Steroid eluting biocompatible stent for subglottic stenosis.

OBJECTIVE: Develop a prototype steroid eluting stent suitable for endoscopic treatment of subglottic stenosis. METHODS: Rectangular-shaped spoke design stents thermally molded into horseshoe-shaped stents were developed using AutoCAD program, and printed on a Lulzbot 3D printer with polycaprolactone (PCL). Kenalog saturated AEROSIL 200 was embedded in the PCL filament. Horizontal radial force measurements were measured at baseline, 1 day, and 1 month when deformation switched from bending to compression. Amount of Kenalog eluted after 1 day, 1 week and 1 month were measured using HPLC. RESULTS: Horizontal pressure applied to the PCL stent corresponding to a 5-0 ET were 1.27 ± 0.38 lb. at baseline, 1.79 ± 0.045 lb. at 1 day, 1.94 ± - 0.22 lb. at 1 week and 2.07 ± 0.11 lb. at 1 month. The horizontal pressure applied to PCL stent corresponding to an 8-0 ET tube were 0.82 ± 0.018 lb. at baseline, 1.008 ± 0.045 lb. at 1 day, 0.95 ± - 0.064 lb. at 1 week and 1.078 ± 0.021 lb. at 1 month. The amount of Kenalog eluted increased from 5.78 µg/mL at 1 day to 15.01 µg/mL at 1 week to 19.35 µg/mL at 1 month. CONCLUSION: This proof-of-concept project is an initial step to demonstrate and create a novel stent in the treatment of subglottic stenosis that applies expansile force on the trachea, elutes steroids and dissolves. Over time the expansile force along the trachea increases allowing the PCL to mucosalize, while it dissolves and continues to elute steroids. The limitations of this in vitro study necessitate experiments on animal models, such as rabbit tracheas to observe for complications and histologic changes. LEVEL OF EVIDENCE: This proof-of-concept project is a Level 5 mechanism-based reasoning study.

Ym155 Induces Oxidative Stress-Mediated DNA Damage and Cell Cycle Arrest, and Causes Programmed Cell Death in Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid cancer (ATC) is one of the most lethal malignancies with a median survival time of about 4 months. Currently, there is no effective treatment, and the development of new therapies is an important and urgent issue for ATC patients. YM155 is a small molecule that was identified as the top candidate in a high-throughput screen of small molecule inhibitors performed against a panel of ATC cell lines by the National Cancer Institute. However, there were no follow-up studies investigating YM155 in ATC. Here, we determined the effects of YM155 on ATC and human primary benign thyroid cell (PBTC) survival with alamarBlue assay. Our data show that YM155 inhibited proliferation of ATC cell lines while sparing normal thyroid cells, suggesting a high therapeutic window. YM155-induced DNA damage was detected by measuring phosphorylation of γ-H2AX as a marker for DNA double-strand breaks. The formamidopyrimidine-DNA glycosylase (FPG)-modified alkaline comet assay in conjunction with reactive oxygen species (ROS) assay and glutathione (GSH)/glutathione (GSSG) assay suggests that YM155-mediated oxidative stress contributes to DNA damage. In addition, we provide evidence that YM155 causes cell cycle arrest in S phase and in the G2/M transition and causes apoptosis, as seen with flow cytometry. In this study, we show for the first time the multiple effects of YM155 in ATC cells, furthering a potential therapeutic approach for ATC.

Cytokeratin-8 in Anaplastic Thyroid Carcinoma: More Than a Simple Structural Cytoskeletal Protein.

Anaplastic thyroid carcinoma (ATC) is almost universally fatal. Elevated keratin-8 (KRT8) protein expression is an established diagnostic cancer biomarker in several epithelial cancers (but not ATC). Several keratins, including KRT8, have been suggested to have a role in cell biology beyond that of structural cytoskeletal proteins. Here, we provide evidence that KRT8 plays a direct role in the growth of ATCs. Genomic and transcriptomic analysis of >5000 patients demonstrates that KRT8 mutation and copy number amplification are frequently evident in epithelial-derived cancers. Carcinomas arising from diverse tissues exhibit KRT8 mRNA and protein overexpression when compared to normal tissue levels. Similarly, in a panel of patient-derived ATC cell lines and patient tumors, KRT8 expression shows a similar pattern. sh-RNA-mediated KRT8 knockdown in these cell lines increases apoptosis, whereas forced overexpression of KRT8 confers resistance to apoptosis under peroxide-induced cell stress conditions. We further show that KRT8 protein binds to annexin A2, a protein known to mediate apoptosis as well as the redox pathway.

