A Prospective Study of Publicly Funded Molecular Testing of Indeterminate Thyroid Nodules: Canada's Experience.

CONTEXT: Indeterminate thyroid nodules (ITNs) lead to diagnostic surgeries in many countries. Use of molecular testing (MT) is endorsed by several guidelines, but costs are limitative, especially in public healthcare systems like in Canada. OBJECTIVES: Primary objective: evaluate the clinical value of Thyroseq® v3 (TSv3) using benign call rate (BCR) in a real-world practice. Secondary objective: assess cost-effectiveness of MT. DESIGN: This is a multicentric prospective study. SETTING: This study was conducted in 5 academic centers in Quebec, Canada. PATIENTS OR OTHER PARTICIPANTS: 500 consecutive patients with Bethesda III (on 2 consecutive cytopathologies) or IV and TIRADS 3 or 4 nodules measuring 1 to 4 cm were included. INTERVENTION: MT was performed between November 2021 and November 2022. Patients with a positive TSv3 were referred to surgery. Patients with a negative TSv3 were planned for follow-up by ultrasonography for a minimum of 2 years. MAIN OUTCOME MEASURE: The BCR, corresponding to the proportion of ITNs with negative TSv3 results, was assessed. RESULTS: 500 patients underwent TSv3 testing, with a BCR of 72.6% (95% CI: 68.5-76.5; p<0.001). 99.7% of patients with a negative result avoided surgery. The positive predictive value of TSv3 was 68.2% (95% CI: 58.5-76.9). The cost-benefit analysis identified that the implementation of MT would yield cost savings of $6.1 million over the next 10 years. CONCLUSIONS: Use of MT (TSv3) in a well-selected population with ITNs led to a BCR of 72.6%. It is cost-effective and prevents unnecessary surgeries in a public healthcare setting.

Inhibition of the CCR6-CCL20 axis prevents regulatory T cell recruitment and sensitizes head and neck squamous cell carcinoma to radiation therapy.

BACKGROUND:  Radioresistance of HNSCCs remains a major challenge for effective tumor control. Combined radiotherapy (RT) and immunotherapy (IT) treatment improved survival for a subset of patients with inflamed tumors or tumors susceptible to RT-induced inflammation. To overcome radioresistance and improve treatment outcomes, an understanding of factors that suppress anti-tumor immunity is necessary. In this regard, regulatory T cells (Tregs) are critical mediators of immune suppression in HNSCCs. In this study, we investigated how radiation modulates Treg infiltration in tumors through the chemokine CCL20. We hypothesized that radiation induces CCL20 secretion resulting in Treg infiltration and suppression of anti-tumor immunity. METHODS:  Human and mouse HNSCC cell lines with different immune phenotypes were irradiated at doses of 2 or 10 Gy. Conditioned media, RNA and protein were collected for assessment of CCL20. qPCR was used to determine CCL20 gene expression. In vivo, MOC2 cells were implanted into the buccal cavity of mice and the effect of neutralizing CCL20 antibody was determined alone and in combination with RT. Blood samples were collected before and after RT for analysis of CCL20. Tumor samples were analyzed by flow cytometry to determine immune infiltrates, including CD8 T cells and Tregs. Mass-spectrometry was performed to analyze proteomic changes in the tumor microenvironment after anti-CCL20 treatment. RESULTS:  Cal27 and MOC2 HNSCCs had a gene signature associated with Treg infiltration, whereas SCC9 and MOC1 tumors displayed a gene signature associated with an inflamed TME. In vitro, tumor irradiation at 10 Gy significantly induced CCL20 in Cal27 and MOC2 cells relative to control. The increase in CCL20 was associated with increased Treg migration. Neutralization of CCL20 reversed radiation-induced migration of Treg cells in vitro and decreased intratumoral Tregs in vivo. Furthermore, inhibition of CCL20 resulted in a significant decrease in tumor growth compared to control in MOC2 tumors. This effect was further enhanced after combination with RT compared to either treatment alone. CONCLUSION:  Our results suggest that radiation promotes CCL20 secretion by tumor cells which is responsible for the attraction of Tregs. Inhibition of the CCR6-CCL20 axis prevents infiltration of Tregs in tumors and suppresses tumor growth resulting in improved response to radiation.

The infraclavicular pedicled flap in head and neck reconstruction: applications and outcomes.

