Targeting Adenoviral Gene Therapy Vectors to Head and Neck Squamous Cell Carcinoma and Heart

Gene therapy aims to treat diseases by introducing genetic material to the diseased tissue. For cancer treatment it is important to destroy cancerous cells; this can be achieved by introducing a gene, which induces cell death or by allowing viral vectors to replicate, which also results in destruction of cancerous cells. For cardiac diseases the approach is more like the former, except the gene produces beneficial effects, like angiogenesis. Adenoviruses have many beneficial qualities, which make the virus an interesting gene therapy vector; it can be produced relatively easily, its manipulation is quite easy and it has naturally broad tropism. By removing or replacing certain genes in the adenoviral genome, it can be made non-replicative. In this study, adenoviral receptor expression patterns were characterized in both head and neck squamous cell carcinoma and the human heart. Adenovirus serotype 5 receptor expression in head and neck cancer cell lines was found to be highly variable between cell lines and overall at lower levels, while Ad35 receptor expression was more uniform and at higher levels in all analyzed cell lines. It was also shown that a hybrid virus Ad5/35 is able to infect cells refractory to Ad5, which correlates with receptor expression in these cells. Furthermore, this difference in infection properties extends to cell killing efficiency in case of conditionally replicative viruses. Expression levels of adenoviral receptors CAR, CD46, CD86 and αv-integrins were found to be high both in normal and dilated cardiomyopathy heart tissue. The receptor levels also correlate with transduction efficiency after intracardiac injection. Ad5 showed superior transduction ability compared with Ad5/35, but evoked also a more profound immune reaction when administered this way. Adenoviral gene therapy vectors are the most used delivery vehicles in clinical trials to date. These vectors have proven to be well tolerated and positive results have been obtained when combined with traditional treatments, although poor transduction efficiency has often been reported due to low-level expression of viral receptors on target cells. In spite of this, the results are encouraging and merit for further research.

Safety and tolerability of controlled-release oxycodone on postoperative pain in patients submitted to the oncologic head and neck surgery

Objective: To evaluate the safety and tolerability of controlled-release oxycodone in the treatment of postoperative pain of head and neck oncologic resections.Methods: We conducted a prospective, observational and open study, with 83 patients with moderate to severe pain after head and neck oncological operations. All patients received general anesthesia with propofol, fentanyl and sevoflurane. Postoperatively, should they have moderate or severe pain, we began controlled-release oxycodone 20 mg 12/12 b.i.d on the first day and 10 mg b.i.d. on the second. We assessed the frequency and intensity of adverse effects, the intensity of postoperative pain by a verbal numeric scale and the use of rescue analgesia from 12 hours after administration of the drug and between 7 and 13 days after the last oxycodone dose.Results: The most common adverse events were nausea, vomiting, dizziness, pruritus, insomnia, constipation and urinary retention, most mild. No serious adverse events occurred. In less than 12 hours after the use of oxycodone, there was a significant decrease in the intensity of postoperative pain, which remained until the end of the study. The rescue medication was requested at a higher frequency when the opioid dose was reduced, or after its suspension.Conclusion: Controlled release oxycodone showed to be safe and well tolerated and caused a significant decrease in post-operative pain.

Prevalence of alterations in chest computerized tomography in patients with head and neck cancer

Objective: to assess the prevalence of abnormalities found by computed tomography (CT) of the chest in patients with squamous cell carcinoma of the head and neck. Methods: we retrospectively analyzed chest CT exams of 209 patients with squamous cell carcinoma of the head and neck. The CT findings were stratified as inflammatory / infectious, parenchymal, nodular uncharacteristic and nodular metastatic / tumoral Results: alterations were diagnosed in 66.6% of patients. Of these, 25.3% represented emphysema; 18.8%, uncharacteristic micronodules; 12.9%, metastases; 11.9%, thoracic lymph node enlargements; and in 6.6% we detected active pulmonary tuberculosis or its sequelae, pneumonia or inflammatory / infectious signs and pleural thickening or effusion. Conclusion: the prevalence of exams with alterations and the considerable rate of detected metastases indicate that chest CT should be required for diagnostic and / or staging in cases of head and neck cancer.