YM155 inhibits topoisomerase function.

YM155 (sepantronium bromide) has been evaluated in clinical trials as a survivin suppressant, but despite positive signals from early work, later studies were negative. Clarification of the mechanism of action of YM155 is important for its further development. YM155 affects cells in a cell cycle-specific manner. When cells are in G1, YM155 prevented their progression through the S phase, leaving the cells at G1/S when exposed to YM155. Passage through mitosis from G2 is also defective following YM155 exposure. In this study, YM155 did not behave like a typical DNA intercalator in viscosity, circular dichroism, and absorption spectroscopy studies. In addition, molecular modeling experiments ruled out YM155 DNA interaction to produce DNA intercalation. We show that YM155 inhibited topoisomerase 2α decatenation and topoisomerase 1-mediated cleavage of DNA, suggesting that YM155 inhibits the enzyme function. Consistent with these findings, DNA double-strand break repair was also inhibited by YM155.

Transgelin increases metastatic potential of colorectal cancer cells in vivo and alters expression of genes involved in cell motility.

BACKGROUND: Transgelin is an actin-binding protein that promotes motility in normal cells. Although the role of transgelin in cancer is controversial, a number of studies have shown that elevated levels correlate with aggressive tumor behavior, advanced stage, and poor prognosis. Here we sought to determine the role of transgelin more directly by determining whether experimental manipulation of transgelin levels in colorectal cancer (CRC) cells led to changes in metastatic potential in vivo. METHODS: Isogenic CRC cell lines that differ in transgelin expression were characterized using in vitro assays of growth and invasiveness and a mouse tail vein assay of experimental metastasis. Downstream effects of transgelin overexpression were investigated by gene expression profiling and quantitative PCR. RESULTS: Stable overexpression of transgelin in RKO cells, which have low endogenous levels, led to increased invasiveness, growth at low density, and growth in soft agar. Overexpression also led to an increase in the number and size of lung metastases in the mouse tail vein injection model. Similarly, attenuation of transgelin expression in HCT116 cells, which have high endogenous levels, decreased metastases in the same model. Investigation of mRNA expression patterns showed that transgelin overexpression altered the levels of approximately 250 other transcripts, with over-representation of genes that affect function of actin or other cytoskeletal proteins. Changes included increases in HOOK1, SDCCAG8, ENAH/Mena, and TNS1 and decreases in EMB, BCL11B, and PTPRD. CONCLUSIONS: Increases or decreases in transgelin levels have reciprocal effects on tumor cell behavior, with higher expression promoting metastasis. Chronic overexpression influences steady-state levels of mRNAs for metastasis-related genes.

Evaluating kinase ATP uptake and tyrosine phosphorylation using multiplexed quantification of chemically labeled and post-translationally modified peptides.

Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease.

Mechanical Modeling of the Human Cricoid Cartilage Using Computer-Aided Design: Applications in Airway Balloon Dilation Research.

OBJECTIVES: Balloon dilation is generally considered first-line treatment for airway stenosis. Some dilation systems utilize a compliant balloon that can conform around rigid structures. Others use a noncompliant balloon that does not conform, allowing for dilation of more rigid stenoses. We hypothesized that subglottic dilation with a noncompliant balloon increases the likelihood of fracture of the cricoid when compared to a compliant balloon. METHODS: Three fresh human cricoid cartilages were placed in a universal testing system to determine the expansile force necessary for cricoid fracture. Using these data, a 3D printer was used to construct a synthetic cricoid model possessing near identical physical characteristics to the human cricoid. Simulated dilation was then performed on the model using a compliant and a noncompliant balloon. RESULTS: Human cricoid fracture occurred at 97.25 N (SD = 8.34), and the synthetic cricoid model fractured at 100.10 N (SD = 7.32). Both balloons fractured the model in every replicate experiment. Mean balloon internal pressure at fracture was 7.67 ATM (SD = 1.21) for the compliant balloon and 11.34 ATM (SD = 1.29) for the noncompliant balloon. CONCLUSIONS: These data show that fracture of the cricoid is a valid concern in balloon dilation procedures where the balloon spans the subglottis. Furthermore, the hypothesis was rejected in that the compliant balloon system was at least as likely to fracture the cricoid model as the noncompliant.