BACKGROUND: An infraclavicular pedicled flap (ICPF) was recently described in the literature. This anatomical region is attractive for the restoration of head and neck oncological defects. This paper is a review of our experience with this versatile flap. METHODS: A retrospective study was conducted by reviewing the records of all the patients operated in a tertiary-care center between August 2013 and January 2019 whose surgery involved an ICPF. RESULTS: Forty-four patients received an ICPF for various indications, including large vessel coverage in neck/parotid recontouring (34.1%), postlaryngectomy reconstruction (34.1%), and fistula closure (25.0%). All flaps survived. Thirteen patients experienced a postoperative complication (29.5%), six of whom (13.9%) required a repeat procedure under general anesthesia. CONCLUSION: ICPF is suitable for several indications and is a useful adjunctive tool in head and neck reconstruction. It proved to have a high survival rate, with complication rates similar to other regional flaps.

Evaluation of PACE4 isoforms as biomarkers in thyroid cancer.

BACKGROUND: To date, no single molecular marker has been demonstrated as clinically useful in differentiating malignant from benign thyroid nodules when a fine needle aspiration falls in the "unknown significance" categories of the Bethesda Classification. PACE4, a member of the proprotein convertase family of enzymes, has been shown to play a major role in the pathogenesis of prostate cancer, through the formation of an oncogenic isoform named PACE4-altCT. PACE4 isoforms have also been suggested to play a role in other cancers, including thyroid cancer, but have never been investigated in a detailed manner. Our objective is to compare the histochemical distribution of the two major PACE4 isoforms in benign and malignant thyroid nodules, in order to determine their potential usefulness as discriminatory biomarkers. METHODS: Thyroid tissues of patients who underwent thyroidectomy were classified according to final pathology. Corresponding tissue sections were immunostained, using two previously validated antibodies raised against the C-terminal end of the two PACE4 isoforms, namely the full-length PACE4 protein (PACE4-FL) and its alternative isoform (PACE4-altCT). Nodules were compared with adjacent normal parenchyma and immunostaining was rated as "low" or "high" by a head and neck pathologist. RESULTS: Non-lesional thyroid parenchyma did not express PACE4-FL (p = 0.002). As a group, malignant (n = 17) nodules expressed PACE4-FL significantly more than benign (n = 24) nodules (percentage of high immunostaining: 52.9% vs 4.2%; p = 0.001). Reciprocally, there was a statistically lower expression of PACE4-altCT in malignant nodules than in adjacent non-lesional parenchyma (p = 0.014). The specificity of a high PACE4-FL immunostaining in determining malignancy was 95.8% (95% CI, 78.9% to 99.9%). CONCLUSION: This study supports the previously described relationship between PACE4-FL and PACE4-altCT through alternative splicing. It also suggests that PACE4-FL is a promising biomarker for thyroid malignancy. Its high specific expression for malignancy could make it an interesting "rule in" test for thyroid cancer. Further prospective, quantitative studies are currently being designed to address how measurements of PACE4 isoforms could be used in a clinical setting. TRIAL REGISTRATION: This study does not report the results of a health care intervention on human participants. It was nonetheless registered on ClinicalTrials.gov under reference number NCT03160482 .

A challenging case of squamous metaplasia in a pleomorphic adenoma: diagnostic and clinical pitfalls.

Squamous metaplasia in a pleomorphic adenoma (PA) has been reported, but rarely has it been documented as being extensive enough to cause significant misdiagnosis. We present a case of a 37-year-old man presenting with a 1.7-cm minor salivary gland PA of the palate. It exhibited extensive squamous metaplasia mimicking a squamous cell carcinoma (SCC) on multiple preoperative biopsies. The final diagnosis was only made after a complete oncological excision with margins and free flap reconstruction. Florid squamous metaplasia in a PA, although rare, should be recognized and distinguished from SCC. Failure to differentiate these entities can lead to patient overtreatment.

Effects of reflux laryngitis on non-nutritive swallowing in newborn lambs.

