Establishment of four head and neck squamous cell carcinoma cell lines: importance of reference DNA for accurate genomic characterisation.

OBJECTIVE: There is significant interest in developing early passage cell lines with matched normal reference DNA to facilitate a precision medicine approach in assessing drug response. This study aimed to establish early passage cell lines, and perform whole exome sequencing and short tandem repeat profiling on matched normal reference DNA, primary tumour and corresponding cell lines. METHODS: A cell culture based, in vitro study was conducted of patients with primary human papillomavirus positive and human papillomavirus negative tumours. RESULTS: Four early passage cell lines were established. Two cell lines were human papillomavirus positive, confirmed by sequencing and p16 immunoblotting. Short tandem repeat profiling confirmed that all cell lines were established from their index tumours. Whole exome sequencing revealed that the matched normal reference DNA was critical for accurate mutational analysis: a high rate of false positive mutation calls were excluded (87.6 per cent). CONCLUSION: Early passage cell lines were successfully established. Patient-matched reference DNA is important for accurate cell line mutational calls.

The prevalence of human papillomavirus in paediatric tonsils in Southwestern Ontario.

OBJECTIVE: To determine the prevalence of human papillomavirus in paediatric tonsils in Southwestern Ontario, Canada. MATERIALS AND METHODS: Patients aged 0-18 years undergoing tonsillectomy were recruited. Two specimens (left and right tonsils) were collected from each participant. Tonsillar DNA was analysed using quantitative polymerase chain reaction to determine the presence of human papillomavirus subtypes 6, 11, 16 or 18. RESULTS: A total of 102 patients, aged 1-18 years (mean age of 5.7 years), were recruited. Ninety-nine surveys were returned. There were 44 females (44.4 per cent) and 55 males (55.6 per cent). Forty patients (40.4 per cent) were firstborn children and 73 (73.7 per cent) were delivered vaginally. Six mothers (6.1 per cent) and one father (1.0 per cent) had prior known human papillomavirus infection, and one mother (1.0 per cent) had a history of cervical cancer. All tonsil specimens were negative for human papillomavirus subtypes 6, 11, 16 and 18. CONCLUSION: No human papillomavirus subtypes 6, 11, 16 or 18 were found in paediatric tonsil specimens from Southwestern Ontario.

Delay in diagnosis of patients with head-and-neck cancer in Canada: impact of patient and provider delay.

Background: Head-and-neck cancers (hncs) often present at an advanced stage, leading to poor outcomes. Late presentation might be attributable to patient delays (reluctance to seek treatment, for instance) or provider delays (misdiagnosis, prolonged wait time for consultation, for example). The objective of the present study was to examine the length and cause of such delays in a Canadian universal health care setting. Methods: Patients presenting for the first time to the hnc multidisciplinary team (mdt) with a biopsy-proven hnc were recruited to this study. Patients completed a survey querying initial symptom presentation, their previous medical appointments, and length of time between appointments. Clinical and demographic data were collected for all patients. Results: The average time for patients to have their first appointment at the mdt clinic was 15.1 months, consisting of 3.9 months for patients to see a health care provider (hcp) for the first time since symptom onset and 10.7 months from first hcp appointment to the mdt clinic. Patients saw an average of 3 hcps before the mdt clinic visit (range: 1-7). No significant differences in time to presentation were found based on stage at presentation or anatomic site. Conclusions: At our tertiary care cancer centre, a patient's clinical pathway to being seen at the mdt clinic shows significant delays, particularly in the time from the first hcp visit to mdt referral. Possible methods to mitigate delay include education about hnc for patients and providers alike, and a more streamlined referral system.

Closure of tracheocutaneous fistula using prefabricated conchal bowl cartilage and a supraclavicular flap.

BACKGROUND: Tracheocutaneous fistula represents one of the most troublesome complications of prolonged tracheostomy. Simple closure of a fistula can be ineffective, particularly in the context of prior surgery and adjuvant radiation. As such, modes of repair have expanded to include locoregional flaps and even free tissue transfers. OBJECTIVE: This paper describes a case of persistent tracheocutaneous fistula in an irradiated patient who had undergone previous unsuccessful attempts at repair. METHOD AND RESULTS: The use of regional fasciocutaneous supraclavicular flap with prefabricated conchal bowl cartilage resulted in successful closure of the tracheocutaneous fistula. CONCLUSION: This represents a novel technique for closure of such fistulas in patients for whom previous attempts have failed. This mode of repair should be added to the surgeon's repertoire of reparative techniques.