Tracheal size variability is associated with sex: implications for endotracheal tube selection.

PURPOSE: Whereas selection of endotracheal tube (ETT) size in pediatric patients benefits from predictive nomograms, adult ETT sizing is relatively arbitrary. We sought to determine associations between cervical tracheal cross-sectional area (CTCSA) and clinical variables. METHODS: One hundred thirty-two consecutive patients undergoing noncontrasted chest computed tomography (CT) at a single tertiary care institution from January 2010 to June 2011 were reviewed. Patients with improper CT technique, endotracheal intubation, and pulmonary/tracheal pathology were excluded. Tracheal luminal diameters in anteroposterior (D1) and transverse (D2) were measured 2 cm inferior to the cricoid and used to determine CTCSA = π*D1*D2*». The demographic variables of age, height, weight, and body mass index (BMI) were tested for association with CTCSA by Spearman correlation. Wilcoxon rank-sum test was used to compare CTCSA by race and sex. Multivariate linear regression was performed including all clinical variables. RESULTS: There were 91 patients who met inclusion criteria. There was no correlation between age, weight, or BMI and CTCSA. There was a significant positive correlation between patient height and CTCSA (P = .001, R = 0.35); however, this was confounded by sex. Female patients had significantly smaller CTCSA (mean = 241 mm(2)) compared to male patients (mean = 349 mm(2), P < .001). Multivariate linear regression stratified by sex revealed that height is correlated with CTCSA only in males (P = .028). Males also had more variability in CTCSA (SD 118.6) compared to females (SD 65.5). CONCLUSION: Our data suggest that selection of ETT size in male patients should include height as a predictive factor. For female patients, it may be appropriate to select a uniformly smaller diameter ETT size.

The effect of voluntary pharyngeal swallowing maneuvers on esophageal swallowing physiology.

The purpose of our study was to evaluate whether swallowing maneuvers designed to impact pharyngeal physiology would also impact esophageal physiology. Healthy volunteers underwent high-resolution manometry while performing three randomized swallowing maneuvers with and without a 5-ml bolus: normal swallowing, Mendelsohn maneuver, and effortful swallowing. We examined esophageal parameters of peristaltic swallows, hypotensive or failed swallows ("nonperistaltic swallows"), distal contractile integral (DCI), contractile front velocity (CFV), intrabolus pressure, and transition zone (TZ) defect. Four females and six males (median age 39 years; range 25-53) were included in the study. The overall number of nonperistaltic swallows was 21/40 (53 %) during normal swallowing, 27/40 (66 %) during the Mendelsohn maneuver, and 13/40 (33 %) during effortful swallowing. There were significantly more overall nonperistaltic swallows with the Mendelsohn maneuver compared with effortful swallowing (p = 0.003). While swallowing a 5-ml bolus, there were more nonperistaltic swallows during the Mendelsohn maneuver (12/20, 60 %) compared to that during normal swallowing (6/20, 30 %) (p = 0.05) and more peristaltic swallows during effortful swallowing as compared to Mendelsohn maneuver (p = 0.003). Intrabolus esophageal pressure was greater during the Mendelsohn maneuver swallows in the bolus-swallowing condition as compared to normal swallowing (p = 0.02). There was no statistical difference in DCI, CFV, or TZ defect between swallowing conditions. The Mendelsohn maneuver may result in decreased esophageal peristalsis while effortful swallowing may improve esophageal peristalsis. Because it is important to understand the implications for the entire swallowing mechanism when considering retraining techniques for our patients, further investigation is warranted.

The genomic and evolutionary landscapes of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations.

Topical bethanechol for the improvement of esophageal dysmotility: a pilot study.

