A Heterotopic Mouse Model for Studying Laryngeal Transplantation.

Laryngeal heterotopic transplantation, although a technically challenging procedure, offers more scientific analysis and cost benefits compared to other animal models. Although first described by Shipchandler et al. in 2009, this technique is not widely used, possibly due to the difficulties in learning the microsurgical technique and time required to master it. This paper describes the surgical steps in detail, as well as potential pitfalls to avoid, in order to encourage effective use of this technique. In this model, the bilateral carotid arteries of the donor larynx are anastomosed to the recipient carotid artery and external jugular vein, allowing for blood flow through the graft. Blood flow can be confirmed intraoperatively by the visualization of blood filling in the graft bilateral carotid arteries, reddening of the thyroid glands of the graft, and bleeding from micro vessels in the graft. The crucial elements for success include delicate preservation of the graft vessels, making the correct size arteriotomy and venotomy, and using the appropriate number of sutures on the arterial-arterial and arterial-venous anastomoses to secure vessels without leakage and prevent occlusion. Anyone can become proficient in this model with sufficient training and perform the procedure in approximately 3 h. If performed successfully, this model allows for immunologic studies to be performed with ease and at low cost.

Safety and Feasibility of a Novel Esophageal Balloon for Circumferential Cytologic Sampling.

OBJECTIVES: A prior publication introduced the Strome-Blitzer balloon's ability to obtain circumferential esophageal cytologic sampling. This GLP study was requisite for FDA approval to determine if equivalent cell capture and cellularity was observed with the balloon compared to surface sampling brushes and to determine the balloon's usability for naive otolaryngologists. METHODS: Three naïve users tested the Hobbs brush and Strome-Blitzer balloon on 4 Yorkshire swine. Four anatomical sites were sampled, beginning distally and ending proximally. In 2 animals, the balloon was used first distally and in the remaining 2, 4 new Hobbs brushes were used distally first. Moving proximally, the balloon and brushes were sequentially alternated. In follow-the-leader fashion, the balloon was introduced trans-orally followed by an endoscope to the desired site. The balloon was inflated exposing the abrasive strips to contact the esophageal mucosa. Moving the balloon 1 to 2 cm superiorly and inferiorly effected circumferential cell capture. The balloon was collapsed and removed, preserving the cellularity. The Hobbs brush was passed through the scope's channel. Four brushes, 1 per quadrant, obtained the samples at an anatomical site. The balloon was rated as pass/fail on the following: delivery, kinking, usability, and malfunction. A blinded veterinary pathologist evaluated the cytology. RESULTS: There was no device malfunction, mucosal trauma, or difficulty with device use. Balloon cytologic samples were comparable in cellularity and quality to the brush. CONCLUSION: A single balloon sampling was comparable to 4 brushes in capturing diagnostically relevant cellular volumes and architecture. Naïve users easily performed the procedures after reading the guidelines. LEVEL OF EVIDENCE: 3.

Validation of the use of a fluorescent PARP1 inhibitor for the detection of oral, oropharyngeal and oesophageal epithelial cancers.

For oral, oropharyngeal and oesophageal cancer, the early detection of tumours and of residual tumour after surgery are prognostic factors of recurrence rates and patient survival. Here, we report the validation, in animal models and a human, of the use of a previously described fluorescently labelled small-molecule inhibitor of the DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP1) for the detection of cancers of the oral cavity, pharynx and oesophagus. We show that the fluorescent contrast agent can be used to quantify the expression levels of PARP1 and to detect oral, oropharyngeal and oesophageal tumours in mice, pigs and fresh human biospecimens when delivered topically or intravenously. The fluorescent PARP1 inhibitor can also detect oral carcinoma in a patient when applied as a mouthwash, and discriminate between fresh biopsied samples of the oral tumour and the surgical resection margin with more than 95% sensitivity and specificity. The PARP1 inhibitor could serve as the basis of a rapid and sensitive assay for the early detection and for the surgical-margin assessment of epithelial cancers of the upper intestinal tract.