Differentiation of Metabolically Distinct Areas within Head and Neck Region using Dynamic 18F-FDG Positron Emission Tomography Imaging

Positron Emission Tomography (PET) using ¹⁸F-FDG is playing a vital role in the diagnosis and treatment planning of cancer. However, the most widely used radiotracer, ¹⁸F-FDG, is not specific for tumours and can also accumulate in inflammatory lesions as well as normal physiologically active tissues making diagnosis and treatment planning complicated for the physicians. Malignant, inflammatory and normal tissues are known to have different pathways for glucose metabolism which could possibly be evident from different characteristics of the time activity curves from a dynamic PET acquisition protocol. Therefore, we aimed to develop new image analysis methods, for PET scans of the head and neck region, which could differentiate between inflammation, tumour and normal tissues using this functional information within these radiotracer uptake areas. We developed different dynamic features from the time activity curves of voxels in these areas and compared them with the widely used static parameter, SUV, using Gaussian Mixture Model algorithm as well as K-means algorithm in order to assess their effectiveness in discriminating metabolically different areas. Moreover, we also correlated dynamic features with other clinical metrics obtained independently of PET imaging. The results show that some of the developed features can prove to be useful in differentiating tumour tissues from inflammatory regions and some dynamic features also provide positive correlations with clinical metrics. If these proposed methods are further explored then they can prove to be useful in reducing false positive tumour detections and developing real world applications for tumour diagnosis and contouring.

Genetic alterations in head and neck squamous cell carcinomas

The genetic alterations observed in head and neck cancer are mainly due to oncogene activation (gain of function mutations) and tumor suppressor gene inactivation (loss of function mutations), leading to deregulation of cell proliferation and death. These genetic alterations include gene amplification and overexpression of oncogenes such as myc, erbB-2, EGFR and cyclinD1 and mutations, deletions and hypermethylation leading to p16 and TP53 tumor suppressor gene inactivation. In addition, loss of heterozygosity in several chromosomal regions is frequently observed, suggesting that other tumor suppressor genes not yet identified could be involved in the tumorigenic process of head and neck cancers. The exact temporal sequence of the genetic alterations during head and neck squamous cell carcinoma (HNSCC) development and progression has not yet been defined and their diagnostic or prognostic significance is controversial. Advances in the understanding of the molecular basis of head and neck cancer should help in the identification of new markers that could be used for the diagnosis, prognosis and treatment of the disease.

Systematic head and neck physical examination as a predictor of obstructive sleep apnea in class III obese patients

Our aim was to determine if anatomical abnormalities of the upper airway (UA) and facial skeleton of class III severely obese patients are related to the presence and severity of obstructive sleep apnea syndrome (OSAS). Forty-five patients (69% females, mean age 46.5 ± 10.8 years) with a body mass index (BMI) over 40 kg/m² underwent UA and facial skeletal examinations as well as polysomnography. Mean BMI was 49 ± 7 kg/m² and mean neck circumference was 43.4 ± 5.1 cm. Polysomnographic findings showed that 22% had a normal apnea-hypopnea index (AHI) and 78% had an AHI over 5. The presence of OSAS was associated with younger age (P = 0.02), larger neck circumference (P = 0.004), presence of a voluminous lateral wall (P = 0.0002), posteriorized soft palate (P = 0.0053), thick soft palate (P = 0.0014), long uvula (P = 0.04), thick uvula (P = 0.0052), and inferior turbinate hypertrophy (P = 0.04). A larger neck circumference (P = 0.02), presence of a voluminous lateral wall (P = 0.04), posteriorized soft palate (P = 0.03), and thick soft palate (P = 0.04) were all associated with OSAS severity. The prevalence of OSAS in this group was high. A larger neck circumference and soft tissue abnormalities of the UA were markers for both the presence and severity of OSAS. Conversely, no abnormalities in the facial skeleton were associated with OSAS in patients with morbid obesity.