Reflux laryngitis in infants may be involved not only in laryngeal disorders, but also in disorders of cardiorespiratory control through its impact on laryngeal function. Our objective was to study the effect of reflux laryngitis on non-nutritive swallowing (NNS) and NNS-breathing coordination. Two groups of six newborn lambs, randomized into laryngitis and control groups, were surgically instrumented for recording states of alertness, swallowing and cardiorespiratory variables without sedation. A mild to moderate reflux laryngitis was induced in lambs from the experimental group. A significant decrease in the number of NNS bursts and apneas was observed in the laryngitis group in active sleep (p=0.03). In addition, lower heart and respiratory rates, as well as prolonged apnea duration (p<0.0001) were observed. No physiologically significant alterations in NNS-breathing coordination were observed in the laryngitis group. We conclude that a mild to moderate reflux laryngitis alters NNS burst frequency and autonomous control of cardiac activity and respiration in lambs.

Effects of simulated reflux laryngitis on laryngeal chemoreflexes in newborn lambs.

It has been suggested that reflux laryngitis (RL) is involved in apneas-bradycardias of the newborn. The aim of the present study was to develop a unique RL model in newborn lambs to test the hypothesis that RL enhances the cardiorespiratory components of the laryngeal chemoreflexes (LCR) in the neonatal period. Gastric juice surrogate (2 ml of normal saline solution with HCl pH 2 + pepsin 300 U/ml) (RL group, n = 6) or normal saline (control group, n = 6) was repeatedly injected onto the posterior aspect of the larynx, 3 times a day for 6 consecutive days, via a retrograde catheter introduced into the cervical esophagus. Lambs instilled with gastric juice surrogate presented clinical signs of RL, as well as moderate laryngitis on histological observation. Laryngeal chemoreflexes were thereafter induced during sleep by injection of 0.5 ml of HCl (pH 2), ewe's milk, distilled water or saline into the laryngeal vestibule via a chronic, transcutaneous supraglottal catheter. Overall, RL led to a significantly greater respiratory inhibition compared with the control group during LCR, including longer apnea duration (P = 0.01), lower minimal respiratory rate (P = 0.002), and a more prominent decrease in arterial hemoglobin saturation (SpO(2)) (P = 0.03). No effects were observed on cardiac variables. In conclusion, 1) our unique neonatal ovine model presents clinical and histological characteristics of RL; and 2) the presence of RL in newborn lambs increases the respiratory inhibition observed with LCR, at times leading to severe apneas and desaturations.

Radiation-induced xerostomia: is octreotide the solution?

OBJECTIVE: In this study, we looked for evidence that octreotide, a drug used specifically in acromegaly and other digestive pathologies, can have a radioprotective effect on salivary glands. This effect has already been proven on the pituitary gland, which is why we postulated that octreotide could act the same way on rat parotid glands. METHOD: A prospective randomized controlled study on animals was conducted. With a noninvasive technique, we collected saliva from the parotid glands of 18 anesthetized rats at time 0 (preirradiation) and 1 month (postirradiation). Each sampling technique lasted 40 minutes, with pilocarpine injection at time 0 and 20 minutes. Saliva was collected bilaterally. Eighteen rats, nine in the saline group and nine in the octreotide group, were randomized. The substance was injected 30 minutes before irradiation. Thirty gray were given with the gamma knife on the left parotid gland of each rat following a computerized targeting method. Each gland was examined after the last saliva collection to determine the percentage of five criteria: fibrosis, ducts, fat, vessels, and acini. RESULTS: Data are available for 17 rats (nine in the octreotide group and eight in the saline group). Statistical analysis was done with a t-test (independent and paired). We noted that the postirradiation secretion in the left (radiated) gland was diminished compared with the right (nonradiated) gland in the saline group (p = .014). Fibrosis was increased in the irradiated (left) gland in both groups (p = .024 in the octreotide group and p = .033 in the saline group). The percentage of duct cells was more important in the left (radiated) gland of the octreotide group (p = .046). A trend appeared for a decrease in acinic cells only in the control group (p = .063). CONCLUSION: Octreotide acted as a radioprotective agent on rat parotid glands 1 month after irradiation with 30 Gy given with the gamma knife.

Laryngeal chemoreflexes induced by acid, water, and saline in nonsedated newborn lambs during quiet sleep.