Abrasion and blunt tissue trauma study of a novel flexible robotic system in the porcine model.

OBJECTIVES: The objective of this study was to determine if a flexible robotic system caused increased tissue reaction when accessing the oropharynx and hypopharynx compared to intubation controls in only 2 scenarios: high speed tissue impact and multiple unit insertions and retractions. The data obtained were submitted as part of the entirety of information submitted for FDA approval. METHODS: This study consisted of 5 groups of Yorkshire pigs (2 animals per group). On Day 0, all animals were intubated. For group 1 (control), a second endotracheal tube was advanced to just above the vocal cords. In abrasion groups 2 and 3, the flexible robotic system was advanced against the oropharyngeal and hypopharyngeal tissues, respectively. In blunt trauma groups 4 and 5, the flexible robotic system was advanced at maximum speed (22mm/s) to collide with oropharyngeal and hypopharyngeal tissues, respectively. Pre- and post-procedure endoscopic assessments of tissue reaction were performed daily for 4 days. An independent reviewer graded tissue reaction using a 0-3 point scale. RESULTS: Tissue reaction scores at each observation time point for all test groups were less than or equal to control scores except for one instance of moderate scoring (2 out of 3) on Day 2 for an animal in the blunt trauma group where reaction was likely intubation-related rather than device impact related. Otherwise, all flexible robotic system-treated animal scores were less than 1 by Day 4. CONCLUSIONS: In this limited study, the flexrobotic system afforded surgical access to the oropharynx and hypopharynx without an increased level of abrasion or tissue trauma when compared to intubation alone.

Metformin Prevents the Progression of Dysplastic Mucosa of the Head and Neck to Carcinoma in Nondiabetic Patients.

OBJECTIVE: Metformin is an oral anti-hyperglycemic agent used to treat type 2 diabetes mellitus (DM). In vitro and animal models have shown that metformin can prevent the progression of oral lesions to carcinoma; however, there is conflicting data in the clinical literature regarding risk reduction for malignancy in head and neck cancer (HNC). STUDY DESIGN: Case series. METHODS: We present 3 cases in which adjuvant metformin therapy was used to treat recurrent and multifocal dysplastic lesions in previously treated nondiabetic HNC patients. RESULTS: Patients included 1 with a history of oral cavity squamous cell carcinoma (SCC) and 2 with a history of laryngeal SCC. Follow-up time ranged between 3 and 33 months. All 3 patients showed complete or partial regression of the remaining mucosal lesions and did not require any additional surgeries. CONCLUSION: We present 3 cases of nondiabetic HNC patients with field cancerization who showed a good response to adjuvant therapy with metformin. The nondiabetic population is not affected by confounding factors such as increased risk of malignancy and decreased overall survival that is itself associated with abnormal glucose metabolism and is therefore an excellent cohort in which to study the use of adjuvant metformin therapy in HNC patients.

Directed balloon cytology of the esophagus: A novel device for obtaining circumferential cytologic sampling.

OBJECTIVE: Current methods of obtaining esophageal cytology include brush biopsy and blind balloon sampling, among others. These methods can be time-consuming if performed in accordance with acknowledged standards. Further, exact site localization can prove to be difficult. We describe a novel device for esophageal sampling using an esophageal balloon with debriding strips contained within the pleats of the balloon. Inflation brings the latter in contact with the surface to be sampled. Cell capture was compared with the commonly used brush technique in a pig model. METHODS: Separate balloon and standard brush cytology samples were collected from a pig model. Smear and cell pellet preparations were compared regarding cell density and total volume. RESULTS: Adequate samples were obtained with both the brush and balloon. On the cell smear preparations, the cell density was greater when obtained with balloon sampling. Further, the cell pellet volume was significantly greater with the latter as well. The intact morphology of individual rafts of squamous epithelial cells also was comparable between the two methods. In addition, the balloon provided precise mapping of the cytology sites in contrast to the standard brush technique. CONCLUSION: We present an innovative new balloon technology for esophageal sampling, which demonstrated a decreased sampling time interval, precise mapping, and increased cellular volume when compared to a commonly used brush technique. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:1032-1035, 2017.

