The burden of gastroesophageal reflux disease on the cost of managing chronic diseases in Australia. The need for a new diagnostic and management paradigm.

INTRODUCTION: Chronic disease poses a major problem for the Australian healthcare system as the leading cost-burden and cause of death. Gastroesophageal reflux disease (GORD) typifies the problems with a growing prevalence and cost. We hypothesise that a scintigraphic test could optimise the diagnosis, especially in problematic extraoesophageal disease. MATERIALS AND METHODS: Data was collected from 2 groups of patients. Patients undergoing fundoplication for severe GORD (n = 30) and those with atypical symptoms (n = 30) were studied by scintigraphy and 24-hour oesophageal pH, impedance and manometry. RESULTS: Mean age of cohort was 55.8 years with 40 females and 20 males. Body mass index was a mean of 28.3. DeMeester score was normal in 12/60 with atypical symptoms and abnormal in the rest. Good correlation was shown between scintigraphy and impedance, manometry and distal pH readings. Pulmonary aspiration was shown in 25/60 (15 with atypical symptoms) and LPR in 20/30. Several impedance, manometric and scintigraphic finding were good predictors of lung aspiration of refluxate. CONCLUSION: Scintigraphy provides a good tool for screening patients with typical and atypical symptoms of GORD. It is well correlated with the standard methods for the diagnosis and provides visual evidence of LPR and lung aspiration.

Variants of EVER1 and EVER2 (TMC6 and TMC8) and human papillomavirus status in patients with mucosal squamous cell carcinoma of the head and neck.

PURPOSE: There is a growing association of human papillomavirus (HPV) with some cases of mucosal squamous cell carcinoma of the head and neck (HNSCC), particularly of the oropharynx. Persistent oral HPV infection is believed to increase the likelihood of malignancy, and it is possible that host genetic factors can determine susceptibility to persistent HPV infection. Polymorphisms in the two EV genes (EVER1 and EVER2, also known as transmembrane channel protein (TMC) 6 and 8) have been identified as strong candidate genes, since a small number of critical mutations in these genes have been shown to cause profound and florid skin HPV infections, and some of them have been linked to susceptibility to cervical cancer. METHODS: We sought to determine whether there was a difference in the frequency of single nucleotide polymorphisms (SNPs) in EVER1 (rs2613516, rs12449858) and EVER2 (rs7205422, rs12452890) between HNSCC patients with HPV-positive and HPV-negative tumors, and healthy controls. We used logistic regression to analyze SNPs in 219 patients with histologically confirmed primary SCC of the oropharynx, oral cavity, hypopharynx, or larynx, and 321 healthy controls. RESULTS: We did not find any associations with the EVER1/EVER2 SNPs and HPV status or being a HNSCC case or a control. CONCLUSIONS: The present data do not provide evidence for a role of genetic variations in EVER1 or EVER2 for HPV status of mucosal HNSCC or between HNSCC patients and controls.

Human papillomavirus status and p16(INK4A) expression in patients with mucosal squamous cell carcinoma of the head and neck in Queensland, Australia.

BACKGROUND: The last decade has seen changes in the epidemiology of mucosal squamous cell carcinomas of the head and neck (HNSCCs), with increasing numbers of cases attributable to human papillomavirus (HPV) infection. We sought to determine the prevalence of HPV and p16(INK4a) expression in Australian HNSCC patients and to identify predictors of HPV-positivity. METHODS: We recruited 248 HNSCC patients with histologically confirmed primary SCC of the oropharynx, oral cavity, hypopharynx or larynx diagnosed between 2004 and 2010. All patients completed a questionnaire. Clinical data were abstracted from medical records. HPV presence in paraffin-embedded tumours was determined by PCR, and expression of p16(INK4a), p21(WAF1), p53, pRB, cyclin D1, and Ki67 by immunohistochemistry. RESULTS: Fifty (20%) patients were HPV-positive, 63 (28%) overexpressed p16(INK4a), and 44 (19%) were positive for HPV and p16(INK4a) (high concordance between HPV-positivity and p16(INK4a) status, κ=0.72). HPV-16 was most common (84%), followed by HPV-18 (10%), HPV-33 (4%) and HPV-69 (2%). HPV and p16(INK4a) prevalence was highest for SCCs of the oropharynx, followed by hypopharynx, larynx and oral cavity (HPV and p16(INK4a)p<0.0001). HPV prevalence and p16(INK4a)-overexpression were significantly higher in younger than older patients (HPV p=0.001; p16 (INK4a)p=0.003). Heavy smokers had lower HPV prevalence than non- or moderate smokers (p=0.017). Gender and alcohol consumption were not associated with HPV or p16(INK4a) status. HPV-positive tumours had significantly lower cyclin D1 and higher p21(WAF1) expression than HPV-negative tumours. CONCLUSION: HPV prevalence and p16(INK4a)-overexpression were highest in oropharyngeal tumours, younger patients, and non-smokers.