OBJECTIVES: We studied a case series to evaluate the effect of topical bethanechol chloride on esophageal function in individuals with ineffective esophageal motility. METHODS: Five subjects with ineffective esophageal motility underwent high resolution esophageal manometry. Ten 5 mL liquid swallows were performed to establish a baseline. Five milligrams of topical bethanechol was then administered. After 10 minutes, the subjects completed 10 additional liquid swallows. This procedure was repeated with 10 mg of bethanechol in 4 subjects. RESULTS: After administration of 5 mg of topical bethanechol, the mean (+/- SD) distal contractile integral, an index of esophageal contractility, increased from 178.3 +/- 83.1 mm Hg x s x cm to 272.3 +/- 216.9 mm Hg x s x cm (p = 0.69). The percentage of failed swallows decreased from 52.8% +/- 33.2% to 29.4% +/- 18.3% (p = 0.14). The percentage of peristaltic swallows increased from 28.0% +/- 26.8% to 67.2% +/- 15.3% (p = 0.04). The contractile front velocity was essentially unchanged. After administration of 10 mg of bethanechol,the distal contractile integral decreased from 349.3 +/- 371.0 mm Hg x s x cm to 261.8 +/- 293.5 mm Hg x s x cm (p = 0.72). The percentage of failed swallows increased from 57.5% +/- 37.7% to 66.8% +/- 24.9% (p = 0.46). The percentage of peristaltic swallows increased from 17.5% +/- 23.6% to 28.3% +/- 19.1% (p = 0.29). The contractile front velocity decreased from 11.6 +/- 5.2 cm/s to 4.9 +/- 3.0 cm/s (p = 0.32). No adverse side effects occurred. CONCLUSIONS: The results of this pilot study support the need for further investigation with larger sample sizes and dose escalation.

The body as a living bioreactor: a feasibility study of pedicle flaps for tracheal transplantation.

Reconstruction of long-segment tracheal stenosis remains problematic. Ex vivo transplantation of stem cell-derived tracheas has been established in humans using external tissue bioreactors. These bioreactors, however, are not widely accessible. Thus, we are developing a rotational flap-based "internal bioreactor" to allow in vivo stem cell engraftment in a pre-vascularized recipient bed. This muscle will also then serve as a carrier for the transplanted trachea during rotation into position for airway reconstruction. Herein, we present a study investigating the feasibility of two pedicle muscle flaps for implantation and subsequent tracheal transplantation. Trapezius and latissimus flaps were raised using established surgical techniques. The length and width of each flap, along with the distance from the pedicle takeoff to the trachea, were measured. The overall ability of the flaps to reach the trachea was assessed. Twelve flaps were raised in 5 fresh adult human cadavers. For the trapezius flap, averages were: flap length of 16.4 cm, flap width of 5.95 cm at the tip, and distance from the pedicle takeoff to the trachea of 11.1 cm. For the latissimus dorsi flap, averages were: flap length of 35.4 cm, flap width of 7.25 cm at the tip, and distance from the pedicle takeoff to the trachea of 27.3 cm. All flaps showed sufficient durability and rotational ability. Our results show that both trapezius and latissimus dorsi flaps can be transposed into the neck to allow tension-free closure of tracheal defects. For cervical tracheal transplantation, both flaps are equally adequate. We believe that trapezius and latissimus dorsi muscle flaps are potential tracheal implantation beds in terms of vascular supply, durability, and rotational ability.

Hemodynamic changes during otolaryngological office-based flexible endoscopic procedures.

OBJECTIVES: A preponderance of literature supports the safety of office-based flexible endoscopic procedures of the upper aerodigestive tract; however, until recently there were no data regarding hemodynamic stability during these procedures. A recent study showed intraprocedure changes in patients' hemodynamic parameters, raising the concern that perhaps patients should be monitored during these procedures. The aim of our study was to determine whether physiologically significant alterations in vital signs occur during office-based flexible endoscopic procedures. METHODS: We performed a retrospective review of 100 consecutive patients who underwent office-based flexible endoscopic procedures of the upper aerodigestive tract from July 2010 to October 2011. Baseline values and the maximal changes in systolic blood pressure, diastolic blood pressure, heart rate, and oxygen saturation were recorded and compared. RESULTS: One hundred consecutive patients were included in the study. Twenty-one patients (21%) had severe hypertension and 40 patients (40%) had tachycardia during the procedure. The mean change overall in systolic blood pressure was 26.2 mm Hg (p < 0.001), the mean change in diastolic blood pressure was 13.9 mm Hg (p < 0.001), the mean change in heart rate was 16.6 beats per minute (p < 0.001), and the mean change in oxygen saturation was 1.6% (p < 0.001). These changes were significant. On further breakdown into groups, patients over 50 years of age and patients who were undergoing esophageal or laser procedures had significant elevations in heart rate (p = 0.01 and p = 0.04, respectively). An elevation in diastolic blood pressure was also significant in patients who were undergoing esophageal or laser procedures (p = 0.04 for both). CONCLUSIONS: These data concur with those of the previous report that found potentially significant hemodynamic changes during office-based procedures. Although preliminary, our findings suggest that it may be wise to monitor vital signs in patients over 50 years of age and patients who are undergoing an esophageal or laser procedure who are at risk for complications that could arise from tachycardia and hypertension.