Total laryngeal transplant explanted: 14 years of lessons learned.

In 1998, the first successful total laryngeal transplant was performed. Outstanding voice quality and swallowing function were achieved, and over the subsequent 14 years, much was learned about tolerance of a transplanted larynx. After approximately a decade, a slowly progressive, chronic rejection process gradually rendered the organ nonfunctional, and the patient and his providers deemed him appropriate for explantation. This is a report of the clinical indications and outcome surrounding the explantation of the first successful total laryngeal transplant.

Ten-month laryngeal allograft survival with use of pulsed everolimus and anti-alphabeta T-cell receptor antibody immunosuppression.

OBJECTIVES: The risks of daily immunosuppression limit the use of laryngeal transplantation as a reconstructive option. Pulsed immunosuppressive dosing can lessen these risks. The study objective was to develop a long-term pulsing regimen that minimizes exposure to immunosuppressive agents. METHODS: Rat laryngeal transplantation was performed. Everolimus (1 mg/kg per day) and anti-c41 T-cell receptor (TCR) antibodies (250 microg) were given for 7 days beginning 1 day before transplantation and for 5 days beginning on day 90 after transplantation. On day 180, group 1 (n = 5) received the initial regimen for 3 days, and group 2 (n = 5) received everolimus (1 mg/kg per day) until euthanization, which occurred when parathyroid hormone (PTH) levels dropped to less than 11 pg/mL or at 300 days. RESULTS: Four of the 5 rats in group 1 had normal PTH levels at 300 days. The PTH level for 1 rat was less than 11 pg/ mL at 270 days. In group 2, none of the 5 rats had normal PTH levels at 300 days. Two had PTH levels below 11 pg/mL at 270 days, and 3 had PTH levels below 11 pg/mL at 300 days. The allografts that survived beyond 300 days had an essentially normal histologic appearance. CONCLUSIONS: Pulsed immunosuppression prevented allograft rejection for 10 months and was more effective than daily everolimus. Short-term perioperative therapy followed by pulsed, tapered dosing is a viable alternative to traditional regimens and may decrease associated risks.

Anti-αß-T-cell receptor antibodies in the setting of laryngeal transplantation.

Inhibiting T-cell activation is critically important to the induction of transplantation tolerance. Monoclonal antibodies directed against the αß-T-cell receptor have been shown to cause selective immunodepletion of this T-cell population and can provide long-term allograft acceptance. This article discusses the role of this promising immunosuppressive agent in scientific research and clinical utilization. Specifically, the article focuses on its efficacy and mechanism of tolerance induction in solid tissue and composite tissue allograft transplantation with a particular focus on laryngeal transplantation.

Long-term laryngeal allograft survival using low-dose everolimus.

OBJECTIVE: The purpose of this study was to explore the mechanism and utility of everolimus as a single-agent therapy in preventing mouse laryngeal allograft rejection. STUDY DESIGN: Prospective animal study. SETTING: Academic research at a tertiary medical center. SUBJECTS AND METHODS: Fifteen recipient mice (five per group) were injected with everolimus (1 mg/kg/d) until euthanized at 15, 30, and 60 days posttransplantation. Five mice received transplants without immunosuppression and were euthanized at day 15. Larynges were graded for rejection severity. Draining lymph nodes and spleens were evaluated by flow cytometry to assess the systemic immunological environment. RESULTS: Each time group demonstrated minor allograft rejection (rejection severity scores: 2.51, 2.46, 2.78; no rejection, 1; severe, 6). This was not significantly different between groups. Everolimus-treated mice had significantly less rejection at all time points compared with non-immunosuppressed mice. Flow cytometry showed a blunted cytotoxic T-cell response, differentiation favoring regulatory T-cells, and decreased number and function of dendritic cells. CONCLUSIONS: Everolimus successfully prevents laryngeal allograft rejection up to 60 days posttransplantation. It appears to increase the production of regulatory T-cells while decreasing cytotoxic T-cell and dendritic cell response. Everolimus alone or in combination with other immunosuppressants may enable laryngeal transplantation to become a viable reconstructive option following laryngectomy for malignancy.