The use of standardized order sets to improve adherence to evidence-based postoperative management in major head and neck surgery.

BACKGROUND: Standardized order sets (SOSs) are clinical tools derived from clinical care pathways that have shown improved patient-recovery and economic benefits. The primary objective was to examine the effect of SOSs on adherence to evidence-based postoperative guidelines for laryngectomy patients. METHODS: A retrospective chart review comparing handwritten and SOS-based postoperative physician orders was conducted for consecutive laryngectomies performed (n=70) within a 3-year time period. Orders were analyzed for errors and deviations from evidence-based guidelines. Secondary outcome included complications such as thromboembolic disease, return to operating room, fistula formation, salivary bypass tube, length of hospital stay and death. RESULTS: Approximately 81% of cases utilizing handwritten orders had at least one error (n=36) compared to 38% in the group that used an SOS (n=34) (P<0.0001). Subgroup analyses demonstrated that errors in mechanical deep vein thrombosis prophylaxis (P<0.0001) and antibiotic prophylaxis (P=0.0173) orders were significantly reduced in the SOS group compared to the handwritten group. No significant differences were observed between the two groups for measured postoperative complications (P>0.05) and length of hospital stay (18.6 days in both SOS and handwritten orders groups). CONCLUSIONS: SOSs are associated with reduced errors in postoperative orders. They are important tools to improve adherence to standardized guidelines for surgeries requiring complex postoperative management. Clinical care pathways and Enhanced Recovery After Surgery protocols can use SOSs to ensure appropriate orders are being made.

The impact of seasonal operating room closures on wait times for oral cancer surgery.

BACKGROUND: Operating room slowdowns occur at specific intervals in the year as a cost-saving measure. We aim to investigate the impact of these slowdowns on the care of oral cavity cancer patients at a Canadian tertiary care centre. METHODS: A total of 585 oral cavity cancer patients seen between 1999 and 2015 at the London Health Science Centre (lhsc) Head and Neck Multidisciplinary Clinic were included in this study. Operating room hours and patient load from 2006 to 2014 were calculated. Our primary endpoint was the wait time from consultation to definitive surgery. Exposure variables were defined according to wait time intervals occurring during time periods with reduced operating room hours. RESULTS: Overall case volume rose significantly from 2006 to 2014 (p < 0.001), while operating room hours remained stable (p = 0.555). Patient wait times for surgery increased from 16.3 days prior to 2003 to 25.5 days in 2015 (p = 0.008). Significant variability in operating room hours was observed by month, with lowest reported for July and August (p = 0.002). The greater the exposure to these months, the more likely patients were to wait longer than 28 days for surgery (odds ratio per day [or]: 1.07, 95% confidence interval [ci]: 1.05 to 1.10, p < 0.001). Individuals seen in consultation preceding a month with below average operating room hours had a higher risk of disease recurrence and/or death (hazard ratio [hr]: 1.59, 95% ci: 1.10 to 2.30, p = 0.014). CONCLUSIONS: Scheduled reductions in available operating room hours contribute to prolonged wait times and higher disease recurrence. Further work is needed to identify strategies maximizing efficient use of health care resources without negatively affecting patient outcomes.

Transoral robotic surgery vs. radiotherapy for management of oropharyngeal squamous cell carcinoma - A systematic review of the literature.

BACKGROUND: Intensity-modulated radiation therapy (IMRT) with or without concurrent chemotherapy is widely utilized for the treatment of oropharyngeal squamous cell carcinoma (OPSCC). However, due to significant acute and late toxicities there has been increasing interest in minimally invasive surgical approaches, particularly transoral robotic surgery (TORS) in an attempt to preserve patient quality of life while maintaining oncologic outcomes. The aim of this study was to review the current literature in order to compare primary IMRT versus TORS in the management of OPSCC. METHODS: A MEDLINE search was conducted to identify studies reporting on the outcomes of TORS or IMRT in the treatment of OPSCC. Reference lists were also reviewed for relevant articles. Oncologic, functional, and quality of life data is summarized and discussed. RESULTS: One hundred-ninety papers were identified through the MEDLINE search. An additional 52 papers were retrieved by hand searching the reference lists. Ultimately, 44 papers were identified that discussed outcomes after IMRT or TORS for OPSCC. No outcomes from randomized trials were identified. CONCLUSION: No randomized trials comparing TORS versus IMRT to each other were identified. Uncontrolled reports from the current literature suggest comparable oncologic outcomes with TORS compared to IMRT and functional outcomes may be superior. However, the median follow-up was relatively short and the TORS studies included patients with earlier stage OPSCC on average compared to IMRT studies. Prospective, randomized controlled trials and direct, well-matched comparisons are needed to further elucidate the role for TORS in the treatment of oropharyngeal squamous cell carcinoma.

