of some increased cardiac morbidity and mortality. Finally, the improved prognosis and treatment responses to chemotherapy and radiotherapy by HPV+ tumours may suggest that HPV status detection is required to better plan and individualize patient treatment regimes.

5-Methyltetrahydrofolate-homocysteine methyltransferase gene polymorphism (MTR) and risk of head and neck cancer

The functional effect of the A>G transition at position 2756 on the MTR gene (5-methyltetrahydrofolate-homocysteine methyltransferase), involved in folate metabolism, may be a risk factor for head and neck squamous cell carcinoma (HNSCC). The frequency of MTR A2756G (rs1805087) polymorphism was compared between HNSCC patients and individuals without history of neoplasias. The association of this polymorphism with clinical histopathological parameters was evaluated. A total of 705 individuals were included in the study. The polymerase chain reaction-restriction fragment length polymorphism technique was used to genotype the polymorphism. For statistical analysis, the chi-square test (univariate analysis) was used for comparisons between groups and multiple logistic regression (multivariate analysis) was used for interactions between the polymorphism and risk factors and clinical histopathological parameters. Using univariate analysis, the results did not show significant differences in allelic or genotypic distributions. Multivariable analysis showed that tobacco and alcohol consumption (P < 0.05), AG genotype (P = 0.019) and G allele (P = 0.028) may be predictors of the disease and a higher frequency of the G polymorphic allele was detected in men with HNSCC compared to male controls (P = 0.008). The analysis of polymorphism regarding clinical histopathological parameters did not show any association with the primary site, aggressiveness, lymph node involvement or extension of the tumor. In conclusion, our data provide evidence that supports an association between the polymorphism and the risk of HNSCC.

Education, tobacco smoking, alcohol consumption, and IL-2 and IL-6 gene polymorphisms in the survival of head and neck cancer

The association of education, tobacco smoking, alcohol consumption, and interleukin-2 (IL-2 +114 and -384) and -6 (IL-6 -174) DNA polymorphisms with head and neck squamous cell carcinoma (HNSCC) was investigated in a cohort study of 445 subjects. IL-2 and IL-6 genotypes were determined by real-time PCR. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (95%CI) of disease-specific survival according to anatomical sites of the head and neck. Mean age was 56 years and most patients were males (87.6%). Subjects with 5 or more years of schooling had better survival in larynx cancer. Smoking had no effect on HNSCC survival, but alcohol consumption had a statistically significant effect on larynx cancer. IL-2 gene +114 G/T (HR = 0.52; 95%CI = 0.15-1.81) and T/T (HR = 0.22; 95%CI = 0.02-3.19) genotypes were associated with better survival in hypopharynx cancer. IL-2 +114 G/T was a predictor of poor survival in oral cavity/oropharynx cancer and larynx cancer (HR = 1.32; 95%CI = 0.61-2.85). IL-2 -384 G/T was associated with better survival in oral cavity/oropharynx cancer (HR = 0.80; 95%CI = 0.45-1.42) and hypopharynx cancer (HR = 0.68; 95%CI = 0.21-2.20), but an inverse relationship was observed for larynx cancer. IL-6 -174 G/C was associated with better survival in hypopharynx cancer (HR = 0.68; 95%CI = 0.26-1.78) and larynx cancer (HR = 0.93; 95%CI = 0.42-2.07), and C/C reduced mortality in larynx cancer. In general, our results are similar to previous reports on the value of education, smoking, alcohol consumption, and IL-2 and IL-6 genetic polymorphisms for the prognosis of HNSCC, but the risks due to these variables are small and estimates imprecise.

Impact of HPV infection on the development of head and neck cancer

Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is considered to be a distinct clinical entity with better prognosis than the classical tobacco- and alcohol-associated tumors. The increasing incidence of this neoplasia during the last decades highlights the need to better understand the role of HPV in the development of these cancers. Although the proportion of HNSCC attributed to HPV varies considerably according to anatomical site, overall approximately 25% of all HNSCC are HPV-DNA positive, and HPV-16 is by far the most prevalent type. In this review we discuss the existing evidence for a causal association between HPV infection and HNSCC at diverse anatomical head and neck subsites.