Extent of neck dissection required after concurrent chemoradiation for stage IV head and neck squamous cell carcinoma.

BACKGROUND: The management of initially bulky nodal disease after primary nonsurgical treatment for stage IV head and neck squamous cell carcinoma (HNSCC) continues to be a subject of debate. METHODS: A retrospective chart review of neck management in patients after chemoradiation was performed. RESULTS: Of the initially positive necks analyzed, 210/329 (65%) had a complete clinical response to treatment and 161 necks underwent neck surgery. Patients were pathologically positive 13.8% and 39.6% of the time after clinical complete or partial response, respectively. Regional recurrence was more frequent in necks with partial clinical (p = .04) or pathologic responses (p < .01) and with primary site recurrences (p < .01). CONCLUSIONS: It is still safest at our institution to perform selective neck dissection on patients with > or = N2 neck disease when initially observed to prevent unsalvageable regional recurrence until more accurate interval assessment tools are confirmed.

A new mouse laryngeal transplantation rejection grading system.

OBJECTIVES/HYPOTHESIS: Development of a rat laryngeal transplantation model allowed for the first total human laryngeal transplantation by the senior author in 1998. In an effort to further our knowledge of the immune system's role in laryngeal rejection, a change to the mouse model was required. Prior to initiating immunosuppressive research protocols, a reliable mouse larynx rejection classification had to be established. STUDY DESIGN: Animal study. METHODS: Thirty-one mouse laryngeal transplants (C57 BL/6 donors to C3H recipients) were performed and allowed to reject. Six time points were evaluated histologically: 1, 3, 5, 7, 9, and 15 days post-transplant. Eight anatomic sites were evaluated and assigned a point value. A linear regression model was constructed using the group number as the response and the scores from the eight histological criteria as predictors. Severity classifications were determined by observing patterns in the sum of scores of variables found to be significant contributors. Group 1 was normal; group 2, minimal rejection; groups 3, 4, and 5, moderate rejection; and group 6, severe rejection. RESULTS: All mice survived the transplants. Of the observed histological changes, cartilage, fat, muscle, and magnitude of lymphocytic infiltration significantly correlated with rejection severity. The rejection model created demonstrated 100% accuracy in predicting the severity classification for the 31 specimens in the study. CONCLUSIONS: The model established provides an accurate and reliable way to classify rejection severity in mice receiving laryngeal allografts. This sets the stage for future advanced study of manipulating the immune system as a mechanism for establishing allograft tolerance.

Decoy NF-kappaB fortified immature dendritic cells maintain laryngeal allograft integrity and provide enhancement of regulatory T cells.

OBJECTIVES/HYPOTHESIS: The increased risk of malignancy associated with post-transplant immunosuppression limits the potential of laryngeal transplantation as a reconstructive option. This risk may be mitigated by utilizing decoy nuclear factor kappa B (NF-kappaB) immature dendritic cells (iDC) to provide donor-specific tolerance. The purpose of this study was to explore whether tolerogenic properties of iDC can be applied to composite tissue transplantation. STUDY DESIGN: Animal study. METHODS: Five iDC-injected mice were euthanized at 15, 30, and 60 days post-laryngeal transplant. Control groups included five transplanted mice without immunosuppression, one iDC-injected mouse euthanized prior to transplantation, one mouse without injection or transplantation, and one mouse administered mature DC to serve as an accelerated rejection control. Larynges were graded for rejection severity according to a grading scale. Draining lymph nodes and spleens were evaluated by flow cytometry to determine immunogenic activity of iDC and T cells locally and peripherally. RESULTS: Each time group demonstrated moderate allograft rejection (rejection severity scores: 4.38, 5.10, 5.29). NF-kappaB iDC-treated mice had significantly less rejection at all time points compared to nonimmunosuppressed mice. Flow cytometry showed inhibition of cytotoxic T cell infiltration and expansion of regulatory T cells at the allograft site. CONCLUSIONS: iDC immunosuppression maintains laryngeal allograft integrity up to 60 days post-transplantation. Regulatory T cells are enhanced at the allograft site, which serves to suppress immune cell activation and induce self-antigen tolerance. iDC injection may lessen post-transplant comorbidities by decreasing the systemic immune response and favorably affecting concurrent immunosuppressive dose sequencing for laryngeal allograft preservation.