Laryngeal chemoreflexes (LCR) are triggered by the contact of assorted liquids with the laryngeal mucosa. In the neonatal period, the immature LCR consist primarily of apnea and bradycardia, which at times can be life threatening. The aim of this study was to assess LCR induction in nonsedated, newborn full-term lambs by several acid solutions, compared with distilled water and saline. Twelve lambs were instrumented for recording of glottal adductor and diaphragm EMG, EEG, eye movements, heart rate, systemic arterial pressure, and respiratory movements. LCR were induced during quiet sleep by the injection (0.5 ml) of saline, distilled water or two acid solutions (HCl and citric acid, pH 2, diluted in either water or saline). A chronic supraglottal catheter was used to inject the solutions in a random order. Distilled water and acid solutions did not induce any significant decrease in heart rate or respiratory rate. However, significant lower airway protective responses (swallowing, cough, and arousal) were observed after distilled water and especially acid solution administration. In conclusion, LCR in full-term lambs, particularly with acid solutions, are merely characterized by lower airway protective responses resembling mature LCR reported in adult mammals.

Active upper airway closure during induced central apneas in lambs is complete at the laryngeal level only.

We tested the hypotheses that active upper airway closure during induced central apneas in nonsedated lambs 1). is complete and occurs at the laryngeal level and 2). is not due to stimulation of the superior laryngeal nerves (SLN). Five newborn lambs were surgically instrumented to record thyroarytenoid (TA) muscle (glottal constrictor) electromyographic (EMG) activity with supra- and subglottal pressures. Hypocapnic and nonhypocapnic central apneas were induced before and after SLN sectioning in the five lambs. A total of 174 apneas were induced, 116 before and 58 after sectioning of the internal branch of the SLN (iSLN). Continuous TA EMG activity was observed in 88% of apneas before iSLN section and in 87% of apneas after iSLN section. A transglottal pressure different from zero was observed in all apneas with TA EMG activity, with a mean subglottal pressure of 4.3 +/- 0.8 cmH2O before and 4.7 +/- 0.7 cmH2O after iSLN section. Supraglottal pressure was consistently atmospheric. Sectioning of both iSLNs had no effects on the results. We conclude that upper airway closure during induced central apneas in lambs is active, complete, and occurs at the glottal level only. Consequently, a positive subglottal pressure is maintained throughout the apnea. Finally, this complete active glottal closure is independent from laryngeal afferent innervation.

Non-nutritive swallowing and respiration coordination in full-term newborn lambs.

Swallowing is a powerful inhibitor of respiratory rhythm in infants. The present study was aimed at investigating the influence of states of alertness on non-nutritive swallowing (NNS) frequency, on NNS and respiration coordination, and on bursts of NNS frequency in newborn lambs. Six full term newborn lambs were instrumented for electroencephalogram, eye movement, diaphragm and thyroarytenoid muscle electromyogram, nasal flow and electrocardiogram. Polysomnographic recordings were performed in non-sedated lambs, using radiotelemetry. NNS frequency was significantly higher during quiet wakefulness (W) and active sleep (AS) than during quiet sleep (QS). NNS mainly interrupted inspiration and the transition phases between expiration and inspiration, especially in W and AS. Bursts of NNS occurred significantly more often during AS. This study highlights the relevance of the ovine model to study ontogeny of NNS during sleep, and documents the influence of sleep on NNS and respiration coordination.

Active glottal closure during central apneas limits oxygen desaturation in premature lambs.

Our laboratory previously reported that active glottal closure was present in 90% of spontaneous central apneas in premature lambs while maintaining a high-apneic lung volume (Renolleau S, Letourneau P, Niyonsenga T, and Praud JP. Am J Respir Crit Care Med 159: 1396-1404, 1999.) The present study aimed at testing whether this mechanism limits postapnea oxygen desaturation. Four premature lambs were instrumented for recording states of alertness, thyroarytenoid muscle and diaphragm electromyographic (EMG) activity, nasal airflow, lung volume changes, and pulse oximetry. One thousand four hundred fifty-two spontaneous central apneas (isolated or during periodic breathing) were analyzed in nonsedated lambs. Apneas, with high lung volume maintained by active glottal closure, were compared with apneas, with a tracheostomy opened at apnea onset. Oxygen desaturation slopes were lower when high-apneic lung volume was actively maintained during both wakefulness and quiet sleep. Furthermore, oxygen desaturation slopes were lower after isolated apneas with continuous thyroarytenoid EMG during wakefulness, compared with apneas with noncontinuous thyroarytenoid EMG (= glottis opened shortly after apnea onset). These results highlight the importance of maintaining high-alveolar oxygen stores during central apneas by active glottal closure to limit desaturation in newborns.