A novel saliva-based microRNA biomarker panel to detect head and neck cancers.

BACKGROUND: MicroRNAs (miRNAs) are known to play an important role in cancer development by post-transcriptionally affecting the expression of critical genes. The aims of this study were two-fold: (i) to develop a robust method to isolate miRNAs from small volumes of saliva and (ii) to develop a panel of saliva-based diagnostic biomarkers for the detection of head and neck squamous cell carcinoma (HNSCC). METHODS: Five differentially expressed miRNAs were selected from miScript™ miRNA microarray data generated using saliva from five HNSCC patients and five healthy controls. Their differential expression was subsequently confirmed by RT-qPCR using saliva samples from healthy controls (n = 56) and HNSCC patients (n = 56). These samples were divided into two different cohorts, i.e., a first confirmatory cohort (n = 21) and a second independent validation cohort (n = 35), to narrow down the miRNA diagnostic panel to three miRNAs: miR-9, miR-134 and miR-191. This diagnostic panel was independently validated using HNSCC miRNA expression data from The Cancer Genome Atlas (TCGA), encompassing 334 tumours and 39 adjacent normal tissues. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic capacity of the panel. RESULTS: On average 60 ng/µL miRNA was isolated from 200 µL of saliva. Overall a good correlation was observed between the microarray data and the RT-qPCR data. We found that miR-9 (P <0.0001), miR-134 (P <0.0001) and miR-191 (P <0.001) were differentially expressed between saliva from HNSCC patients and healthy controls, and that these miRNAs provided a good discriminative capacity with area under the curve (AUC) values of 0.85 (P <0.0001), 0.74 (P < 0.001) and 0.98 (P < 0.0001), respectively. In addition, we found that the salivary miRNA data showed a good correlation with the TCGA miRNA data, thereby providing an independent validation. CONCLUSIONS: We show that we have developed a reliable method to isolate miRNAs from small volumes of saliva, and that the saliva-derived miRNAs miR-9, miR-134 and miR-191 may serve as novel biomarkers to reliably detect HNSCC.

DNA Methylation at the Novel CpG Sites in the Promoter of MED15/PCQAP Gene as a Biomarker for Head and Neck Cancers.

Head and neck cancers (HNCs) represent a significant and ever-growing burden to the modern society, mainly due to the lack of early diagnostic methods. A significant number of HNCs is often associated with drinking, smoking, chewing beetle nut, and human papilloma virus (HPV) infections. We have analyzed DNA methylation patterns in tumor and normal tissue samples collected from head and neck squamous cell carcinoma (HNSCC) patients who were smokers. We have identified novel methylation sites in the promoter of the mediator complex subunit 15 (MED15/PCQAP) gene (encoing a co-factor important for regulation of transcription initiation for promoters of many genes), hypermethylated specifically in tumor cells. Two clusters of CpG dinucleotides methylated in tumors, but not in normal tissue from the same patients, were identified. These CpG methylation events in saliva samples were further validated in a separate cohort of HNSCC patients (who developed cancer due to smoking or HPV infections) and healthy controls using methylation-specific PCR (MSP). We used saliva as a biological medium because of its non-invasive nature, close proximity to the tumors, easiness and it is an economically viable option for large-scale screening studies. The methylation levels for the two identified CpG clusters were significantly different between the saliva samples collected from healthy controls and HNSCC individuals (Welch's t-test returning P < 0.05 and Mann-Whitney test P < 0.01 for both). The developed MSP assays also provided a good discriminative ability with AUC values of 0.70 (P < 0.01) and 0.63 (P < 0.05). The identified novel CpG methylation sites may serve as potential non-invasive biomarkers for detecting HNSCC.