H19-DMR allele-specific methylation analysis reveals epigenetic heterogeneity of CTCF binding site 6 but not of site 5 in head-and-neck carcinomas: A pilot case-control analysis

Aberrant methylation of seven potential binding sites of the CTCF factor in the differentially methylated region upstream of the H19 gene (H19-DMR) has been suggested as critical for the regulation of IGF2 and H19 imprinted genes. In this study, we analyzed the allele-specific methylation pattern of CTCF binding sites 5 and 6 using methylationsensitive restriction enzyme PCR followed by RFLP analysis in matched tumoral and lymphocyte DNA from head-and-neck squamous cell carcinoma (HNSCC) patients, as well as in lymphocyte DNA from control individuals who were cancer-free. The monoallelic methylation pattern was maintained in CTCF binding site 5 in 22 heterozygous out of 91 samples analyzed. Nevertheless, a biallelic methylation pattern was detected in CTCF binding site 6 in a subgroup of HNSCC patients as a somatic acquired feature of tumor cells. An atypical biallelic methylation was also observed in both tumor and lymphocyte DNA from two patients, and at a high frequency in the control group (29 out of 64 informative controls). Additionally, we found that the C/T transition detected by HhaI RFLP suppressed one dinucleotide CpG in critical CTCF binding site 6, of a mutation showing polymorphic frequencies. Although a heterogeneous methylation pattern was observed after DNA sequencing modified by sodium bisulfite, the biallelic methylation pattern was confirmed in 9 out of 10 HNSCCs. These findings are likely to be relevant in the epigenetic regulation of the DMR, especially in pathological conditions in which the imprinting of IGF2 and H19 genes is disrupted.

DNA methylation in the CTCF-binding site I and the expression pattern of the H19 gene: Does positive expression predict poor prognosis in early stage head and neck carcinomas?

Loss of allele-specific expression by the imprinted genes IGF2 and H19 has been correlated with a differentially methylated region (DMR) upstream to the H19 gene. The H19-DMR contains seven potential CCCTC-binding factor (CTCF) binding sites. CTCF is a chromatin insulator and a multifunctional transcription factor whose binding to the H19-DMR is suppressed by DNA methylation. Our study included a group of 41 head and neck squamous cell carcinoma (HNSCC) samples. The imprinting status of the H19 gene was analyzed in 11 out of 35 positive cases for H19 gene expression, and only 1 of them showed loss of imprinting. We detected a significant correlation (P=0.041, Fisher's exact test) between H19 expression and tumor recurrence. Among H19 positive cases, six were T2, in which five developed recurrence and/or metastasis. Inversely, in the group of tumors that showed no H19 gene expression, 5 out of 24 were T2 and only I presented regional recurrence. These data support the hypothesis that H19 expression could be used as a prognostic marker to indicate recurrence in early stage tumors. We also examined the methylation of the CTCF binding site 1 in a subgroup of these samples. The H19 gene silencing and loss of imprinting were not correlated with the methylation pattern of the CTCF binding site 1. However, the significant correlation between H19 expression and tumor recurrence suggest that this transcript could be a marker for the progression of HNSCC. (c) 2005 Wiley-Liss, Inc.

LHX6 is a sensitive methylation marker in head and neck carcinomas

Head and neck cancer remains a morbid and often fatal disease and at the present time few effective molecular markers have been identified. The purpose of the present work was to identify new molecular markers for head and neck squamous cell carcinoma (HNSCC). We applied methylation-sensitive arbitrarily primed PCR (MS/APPCR) to isolate sequences differentially methylated in HNSCC. The most frequently hypermethylated fragment we found maps close to a cytosine guanine dinucleotide (CpG) island on chromosome 9q33.2, and hypermethylation of this CpG island was associated with transcriptional silencing of an alternative transcript of the LHX6 gene. Using combined bisulfite restriction analysis (COBRA), hypermethylation of this fragment was detected in 13 of 14 (92.8%) HNSCC cell lines studied and 21 of 32 (65.6%) primary tumors, whereas little or no methylation was seen in 10 normal oral mucosa samples. We extended this investigation to other cancer cell lines and methylation was found in those derived from colon, breast, leukemia and lung, and methylation was also found in 12/14 primary colon tumors. These findings suggest that differentially methylated (DIME)-6 hypermethylation is a good cancer marker in HNSCC as well as in other kinds of neoplasias and confirm the importance of searching for markers of epigenetic dysregulation in cancer.