New mouse model for studying laryngeal transplantation.

OBJECTIVES: Laryngeal transplantation research has been studied in various animal models. For in-depth, immunology-based transplantation research, however, a thoroughly studied animal model must exist. The purpose of this study was to develop a reliable surgical technique in mice to serve as a model for further study of laryngeal transplantation. METHODS: Heterotopic laryngeal transplantation was attempted in 15 immunocompetent mice by use of modifications of previously described techniques established in rats. RESULTS: Various microvascular techniques were used that led to 8 successful transplants (of 15) with patent vascularity at the time of sacrifice. The first 7 attempts at transplantation were unsuccessful because of technical difficulties related to vessel size, soft tissue traumatic injury, and venous congestion. Subsequently, 8 transplantation procedures were successfully performed after modifications of the surgical technique. CONCLUSIONS: This pilot study describes the reproducible surgical techniques performed in using mice for studying laryngeal transplantation. Mice are cost-effective and immunologically well studied, and are thus ideal for further laryngeal transplantation research.

Does vocal cord fixation preclude nonsurgical management of laryngeal cancer?

OBJECTIVES/HYPOTHESIS: To determine whether vocal cord fixation precludes nonsurgical management of T3/T4 laryngeal carcinoma. STUDY DESIGN: A retrospective chart review. METHODS: Between 1989 and 2005 patient records with T3/T4 squamous cell carcinoma of the larynx with vocal cord fixation at presentation were reviewed. All were treated with a concomitant cisplatin-based chemoradiotherapy protocol and were part of the institutional head and neck cancer chemoradiotherapy registry. Only patients with adequate pre- and post-treatment fiberoptic evaluations were included. Charts were reviewed for demographics and tumor characteristics; return of vocal cord function; local, regional, or distant recurrence after treatment; and need for salvage surgery. The Kaplan-Meier method was used to estimate outcomes, and the log-rank test was used to compare those patients whose vocal cords remained fixed to those with recovery of function. RESULTS: Twenty-three patients met the inclusion criteria, 19 males and 4 females. The median age was 59 years (range, 39-73). Fourteen patients had T3 and nine had T4 tumors. Twelve patients recovered full range of mobility, three had partial recovery, and eight did not recover motion. The median follow-up was 68 months (range, 34-191). Comparing patients with post-treatment partial or fully mobile cords to those with persistent fixation revealed the following: A projected five-year overall survival of 100% versus 25%, (P < .001), freedom from recurrence of 86.7 versus 25% (P < .001), local control without surgery of 86.7% versus 30% (P = .003), and survival with functional larynx of 86.7% versus 25% (P = .008), respectively. CONCLUSIONS: Nonsurgical therapy in patients with pretreatment vocal cord fixation is feasible. However, persistence of vocal cord fixation after definitive chemoradiotherapy is a poor prognostic sign and early surgical intervention should be considered. Laryngoscope, 2009.

Laryngeal transplantation in the setting of cancer: a rat model.