Alpha B-crystallin - a validated prognostic factor for poor prognosis in squamous cell carcinoma of the oral cavity.

CONCLUSION: Alpha B-crystallin was found to be an independent prognostic marker for poor prognosis in oral cavity tumours. For oropharyngeal cancer, alpha B-crystallin had no prognostic value. OBJECTIVE: The aim of this study was to see if earlier findings of alpha B-crystallin as an independent prognostic marker, and SPARC/osteonectin, PAI-1 and uPA as a prognostic combination for poor outcome in squamous cell carcinoma (SCC) of the head and neck could be confirmed in a new set of tumours. METHODS: In a consecutive series of patients, assessed and primarily treated at a tertiary referral centre, histological sections from 55 patients with oral and SCC (OOPHSSC) with complete clinical data and follow-up were obtained. Oral and oropharyngeal tumours were studied separately. Immunohistochemical detection of alpha B-crystallin, SPARC/osteonectin, PAI-1 and uPA expression was performed. RESULTS: Thirty-five patients had an oral tumour and 20 patients an oropharyngeal tumour. Twenty-five oral tumours stained negatively and 10 positively for alpha B-crystallin. For oropharyngeal tumours the figures were 15 negatively and 5 positively. Median disease-specific survival (DSS) for both sites was 33.8 and 11.9 months, for negative and positive alpha B-crystallin staining, respectively (p = 0.046). For the oral cavity, median DSS was 27.3 months for negative tumours and 7.5 months for positive tumours (p = 0.012). Corresponding figures for oropharyngeal tumours were 33.8 and 34.1 months (p = 0.95). Thus, significance in survival was only found in oral cavity tumours. In multivariate analyses there were no significant differences in DSS in the oropharyngeal group when adjusted for tumour size (T status) and presence of neck node metastasis (N status). In the oral cavity group, the significantly better DSS for negative tumours became even stronger when adjusted for T and N status. No statistical difference was found in DSS between positive and negative staining for SPARC/osteonectin, PAI-1 or uPA.

Cytotoxic T cell adoptive immunotherapy as a treatment for nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC). We assess the safety and tolerability of adoptive transfer of autologous cytotoxic T lymphocytes (CTLs) specific for the EBV latent membrane protein (LMP) in a patient with recurrent NPC. After infusion, the majority of pulmonary lesions were no longer evident, although the primary tumor did not regress.

miRNAs in head and neck cancer revisited.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cause of cancer mortality in the world and the 5th most commonly occurring cancer. Tobacco smoking, alcohol consumption and human papilloma virus (HPV) infections have been associated with the occurrence of HNSCC. Despite advances that have been made in HNSCC treatment, smoking-associated HNSCC patients still exhibit a poor 5 year survival rate (30-50 %) and a concomitant poor quality of life. The major clinical challenge to date lies in the early detection of dysplastic lesions,which can progress to malignancy. In addition, there are currently no tools available to monitor HNSCC patients for early stages of local recurrences or distant metastases. In the recent past, micro-RNAs (miRNA) have been assessed for their role in cancer initiation and progression, including HNSCC. It is now well-established that deregulation of these single stranded, small non-coding, 19-25 nt RNAs can e.g. enhance the expression of oncogenes or subdue the expression of tumor suppressor genes. The aims of this review are three-fold: first to retrieve from the literature miRNAs that have specifically been associated with HNSCC, second to group these miRNAs into those regulating tumor initiation, progression and metastasis, and third to discern miRNAs related to smoking-associated HNSCC versus HPV-associated HNSCC development. CONCLUSIONS: This review gives an overview on the miRNAs regulating the development of head and neck cancers. The ultimate establishment of miRNA expression profiles that are HNSCC specific, and miRNAs that orchestrate altered gene and protein expression levels in HNSCC, could pave the way for a better understanding of the mechanism underlying its pathogenesis and the development of novel, targeted therapies.

Tumor-suppressor Gene Promoter Hypermethylation in Saliva of Head and Neck Cancer Patients.