Epigenetic Silencing of CRABP2 and MX1 in Head and Neck Tumors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Salivary and serum proteomics in head and neck carcinomas before and after surgery and radiotherapy

Several body fluids have been evaluated as new sources for cancer biomarker discovery. In this context, salivary and serum proteomics seem promising diagnostic and predictive tools for head and neck diseases. In the present study, we performed a proteomic analysis of saliva and serum from patients presenting head and neck squamous cell carcinoma (HNSCC) and compared the results before and after therapy. In saliva of cancer patients, we observed an altered protein profile, including over-expression of PLUNC and zinc-alpha-2-glycoprotein. Both proteins may contribute to control tumor growth and, therefore, represent targets for new analysis. We also detected serotransferrin and a modified transthyretin form with altered levels in serum from patients. Comparing preoperative and postreatment samples, the results showed that the protein profile after treatment reverted to a pattern closer to those observed for controls. These results add information on the role of secreted proteins in the cancer process and emphasize the potential of saliva and serum analysis for diagnosis and monitoring of HNSCC.

Profiling invasiveness in head and neck cancer: recent contributions of genomic and transcriptomic approaches.

High-throughput molecular profiling approaches have emerged as precious research tools in the field of head and neck translational oncology. Such approaches have identified and/or confirmed the role of several genes or pathways in the acquisition/maintenance of an invasive phenotype and the execution of cellular programs related to cell invasion. Recently published new-generation sequencing studies in head and neck squamous cell carcinoma (HNSCC) have unveiled prominent roles in carcinogenesis and cell invasion of mutations involving NOTCH1 and PI3K-patwhay components. Gene-expression profiling studies combined with systems biology approaches have allowed identifying and gaining further mechanistic understanding into pathways commonly enriched in invasive HNSCC. These pathways include antigen-presenting and leucocyte adhesion molecules, as well as genes involved in cell-extracellular matrix interactions. Here we review the major insights into invasiveness in head and neck cancer provided by high-throughput molecular profiling approaches.

Head and neck cancer: past, present and future

Head and neck cancer consists of a diverse group of cancers that ranges from cutaneous, lip, salivary glands, sinuses, oral cavity, pharynx and larynx. Each group dictates different management. In this review, the primary focus is on head and neck squamous cell carcinoma (HNSCC) arising from the mucosal lining of the oral cavity and pharynx, excluding nasopharyngeal cancer. Presently, HNSCC is the sixth most prevalent neoplasm in the world, with approximately 900,000 cases diagnosed worldwide. Prognosis has improved little in the past 30 years. In those who have survived, pain, disfigurement and physical disability from treatment have had an enormous psychosocial impact on their lives. Management of these patients remains a challenge, especially in developing countries where this disease is most common. Of all human cancers, HNSCC is the most distressing since the head and neck is the site of the most complex functional anatomy in the human body. Its areas of responsibility include breathing, the CNS, vision, hearing, balance, olfaction, taste, swallowing, voice, endocrine and cosmesis. Cancers that occur in this area impact on these important human functions. Consequently, in treating cancers of the head and neck, the effects of the treatment on the functional outcome of the patient need the most serious consideration. In assessing the success of HNSCC treatment, consideration of both the survival and functional deficits that the patient may suffer as a consequence of their treatment are of paramount importance. For this reason, the modern-day management of head and neck patients should be carried out in a multidisciplinary head and neck clinic.

MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA-10b dysregulation implicated in cell proliferation

Abstract Background Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. Methods MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. Results Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. Conclusions Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA alterations in HNSCC is an essential step to the mechanistic understanding of tumor formation and could lead to the discovery of clinically relevant biomarkers.