Defining the genomic landscape of head and neck cancers through next-generation sequencing.

Next-generation sequencing (NGS) has revolutionized the field of genomics and improved our understanding of cancer biology. Advances have been achieved by sequencing tumor DNA and using matched normal DNA to filter out germ line variants to identify cancer-specific changes. The identification of high incidences of activating mutations in head and neck squamous cell carcinoma (HNSCC) amenable to drug targeting has been made, with clear distinctions between the mutational profile of HPV-positive and HPV-negative tumors. This wealth of new understanding undoubtedly ameliorates our understanding of HNSCC cancer biology and elucidates clear targets for drug targeting which will guide future personalized medicine.

The epidemic of human papillomavirus and oropharyngeal cancer in a Canadian population.

BACKGROUND: Sexually transmitted infection with the human papillomavirus (hpv) is responsible for a significant burden of human cancers involving the cervix, anogenital tract, and oropharynx. Studies in the United States and Europe have demonstrated an alarming increase in the frequency of hpv-positive oropharyngeal cancer, but the same direct evidence does not exist in Canada. METHODS: Using the London Health Sciences Centre pathology database, we identified tonsillar cancers diagnosed between 1993 and 2011. Real-time polymerase chain reaction was then used on pre-treatment primary-site biopsy samples to test for dna from the high-risk hpv types 16 and 18. The study cohort was divided into three time periods: 1993-1999, 2000-2005, and 2006-2011. RESULTS: Of 160 tumour samples identified, 91 (57%) were positive for hpv 16. The total number of tonsillar cancers significantly increased from 1993-1999 to 2006-2011 (32 vs. 68), and the proportion of cases that were hpv-positive substantially increased (25% vs. 62%, p < 0.002). Those changes were associated with a marked improvement in 5-year overall survival (39% in 1993-1999 vs. 84% in 2006-2011, p < 0.001). When all factors were included in a multivariable model, only hpv status predicted treatment outcome. INTERPRETATION: The present study is the first to provide direct evidence that hpv-related oropharyngeal cancer is increasing in incidence in a Canadian population. Given the long lag time between hpv infection and clinically apparent malignancy, oropharyngeal cancer will be a significant clinical problem for the foreseeable future despite vaccination efforts.

The use of autologous platelet and plasma products in salvage neck dissections: a prospective clinical study evaluating early and late wound healing.

OBJECTIVES: To evaluate the effect of autologous platelet and plasma adhesives (APA) on postoperative drainage and soft-tissue fibrosis following neck dissections. DESIGN: This was a blinded comparative prospective cohort study done as two parts: part one evaluated early post-surgical outcomes and part two evaluated late tissue fibrosis. METHOD: Salvage neck dissections were stratified into two groups based on severity of prior treatment. High risk patients were defined as those who had previously undergone chemoradiation therapy and autologous platelet adhesives were administered to the surgical wound intraoperatively. The low risk group consisted of patients undergoing salvage neck dissections following radiation only and acted as controls. Part one evaluated postsurgical wound drainage as the primary outcome as well as length of hospital stay and complications. Part two evaluated late postoperative tissue fibrosis by comparing neck skin using the Cutometer. R2 and F0 were the specific Cutometer parameters for quantifying the viscoelastic properties of the skin. RESULTS: Postoperative wound drainage was significantly less (253.7 vs. 345.8) in the autologous platelet adhesive group as compared to the control group (p less than 0.03). Length of stay in the APA group versus the control group was 3.13 and 3.86 days respectively (p less than 0.004). Both R2 and F0 measurements showed improved viscoelastic properties of the skin in the APA group (R2 p less than 0.05, F0 p less than 0.05). CONCLUSIONS: APA application following salvage neck dissections may reduce early postperative wound drainage and improve long-term skin quality.

The effects of chronic mercuric chloride ingestion in female Sprague-Dawley rats on fertility and reproduction.