Genetic variability of vascular endothelial growth factor and prognosis of head and neck cancer in a Brazilian population

Vascular endothelial growth factor (VEGF) is one of the most potent endothelial cell mitogens and plays a critical role in angiogenesis. Polymorphisms in this gene have been evaluated in patients with several types of cancer. The objectives of this study were to determine if there was an association of the -1154G/A polymorphism of the VEGF gene with head and neck cancer and the interaction of this polymorphism with lifestyle and demographic factors. Additionally, the distribution of the VEGF genotype was investigated with respect to the clinicopathological features of head and neck cancer patients. The study included 100 patients with histopathological diagnosis of head and neck squamous cell carcinoma. Patients with treated tumors were excluded. A total of 176 individuals 40 years or older were included in the control group and individuals with a family history of neoplasias were excluded. Analysis was performed after extraction of genomic DNA using the real-time PCR technique. No statistically significant differences between allelic and genotype frequencies of -1154G/A VEGF polymorphism were identified between healthy individuals and patients. The real-time PCR analyses showed a G allele frequency of 0.72 and 0.74 for patients and the control group, respectively. The A allele showed a frequency of 0.28 for head and neck cancer patients and 0.26 for the control group. However, analysis of the clinicopathological features showed a decreased frequency of the A allele polymorphism in patients with advanced (T3 and T4) tumors (OR = 0.36; 95%CI = 0.14-0.93; P = 0.0345). The -1154A allele of the VEGF gene may decrease the risk of tumor growth and be a possible biomarker for head and neck cancer. This polymorphism is associated with increased VEGF production and may have a prognostic importance.

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.

Loss of Y-chromosome does not correlate with age at onset of head and neck carcinoma: a case-control study

Loss of Y-chromosome has been correlated with older age in males. Furthermore, current evidence indicates that Y-chromosome loss also occurs in several human tumors, including head and neck carcinomas. However, the association between Y nullisomy and the occurrence of neoplasias in elderly men has not been well established. In the present study, the association between Y-chromosome loss and head and neck carcinomas was evaluated by comparison to cells from peripheral blood lymphocytes and normal mucosa of cancer-free individuals matched for age using dual-color fluorescence in situ hybridization. Twenty-one patients ranging in age from 28 to 68 years were divided into five-year groups for comparison with 16 cancer-free individuals matched for age. The medical records of all patients were examined to obtain clinical and histopathological data. None of the patients had undergone radiotherapy or chemotherapy before surgery. In all groups, the frequency of Y-chromosome loss was higher among patients than among normal reference subjects (P < 0.0001) and was not age-dependent. These data suggest that Y-chromosome loss is a tumor-specific alteration not associated with advanced age in head and neck carcinomas.

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.

Relationship of inflammatory markers and pain in patients with head and neck cancer prior to anticancer therapy

Pain is a common symptom in patients with cancer, including those with head and neck cancer (HNC). While studies suggest an association between chronic inflammation and pain, levels of inflammatory cytokines, such as C-reactive protein (CRP) and tumor necrosis factor-alpha (TNF-α), have not been correlated with pain in HNC patients who are not currently undergoing anticancer treatment. The purpose of this study was to examine the relationship between these inflammatory markers and perceived pain in HNC patients prior to anticancer therapy. The study group consisted of 127 HNC patients and 9 healthy controls. Pain was assessed using the Brief Pain Inventory (BPI), and serum levels of CRP and TNF-α were determined using the particle-enhanced turbidimetric immunoassay (PETIA) and ELISA techniques, respectively. Patients experiencing pain had significantly higher levels of CRP (P<0.01) and TNF-α (P<0.05) compared with controls and with patients reporting no pain. There were significantly positive associations between pain, CRP level, and tumor stage. This is the first study to report a positive association between perceived pain and CRP in HNC patients at the time of diagnosis. The current findings suggest important associations between pain and inflammatory processes in HNC patients, with potential implications for future treatment strategies.