Head and neck squamous cell carcinoma (HNSCC) accounts for a bulk of the oral and laryngeal cancers, the majority (70%) of which are associated with smoking and excessive drinking, major known risk factors for the development of HNSCC. In contrast to reports that suggest an inverse relationship between smoking and global DNA CpG methylation, hypermethylation of promoters of a number of genes was detected in saliva collected from patients with HNSCC. Using a sensitive methylation-specific polymerase chain reaction (MSP) assay to determine specific methylation events in the promoters of RASSF1A, DAPK1, and p16 genes, we demonstrate that we can detect tumor presence with an overall accuracy of 81% in the DNA isolated from saliva of patients with HNSCC (n = 143) when compared with the DNA isolated from the saliva of healthy nonsmoker controls (n = 31). The specificity for this MSP panel was 87% and the sensitivity was 80% (with a Fisher exact test P < .0001). In addition, the test panel performed extremely well in the detection of the early stages of HNSCCs, with a sensitivity of 94% and a specificity of 87%, and a high κ concordance value of 0.8, indicating an excellent overall agreement between the presence of HNSCC and a positive MSP panel result. In conclusion, we demonstrate that the promoter methylation of RASSF1A, DAPK1, and p16 MSP panel is useful in detecting hypermethylation events in a noninvasive manner in patients with HNSCC.

Effective treatment of metastatic forms of Epstein-Barr virus-associated nasopharyngeal carcinoma with a novel adenovirus-based adoptive immunotherapy.

Nasopharyngeal carcinoma (NPC) is endemic in China and Southeast Asia where it is tightly associated with infections by Epstein-Barr virus (EBV). The role of tumor-associated viral antigens in NPC renders it an appealing candidate for cellular immunotherapy. In earlier preclinical studies, a novel adenoviral vector-based vaccine termed AdE1-LMPpoly has been generated that encodes EBV nuclear antigen-1 (EBNA1) fused to multiple CD8(+) T-cell epitopes from the EBV latent membrane proteins, LMP1 and LMP2. Here, we report the findings of a formal clinical assessment of AdE1-LMPpoly as an immunotherapeutic tool for EBV-associated recurrent and metastatic NPC. From a total of 24 patients with NPC, EBV-specific T cells were successfully expanded from 16 patients with NPC (72.7%), whereas six patients with NPC (27.3%) showed minimal or no expansion of virus-specific T cells. Transient increase in the frequencies of LMP1&2- and EBNA1-specific T-cell responses was observed after adoptive transfer to be associated with grade I flu-like symptoms and malaise. The time to progression in these patients ranged from 38 to 420 days with a mean time to progression of 136 days. Compared with patients who did not receive T cells, the median overall survival increased from 220 to 523 days. Taken together, our findings show that adoptive immunotherapy with AdE1-LMPpoly vaccine is safe and well tolerated and may offer clinical benefit to patients with NPC.

Results of a prospective study of positron emission tomography-directed management of residual nodal abnormalities in node-positive head and neck cancer after definitive radiotherapy with or without systemic therapy.

BACKGROUND: The purpose of this study was to present our prospectively evaluated positron emission tomography (PET)-directed policy for managing the neck in node-positive head and neck squamous cell carcinoma (N+HNSCC) after definitive radiotherapy (RT) with or without concurrent systemic therapy. METHODS: One hundred twelve consecutive patients who achieved a complete response at the primary site underwent a 12-week posttherapy nodal response assessment with PET and diagnostic CT. Patients with an equivocal PET underwent a repeat PET 4 to 6 weeks later. Patients with residual CT nodal abnormalities deemed PET-negative were uniformly observed regardless of residual nodal size. RESULTS: Median follow-up from commencement of RT was 28 months (range, 13-64 months). Residual CT nodal abnormalities were present in 50 patients (45%): 41 PET-negative and 9 PET-positive. All PET-negative residual CT nodal abnormalities were observed without subsequent isolated nodal failure. CONCLUSION: PET-directed management of the neck after definitive RT in node-positive HNSCC appropriately spares neck dissections in patients with PET-negative residual CT nodal abnormalities.