OBJECTIVE: Traditional immunosuppressive regimens make laryngeal transplantation in cancer patients prohibitive because of the increased risk of recurrence. Everolimus, a recently developed immunosuppressant, has demonstrated significant antitumor properties. The purpose of this study was to examine the effects of everolimus alone and in combination with other immunosuppressants on tumor growth in a combined laryngeal transplantation and tumor model. STUDY DESIGN: Animal, prospective, randomized, controlled, and blinded. METHODS: One million squamous cell carcinoma cells (SCC-158) were injected intravenously into a total of 40 rats 1 day before laryngeal transplantation. Rats were divided into four groups differing by immunosuppressive regimens. Lung surface metastases were counted 21 days after inoculation, and numerical transplantation rejection scores were recorded. A separate experiment for comparison was performed with no transplant on 24 rats, but with the same immunosuppressive treatment groups. RESULTS: The median number of lung surface metastases were: a) control (i.e., no immunosuppression): 85; b) everolimus 1.0 mg/kg: 25; c) tacrolimus 1.2 mg/kg: 1650; d) everolimus 1.0 mg/kg + tacrolimus 0.05 mg/kg: 1300. Rats receiving everolimus alone showed a statistically significant decrease in pulmonary surface metastases compared with the other groups. Transplanted rats had no difference in their outcomes when compared with non-transplanted rats. CONCLUSION: Everolimus significantly decreases SCC-158 growth in our combined transplantation and tumor model compared with controls and other immunosuppressants.

Use of in situ hybridization to detect human papillomavirus in head and neck squamous cell carcinoma patients without a history of alcohol or tobacco use.

CONTEXT: Head and neck squamous cell carcinoma is commonly associated with tobacco and alcohol use. There are, however, a group of patients without a significant history of tobacco or alcohol use, and the etiology of these tumors is incompletely understood. OBJECTIVE: To examine tumors in this subpopulation for association with human papillomavirus (HPV) using newly available in situ hybridization probes. DESIGN: Between October 2004 and October 2005, 22 patients who did not use alcohol or tobacco were included. Formalin-fixed, paraffin-embedded tissue sections were used to perform in situ hybridization using newly available probe sets (Ventana Medical Systems, Tucson, Ariz). The slides were examined for the presence of integrated HPV using light microscopy. Positive and negative xenograft controls were run with the assay. Results.-The mean age of the patients was 64 years. There were 14 men and 8 women. The most common anatomic sites included tongue (n = 8), tonsil (n = 7), and larynx (n = 7). All cases and controls were successfully stained. Only 2 cases were positive for high-risk HPV, and both demonstrated an integrated pattern. Both cases were tumors of the tonsil. No cases were positive for low-risk HPV. CONCLUSIONS: These results demonstrate that the new probe sets for HPV can be used very efficiently in clinical pathology material of head and neck squamous cell carcinoma. Our data show that high-risk HPV is an uncommon finding in head and neck squamous cell carcinoma from patients who do not have a history of tobacco or alcohol use; low-risk HPV was not seen in any case.

Clinical predictors of larynx preservation after multiagent concurrent chemoradiotherapy.

BACKGROUND: Determining which patients benefit from larynx preservation strategies remains problematic. We reviewed our experience using multiagent concurrent chemoradiotherapy to identify clinical predictors for success. METHODS: Cisplatin and fluorouracil were given during weeks 1 and 4 of radiation to 115 patients with locoregionally advanced larynx or hypopharynx squamous cell cancer without cartilage invasion or laryngeal destruction. Laryngectomy was reserved for local failure. RESULTS: The 5-year Kaplan-Meier projected overall survival was 58%, survival with larynx preservation 52%, local control without surgery 82%, local control (including surgical salvage) 94%, and survival with functional larynx 49%. Local control without surgery was superior in patients with T1-2 versus T3-4 tumors (97% vs 77%, p = .032). No other clinical parameters proved predictive of local control. CONCLUSION: Larynx preservation was successful in all subsets of appropriately selected patients. Although local failure was more likely in patients with T3-4 tumors, it was infrequent and surgical salvage was effective.