Metal Allergy in Tracheostomy Tube Placement Resulting in Complete Subglottic Stenosis: A Case Report.

OBJECTIVES: Metal hypersensitivity reaction to surgical implants is a well- known phenomenon that is associated with pain, swelling, inflammation, and decreased efficacy of the implant. We present a unique case of a patient with placement a metal Jackson tracheostomy tube that led to expeditious total subglottic stenosis. METHODS: The patient was a 33-year old, severely atopic woman with history of asthma exacerbations requiring several intubations for acute respiratory failure with several subsequent tracheal dilations with steroid injections, and eventual tracheostomy placement with a metal Jackson tracheostomy tube that led to expeditious total subglottic stenosis. RESULTS: Initial intervention included performing an airway evaluation, CO2 laser, and steroid injection of the area of complete subglottic stenosis. Follow up several months later revealed little improvement in level of tracheal narrowing proximal to the tracheostomy tube. Patient did not have shortness of breath but continued to be aphonic. Cricotracheal versus tracheal resection have been proposed but surgical morbidity was deemed too high due to patient's obesity. CONCLUSIONS: Metal hypersensitivity reactions are well known phenomena as it relates to surgical implants in other surgical specialties but are seldom reported within the ear, nose and throat literature. Oftentimes, it takes astute observation to diagnose and establish a connection. Prompt recognition and treatment can be acquired from interdisciplinary collaboration with allergy.

Pilot Study: Pneumatic Compression Garment Therapy for Postradiotherapy Laryngopharyngeal Edema.

OBJECTIVES: Pneumatic compression garment therapy (PCGT) has been established as treatment for postradiotherapy lymphedema, and its use in head and neck patients is becoming more common. Although effects on interstitial edema of the cervical soft tissues have been studied, effects on internal laryngopharyngeal edema, as well as associated symptoms of dysphagia and dysphonia, have yet to be published. METHODS: We surveyed 7 patients treated with radiation for head and neck cancer (HNC) who had also been prescribed PCGT for cervical lymphedema. Patients were asked about subjective experience with the device, and also administered the Eating Assessment Tool-10 (EAT-10) and Voice Handicap Index-10 (VHI-10) surveys regarding their symptoms after using PCGT. Laryngoscopy videos from these same periods were also reviewed and scored using a validated tool for assessing laryngopharyngeal edema. RESULTS: 85% of patients reported at least some improvement in dysphagia and dysphonia following PCGT. Average EAT-10 score after PCGT was 11.4 and average VHI-10 score after PCGT was 8.7. These compare more favorably to historical scores for the same questionnaires in similar patient populations. Laryngeal edema scores on endoscopic examination were not significantly different after at least 3 months of therapy (pre: 20.15, post: 20.21, P = .975); however, the utility of this result is limited by a low inter-rater reliability (Krippendorff α = .513). CONCLUSIONS: While we are unable to show any difference in objective assessment of laryngopharyngeal edema on endoscopic examination in this small pilot study, patients report substantial subjective improvement in postradiotherapy dysphagia and dysphonia following cervical PCGT that warrants more formal investigation.

YM155 Induces DNA Damage and Cell Death in Anaplastic Thyroid Cancer Cells by Inhibiting DNA Topoisomerase IIα at the ATP-Binding Site.

Anaplastic thyroid cancer (ATC) is among the most aggressive of human cancers, and currently there are few effective treatments for most patients. YM155, first identified as a survivin inhibitor, was highlighted in a high-throughput screen performed by the National Cancer Institute, killing ATC cells in vitro and in vivo. However, there was no association between survivin expression and response to YM155 in clinical trials, and YM155 has been mostly abandoned for development despite favorable pharmacokinetic and toxicity profiles. Currently, alternative mechanisms are being explored for YM155 by a number of groups. In this study, ATC patient samples show overexpression of topoisomerase Top2α compared with benign thyroid samples and to differentiated thyroid cancers. ATC cell lines that overexpress Top2α are more sensitive to YM155. We created a YM155-resistant cell line, which shows decreased expression of Top2α and is resensitized with Top2α overexpression. Molecular modeling predicts binding for YM155 in the Top2α ATP-binding site and identifies key amino acids for YM155-Top2α interaction. A Top2α mutant abrogates the effect of YM155, confirming the contribution of Top2α to YM155 mechanism of action. Our results suggest a novel mechanism of action for YM155 and may represent a new therapeutic approach for the treatment of ATC.