Pathologic occult neck disease in patients with metastatic cutaneous squamous cell carcinoma to the parotid.

OBJECTIVE: The aim of this study was to document the rate of pathologic neck disease in patients presenting with metastatic cutaneous squamous cell carcinoma (CSCC) to the parotid gland following parotidectomy and neck dissection in the clinically and radiologic negative neck. STUDY DESIGN: Case series with chart review. SETTING: Tertiary referral center. SUBJECTS AND METHODS: The study involved a retrospective chart review from 1999 to 2008 of patients presenting with metastatic CSCC to the parotid at the Princess Alexandra Hospital, Brisbane, Australia. RESULTS: Eighty-one patients with metastatic parotid disease were identified. A total of 51 (63%) patients had no clinical or radiological evidence of cervical nodal disease. Forty-five patients (88%) were male, median age was 69 (range, 42-91) years, and the median follow-up was 16 (interquartile range, 9-44) months. Thirty-four of these patients underwent a parotidectomy and neck dissection with/without postoperative radiotherapy (RT). Occult pathological cervical nodal disease was found in 5 (14.7%) patients. Of those who received a neck dissection, 3 patients relapsed in the parotid, 1 in the neck alone, and 1 distantly. CONCLUSION: This series has shown that the rate of pathologically involved neck nodes in patients with metastatic CSCC to the parotid in the clinically node negative neck is low. Given many of these patients warrant postoperative RT to the parotid bed, an elective neck dissection may not be warranted as the parotid and neck may be treated in continuity with RT.

Nasal septum malignancy.

INTRODUCTION: Malignancies of the nasal septum are rare diseases and fewer than 400 cases were reported. The understanding of the disease is limited due to its rarity. METHODS: We present a series of patients with nasal septum malignancies, who were referred to the Princess Alexandra Hospital, Ear, Nose and Throat Department from 2007 to 2010. RESULTS: Seventeen patients were found to have nasal septum malignancies. The average age was 59.5 years old (range: 36 to 83 years old). The commonest initial symptom on presentation was nasal obstruction (nine out of 17, 53%), seconded by epistaxis (eight out of 17, 47%). The average time from the initial onset of symptoms to presentation averaged 18.8 months (range: 1 to 48 months). The commonest physical finding on presentation was nasal masses (11 out of 17, 65%), followed by nasal septum ulcers (four out of 17, 24%). The histology of the lesions was predominantly squamous cell carcinoma. The mean duration of follow-up was 24.7 months. The overall 3-year survival was 81.9% with the relapse free survival 66.7%. DISCUSSION: Nasal septum malignancies are highly treatable with good prognoses when in early stages. They required high degree of suspicion to be detected early. Treatment options include surgical resection and radiotherapy and they offered similar 3-year survival rate. Combined therapy is adopted in larger tumours; however, it is not verified with randomized trials. Vigilant follow-up is vital to detect early recurrence, which is common in advanced stage lesions.

Manipulation of nasal fractures with local anaesthetic: a 'how to do it' with online video tutorial.

Nasal fractures are the most common facial fractures and displaced fractures may cause considerable cosmetic concern. Traditionally, displaced nasal fractures have been manipulated under general anaesthesia (GA) performed within 2 weeks of the injury. Despite evidence for the benefit of local anaesthesia (LA), nasal fractures are still most commonly reduced under GA. We have presented a method of reduction of simple nasal fractures under LA in an outpatient setting. This has the advantage of being painless, simple to attempt and cost-effective. If reduction is inadequate then a general anaesthetic reduction is still possible. A recent comprehensive systematic review of all the available evidence did not show any significant difference (in terms of cosmesis, pain or nasal obstruction) between using LA and GA methods and highlighted the evidence base to support LA. We describe our method of assessment and treatment of displaced nasal fractures and provide an online tutorial (http://sciencestage.com/v/22194/local-anaesthetic-nasal-fracture-reduction.html). It is important to keep in mind that any concerns should be referred to an otolaryngology specialist for further management and that practitioners attempting this technique should first receive training from an otolaryngologist.

Long-term functional speech and swallowing outcomes following pharyngolaryngectomy with free jejunal flap reconstruction.