Strategies to promote translational research within the European Organisation for Research and Treatment of Cancer (EORTC) Head and Neck Cancer Group: a report from the Translational Research Subcommittee.

Head and neck squamous cell cancer (HNSCC) is the sixth leading cause of cancer-related deaths worldwide. These tumors are commonly diagnosed at advanced stages and mortality rates remain high. Even cured patients suffer the consequences of aggressive treatment that includes surgery, chemotherapy, and radiotherapy. In the past, in clinical trials, HNSCC was considered as a single disease entity. Advances in molecular biology with the development of genomic and proteomic approaches have demonstrated distinct prognostic HNSCC patient subsets beyond those defined by traditional clinical-pathological factors such as tumor subsite and stage [Cho W (ed). An Omics Perspective on Cancer Research. New York/Berlin: Springer 2010]. Validation of these biomarkers in large prospective clinical trials is required before their clinical implementation. To promote this research, the European Organisation for Research and Treatment of Cancer (EORTC) Head and Neck Cancer Program will develop the following strategies-(i) biobanking: prospective tissue collection from uniformly treated patients in the setting of clinical trials; (ii) a group of physicians, physician-scientists, and EORTC Headquarters staff devoted to patient-oriented head and neck cancer research; (iii) a collaboration between the basic scientists of the Translational Research Division interested in head and neck cancer research and the physicians of the Head and Neck Cancer Group; and (iv) funding through the EORTC Grant Program and the Network Core Institutions Consortium. In the present report, we summarize our strategic plans to promote head and neck cancer research within the EORTC framework.

SET protein accumulates in HNSCC and contributes to cell survival: Antioxidant defense, Akt phosphorylation and AVOs acidification

Objectives: Determination of the SET protein levels in head and neck squamous cell carcinoma (HNSCC) tissue samples and the SET role in cell survival and response to oxidative stress in HNSCC cell lineages. Materials and Methods: SET protein was analyzed in 372 HNSCC tissue samples by immunohistochemistry using tissue microarray and HNSCC cell lineages. Oxidative stress was induced with the pro-oxidant tert-butylhydroperoxide (50 and 250 mu M) in the HNSCC HN13 cell lineage either with (siSET) or without (siNC) SET knockdown. Cell viability was evaluated by trypan blue exclusion and annexin V/propidium iodide assays. It was assessed caspase-3 and -9, PARP-1, DNA fragmentation, NM23-H1, SET, Akt and phosphorylated Akt (p-Akt) status. Acidic vesicular organelles (AVOs) were assessed by the acridine orange assay. Glutathione levels and transcripts of antioxidant genes were assayed by fluorometry and real time PCR, respectively. Results: SET levels were up-regulated in 97% tumor tissue samples and in HNSCC cell lineages. SiSET in HN13 cells (i) promoted cell death but did not induced caspases, PARP-1 cleavage or DNA fragmentation, and (ii) decreased resistance to death induced by oxidative stress, indicating SET involvement through caspase-independent mechanism. The red fluorescence induced by siSET in HN13 cells in the acridine orange assay suggests SET-dependent prevention of AVOs acidification. NM23-H1 protein was restricted to the cytoplasm of siSET/siNC HN13 cells under oxidative stress, in association with decrease of cleaved SET levels. In the presence of oxidative stress, siNC HN13 cells showed lower GSH antioxidant defense (GSH/GSSG ratio) but higher expression of the antioxidant genes PRDX6, SOD2 and TXN compared to siSET HN13 cells. Still under oxidative stress, p-Akt levels were increased in siNC HN13 cells but not in siSET HN13, indicating its involvement in HN13 cell survival. Similar results for the main SET effects were observed in HN12 and CAL 27 cell lineages, except that HN13 cells were more resistant to death. Conclusion: SET is potential (i) marker for HNSCC associated with cancer cell resistance and (ii) new target in cancer therapy. (C) 2012 Elsevier Ltd. All rights reserved.