Point-of-care rapid intraoperative parathyroid hormone assay of needle aspirates from parathyroid tissue: a substitute for frozen sections.

BACKGROUND: The intraoperative parathyroid hormone (IOPTH) assay is an important tool that facilitates targeted parathyroidectomy and may increase surgical cure rates. We sought to evaluate the utility of a point-of-care assay to distinguish parathyroid adenomas from nonparathyroid tissues, which can sometimes pose a challenge during parathyroidectomy and is commonly achieved with the use of frozen sections. We hypothesized that point-of-care rapid IOPTH assays of needle aspirates from suspected adenomas would be faster and equal in accuracy to frozen sections. METHODS: Parathyroid and nonparathyroid lesions were excised, and the tissues were needle aspirated, diluted in saline, and submitted to a rapid IOPTH assay located in the operating room. Frozen sections were simultaneously sent for analysis. The time intervals to result availability were tracked and compared using a paired t test. RESULTS: Point-of-care IOPTH assays of needle aspirates were available in a mean (±SD) of 11.6 ± 1.5 minutes compared to 18.7 ± 4.0 minutes for frozen sections (P = .005). The findings were concordant 100% of the time for both parathyroid (mean parathyroid hormone [PTH] > 3338.9 pg/mL) and parathyroid tissues (mean PTH = 8.7 pg/mL). CONCLUSION: Point-of-care IOPTH assay of needle aspirates is an accurate method of distinguishing parathyroid from nonparathyroid tissues. It is suggested that this would be particularly useful in instances where use of IOPTH is planned for assessment of a drop in serum PTH.

R-Loop Physiology and Pathology: A Brief Review.

Physiological and pathological roles for R-loop structures continue to be discovered, and studies suggest that R-loops could contribute to human disease. R-loops are nucleic acid structures characterized by a DNA:RNA hybrid and displaced single-stranded DNA that occur in connection with transcription. R-loops form naturally and have been shown to be important for a number of physiological processes such as mitochondrial replication initiation, class switch recombination, DNA repair, modulating DNA topology, and regulation of gene expression. However, subsets of R-loops or persistent R-loops lead to DNA breaks, chromosome rearrangement, and genome instability. In addition, R-loops have been linked to human diseases, specifically neurological disorders and cancer. Of the large amount of research produced recently on R-loops, this review covers evidence for R-loop involvement in normal cellular physiology and pathophysiology, as well as describing factors that contribute to R-loop regulation.

Defining molecular phenotypes of human papillomavirus-associated oropharyngeal squamous cell carcinoma: validation of three-class hypothesis.

OBJECTIVE: The purpose of this study was to determine if oropharyngeal squamous cell carcinoma (OSCC) classified into three groups based on human papillomavirus (HPV) 16 DNA presence and p16 expression display different protein expression patterns. STUDY DESIGN: Cross-sectional study. SETTING: A laboratory-based study of patients with OSCC treated at a tertiary care academic medical center. SUBJECTS AND METHODS: Paraffin-embedded OSCC specimens from 77 patients classified into the three-class model (HPV negative, HPV inactive [HPV16+/p16-], and HPV active [HPV16+/p16+]) were queried for the expression of 14 tumor progression proteins using AQUA (HistoRx, New Haven CT). Protein expression between groups was assessed by analysis of variance. Global expression patterns were determined by unsupervised hierarchical clustering. RESULTS: There were significant differences in expression of beta-catenin (P = 0.009), epidermal growth factor receptor (P = 0.009), and vascular endothelial growth factor (P = 0.028) between groups. HPV-active tumors had overexpression of beta-catenin. Hierarchical clustering showed HPV-negative and HPV-inactive tumors displayed association patterns distinct from HPV-active tumors. CONCLUSIONS: Tumors classified by HPV DNA presence and p16 expression have different molecular phenotypes. This is the first demonstration of overexpression of beta-catenin (also found in HPV-caused cervical cancer) in HPV-active OSCC. HPV-active OSCC may share a similar ontogeny to HPV-caused cervical cancer.