Thirty-days-old female rats were chronically exposed, for 60 days, to 1or 2mg/kg/day of mercuric chloride or an equivalent volume of water, via gavage. At 90 days of age they were mated with unexposed males. At approximately day 13 of gestation necropsies were performed on the females. Data were collected on the number of implantations and non-viable implantations in the uterus. No physical signs of Hg intoxication were seen except in weight gain. There were significantly fewer implantations in the high HgCl2 group, with significantly more non-viable implantations in the low and high HgCl2 groups, compared to controls. Lower levels of progesterone and higher levels of pituitary luteinizing hormone (LH) were found in the high HgCl2 group compared to controls, whereas pituitary follicle stimulating hormone levels (FSH), while not significant, showed a dose-response relationship to HgCl2 levels. No difference was found in the number of corpora lutea. The experiment indicated low level chronic ingestion of mercuric chloride, in female rats, while not effecting ovulation, produced disruption of implantation and fetal viability. Lower progesterone levels, higher LH, and possibly FSH levels, indicate that mercuric chloride may have a disruptive effect in the corpora lutea which manifests itself after ovulation.

A chlorodiazirine analog of pentamidine with anti-trypanosomal activity.

A chlorodiazirine derivative of pentamidine was synthesized and tested for anti-trypanosomal activity using EATRO stock 164 trypanosomes in cell culture. Anti-trypanosomal activity was measured as a decrease in [3H]hypoxanthine incorporation by the organisms. The derivative, 3,3'-[1,5-pentanediylbis(oxy-4,1-phenylene)]bis(3-chloro-3H-diazir ine), at a treatment level of 0.1 microM inhibited isotope incorporation by 40-50% compared to nontreated controls. At this concentration, pentamidine inhibited incorporation only 10-15%. The derivative is a nonionic molecule with much different solubility properties than the parent compound and should readily cross the blood-brain barrier.

Nasalance statistics for two Mexican populations.

OBJECTIVES: This study was undertaken (1) to obtain nasalance statistics for 152 Spanish-speaking normal males and females in three age groups (young children, older children, and adults) from two cities in central Mexico, (2) to determine the reliability of nasalance statistics, and (3) to test whether cities, age group, and/or gender significantly affected nasalance scores. DESIGN/PARTICIPANTS: Randomly selected volunteers, screened for normal speech and hearing, in two middle-class neighborhoods in each city participated. SETTINGS: Measures were made in private homes. MEASURES: Eight nasalance measures were obtained: means, "deviations," maxima, and minima for two sets of sentences; one set that contained no nasals (NN); and another set that included sentences containing several words with nasal sounds (N). RESULTS: Reliability (Cronbach's alpha) of the measures was demonstrated to be very high (0.87 to 0.95). Eight multivariate analyses of variance, one for each type of measure, were performed. Significant differences among subgroups were limited to four: (1) cities differed in their NN means, (2) age groups differed in their NN minima, (3) cities differed in their N minima, and (4) genders differed in their N minima. (Bonferroni adjusted alphas = 0.05).

Anticonvulsant activity of 4-urea-5,7-dichlorokynurenic acid derivatives that are antagonists at the NMDA-associated glycine binding site.

Twelve 4-urea-5,7-dichlorokynurenic acid derivatives were synthesized by reacting the 4-tosylimino derivative of 5,7-dichlorokynurenate methyl ester first with triphosgene and then with a secondary amine. Compounds were screened in mice for anticonvulsant activity using maximal electroshock (MES), subcutaneous pentylenetetrazole (Met), and threshold tonic extension (TTE) tests. A rotorod test was used to determine neurotoxicity. Seven of the derivatives had anticonvulsant activity in TTE testing at 100 mg/kg. One compound, 2-methyl carboxylate-5,7-dichloro-4-([¿diphenylamino¿-carbonyl]amino)-quino line, had an ED50 value of 134 mg/kg (95% conf. int.: low-78.5, high-205.7; slope 1.9, SE = 0.44) in TTE testing. Two derivatives had MES activity. Only one compound, an N,N-diethylamino derivative, was neurotoxic in the rotorod test. Compounds were screened at a 10-microM concentration for activity in displacing 5,7-dichlorokynurenic acid from synaptosomal membrane fragments. Since 9 of the 12 compounds synthesized and tested have demonstrated anticonvulsant activity, this class of chemicals offers promise for the production of useful therapeutic agents.