Surgery for advanced cancer of the hypopharynx is a complex issue. Surgical intervention needs to take into consideration the resultant quality of life, in particular fundamental functional outcomes such as speech and swallowing. The aim of this study is to look at these long-term functional outcomes, following pharyngolaryngectomy and free jejunal reconstruction. A total of 19 patients, each undergoing a pharyngolaryngectomy with free jejunal graft was included. Each had a primary tracheoesophageal puncture for insertion of an indwelling voice prosthesis for speech. Functional outcomes of speech and swallow were assessed by a qualified speech pathologist. The impact on patients' quality of life was assessed under 4 domains: impairment, disability, handicap, and well being. The mean time period to follow-up was 4 years. Eighteen of the 19 patients were tolerating an oral diet, with one patient reliant on percutaneous endoscopic gastrostomy feeds. Seventeen patients (89%) were assessed as either having either no--or only a mild degree--of dysphagia, with no evidence of aspiration. Of the 19 patients, 15 were utilizing tracheosophageal speech for communication with 11 (73%) having no--or only a mild degree--of dsyphonia. Patients assessed as having no evidence of dysphagia or dysphonia also reported reduced levels of handicap and distress compared with patients experiencing any degree of dysphagia (P = 0.46) or dysphonia (P = 0.01). While rates of pharyngolaryngectomy increase, most patients have a poor long-term prognosis, heightening the significance of postoperative outcomes. The results of this study highlight the importance of speech and swallow outcomes, and demonstrate the direct correlation between these functions and resultant quality of life.

Novel approach to the formulation of an Epstein-Barr virus antigen-based nasopharyngeal carcinoma vaccine.

Epstein-Barr virus (EBV) is associated with several malignant diseases including nasopharyngeal carcinoma (NPC), a common neoplasm throughout southeast Asia. Radiotherapy and chemotherapy can achieve remission, but a reemergence of disease is not uncommon. Therefore, there is a need for specific therapies that target the tumor through the recognition of EBV antigens. In NPC, latent membrane protein 1 (LMP1) and LMP2 offer the best opportunity for specific targeting since they are typically expressed and T-cell determinants in each of these proteins have been defined. We have attempted to maximize the opportunity of incorporating every possible CD4 and CD8 determinant in a single formulation. We have achieved this by generating a scrambled protein incorporating random overlapping peptide sets from EBNA1, LMP1, and LMP2, which was then inserted into a replication-deficient strain of adenovirus (adenovirus scrambled antigen vaccine [Ad-SAVINE]). This report describes the construction of this Ad-SAVINE construct, its utility in generating LMP1 and LMP2 responses in healthy individuals as well as NPC patients, and its capacity to define new epitopes. This formulation could have a role in NPC immunotherapy for all ethnic groups since it has the potential to activate all possible CD4 and CD8 responses within EBNA1 and LMPs.

Optimization of LMP-specific CTL expansion for potential adoptive immunotherapy in NPC patients.

Nasopharyngeal carcinoma (NPC) is Epstein-Barr virus (EBV) positive in all undifferentiated cases, expressing the latency II phenotype of latent membrane proteins (LMPs) 1 and 2, in addition to EBV nuclear antigen (EBNA) 1. Several studies have attempted to treat NPC with EBV-specific cytotoxic T lymphocyte (CTL) with a partial response. To improve this therapy, there is a need to expand CTL targeted to the latency II antigens of EBV, rather than the immunodominant EBV nuclear antigens 3-6 peptides typically expanded by lymphoblastoid cells. In order to maximize the expansion of LMP-specific CTL in vitro for use in adoptive immunotherapy of nasopharyngeal carcinoma patients, we used lymphoblastoid cell lines coated with synthetic peptides corresponding to CTL determinants from the LMP proteins. We investigated several issues pertaining to the expansion of an immunologically weak CTL response, including peptide and interleukin-2 concentration, and screening assays for selecting the optimal peptide for use in expansion of LMP-specific CTL. Although screening of ex vivo peripheral blood mononuclear cells did not prove to be useful in the selection of an LMP peptide for use in CTL cultures, the peptide and interleukin-2 concentrations were critical for the maximum expansion of CTL. Therefore, it is imperative that stimulation protocols are optimized for the expansion of LMP-specific CTL.