O6-Methylguanine-DNA-Methyltransferase methylation: prevalence and predictive value in head and neck squamous cell carcinoma

Introduction: Le gène O6-méthylguanine-ADN méthyltransferase (MGMT) code pour une enzyme spécifique réparatrice de l’ADN qui protège les cellules de la toxicité des agents alkylants. Ainsi, l’activité du MGMT est un mécanisme majeur de résistance aux agents alkylants. Il a été démontré qu’une diminution de l’expression du gène MGMT par une hyperméthylation du promoteur résulte en une amélioration de la survie chez les patients avec certains types de tumeurs qui sont traitées avec des agents chimiothérapeuthique alkylants. Objectifs: Déterminer la prévalence de la méthylation du gène MGMT chez des patients avec des cancers épidermoïdes localement avancés de la sphère ORL traités avec chimioradiothérapie et évaluer l’impact de cette méthylation sur la survie. Méthodes: Sur 428 patients consécutifs, traités avec chimioradiothérapie à notre institution et suivis pour un période médiane de 37 mois, 199 spécimens chirurgicaux paraffinés ont été récupérés. L’ADN était extrait et modifié par le traitement au bisulfite. Une réaction en chaîne de la polymérase, spécifique à la méthylation était entreprise pour évaluer l’état de méthylation du promoteur du gène du MGMT. Les résultats de laboratoire étaient corrélés avec la réponse clinique. L’analyse statistique était exécutée à l’aide du test de Fisher pour les données catégoriques et à l’aide des courbes de Kaplan-Meier pour les échecs au traitement. Résultats : Des 199 extraits d’ADN initiaux, 173 (87%) étaient modifiés au bisulfite avec succès. Des ces spécimens modifiés, 71 (41%) ont démontré une hyperméthylation du MGMT. Pour les cas de méthylation et nonméthylation du MGMT, les caractéristiques des patients n’étaient pas significativement différentes. Les taux de réponse étaient 71 et 73% (p=NS) respectivement. Le contrôle locorégional était respectivement 87 et 77% (p=0.26), la survie sans maladie était 80 et 60% (p=0.38), la survie sans métastase à distance était 92 et 78% (p=0.08) et la survie globale était 64 et 62% (p=0.99) à 3 ans. Conclusions : L’état de méthylation du MGMT est fortement prévalent (41%) et semble avoir un possible impact bénéfique sur la survie quand la chimioradiothérapie est administrée aux patients avec des stades avancés de cancers tête et cou.

Loss of Y-chromosome does not correlate with age at onset of head and neck carcinoma: a case-control study

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cytogenetic damage in circulating lymphocytes and buccal mucosa cells of head-and-neck cancer patients undergoing radiotherapy

This study evaluated cytogenetic damage by measuring the frequency of micronucleated cells (MNC) in peripheral blood and buccal mucosa of head-and-neck cancer patients undergoing radiotherapy.MNC frequencies were assessed in 31 patients before, during, and after radiotherapy, and in 17 C, healthy controls matched for gender, age, and smoking habits. Results showed no statistically significant difference between patients and controls prior to radiotherapy in cytokinesis-blocked lymphocytes or buccal mucosa cells. During treatment, increased MNC frequencies were observed in both cell types. Micronucleated lymphocyte levels remained high in samples collected 30 to 140 days after the end of treatment, while MNC frequency in buccal mucosa decreased to values statistically similar to baseline values. There is controversy over the effects of age, smoking habit, tumor stage, and/or metastasis on MNC frequency. However, increased frequency of micronucleated buccal mucosa cells was seen in patients under 60 years old and in those with tumors >4cm.In conclusion, the data show that radiotherapy has a potent clastogenic effect in Circulating lymphocytes and buccal mucosa cells of head-and-neck cancer patients, and that the baseline MNC frequency in these two tissues is not a sensitive marker for head-and neck neoplasm.