Anticonvulsant activity of antagonists for the NMDA-associated glycine binding site.

Coupled to the N-methyl-D-aspartate (NMDA) receptor-channel complex is a strychnine-insensitive binding site for glycine. Pharmacological antagonism of glycine binding at this site can produce anticonvulsant activity. Derivatives of the glycine antagonists kynurenic acid and 2-carboxy-indole were synthesized and evaluated for anticonvulsant effects. Compounds were tested in mice against seizures induced by electroshock and pentylenetetrazole, and in the rotorod assay for neurological deficit. The derivatives were also assayed for binding at the NMDA-associated glycine site. The most potent anticonvulsant was ethyl 4-methylamino-5,7-dichloro-2-quinoline carboxylate. This compound provided protection against maximal electroshock (MES) induced seizures at a dose level including 5-fluoro-2-indole carboxylic acid and the diethyl ester of 2,6-pyridine dicarboxylic acid.

gamma-Hydroxybutyric acid binding sites: interaction with the GABA-benzodiazepine-picrotoxin receptor complex.

The effect of three compounds known to allosterically modulate binding to the GABA/benzodiazepine/picrotoxin receptor complex on 4-hydroxy-2,3 [3H]butyric acid (GHB) binding was investigated. Pentobarbital, pentylenetetrazole, and picrotoxin enhanced [3H]GHB binding in a dose dependent fashion. Pentobarbital enhanced 4-hydroxy-2,3 [3H]butyric acid binding was associated with an increase in Bmax while pentylenetetrazole and picrotoxin altered the affinity of GHB for its binding site producing a decrease in Kd. These findings suggest that the GHB and GABA receptor complex may share certain moieties in common.

The development of procedures for the measurement of vocal loudness behaviors.

Vocal loudness productions of five similar groups of speakers were analyzed. The groups differed significantly in "comfortable" loudness, in multiples (2x, 4x, and 0.5x) of comfortable loudness, and in ranges (4x -0.5x) of comfortable loudness. Interclass reliabilities for sets of measures were high and highly significant. Implications for the specification of vocal loudness behaviors are discussed.

Esophageal intelligibility training: back consonants and clusters.

Seven esophageal speakers recorded multiple choice intelligibility lists loaded with words beginning with +BACK consonants and clusters. (A third of the items began with -BACK consonants and clusters). After recording several lists, they played them back and scored them, noting their errors for independent practice. After eight sessions (four weeks) of practice, prepractice and postpractice recordings were randomized and presented to a group of naive listeners. The group scores for the +BACK words improved significantly from prepractice to postpractice (84.1% to 90.6%). The average gain per session for +BACK practice was 0.81%, a result that was in close agreement with prior research. The average gain for the less-practiced -BACK items was 0.46%.

gamma-Hydroxybutyric acid binding sites: evidence for coupling to a chloride anion channel.

The effect of eight anions, including chloride, on the binding of gamma-hydroxy[2,3-3H]butyric acid (GHB) to synaptosomal membranes of rat and human brain was ascertained, as was the effect of a number of other allosteric modulators of the GABA/benzodiazepine/picrotoxin complex. All ions which were active at the chloride ion channel, inhibited the binding of [3H]GHB in a dose-dependent manner, with maximum inhibition of binding being 60% of 300 mM concentration of anion. Inactive ions in this binding system included sulfate, acetate and fluoride, all impermeable to the chloride ion channel. The inhibition of binding was temperature-dependent, being abolished at 37 degrees C and was independent of the cation used. The binding of [3H]GHB was also enhanced by pentobarbital, picrotoxin and diazepam but unchanged in the presence of GABA, muscimol, bicuculline, baclofen or strychnine. These data raise the possibility that the epileptogenic effect of GHB may be modulated by an action on the chloride ion channel, that is tightly coupled to the GABA/benzodiazepine/picrotoxin and/or GHB receptor complex.

The vocal tract model of placement: II. Acoustic aspects.

The technique of "placing" the voice in the throat, head, or elsewhere is used in training singers of operatic quality and in vocal rehabilitation. A vocal tract model of placement has been shown to have perceptual validity (Nichols and Shellenberger, 1985). This study tested its acoustic validity. Line spectra of vowels at different vocal placements were analyzed. We found that Formant 2 reflected changes in vocal placement for both male and female speakers.