A distinct human cell type expressing MHCII and RORγt with dual characteristics of dendritic cells and type 3 innate lymphoid cells.

Recent studies have characterized various mouse antigen-presenting cells (APCs) expressing the lymphoid-lineage transcription factor RORγt (Retinoid-related orphan receptor gamma t), which exhibit distinct phenotypic features and are implicated in the induction of peripheral regulatory T cells (Tregs) and immune tolerance to microbiota and self-antigens. These APCs encompass Janus cells and Thetis cell subsets, some of which express the AutoImmune REgulator (AIRE). RORγt+ MHCII+ type 3 innate lymphoid cells (ILC3) have also been implicated in the instruction of microbiota-specific Tregs. While RORγt+ APCs have been actively investigated in mice, the identity and function of these cell subsets in humans remain elusive. Herein, we identify a rare subset of RORγt+ cells with dendritic cell (DC) features through integrated single-cell RNA sequencing and single-cell ATAC sequencing. These cells, which we term RORγt+ DC-like cells (R-DC-like), exhibit DC morphology, express the MHC class II machinery, and are distinct from all previously reported DC and ILC3 subsets, but share transcriptional and epigenetic similarities with DC2 and ILC3. We have developed procedures to isolate and expand them in vitro, enabling their functional characterization. R-DC-like cells proliferate in vitro, continue to express RORγt, and differentiate into CD1c+ DC2-like cells. They stimulate the proliferation of allogeneic T cells. The identification of human R-DC-like cells with proliferative potential and plasticity toward CD1c+ DC2-like cells will prompt further investigation into their impact on immune homeostasis, inflammation, and autoimmunity.

Novel cellular systems unveil mucosal melanoma initiating cells and a role for PI3K/Akt/mTOR pathway in mucosal melanoma fitness.

BACKGROUND: Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. METHODS: Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. RESULTS: This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. CONCLUSIONS: Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

Functional outcomes after transoral CO2 laser treatment for posterior glottic stenosis: a bicentric case series.

PURPOSE: The aim of this study is to evaluate functional outcomes in terms of decannulation rate and quality of life of patients affected by PGS (Grades I-IV) treated only by transoral CO2 laser microsurgery (TOLMS) in two tertiary centers. METHODS: An observational retrospective study was carried out, enrolling 22 patients affected by PGS who were treated by a transoral approach at two tertiary referral centers. Surgical treatment included TOLMS with tailored laser resection of the scar tissue combined with posterior cordotomy, resurfacing of the raw area with mucosal microflap, or placement of a Montgomery T-tube or Keel stent. All patients were evaluated and staged preoperatively and postoperatively, at least 6 months after the surgery. Functional outcomes were objectively evaluated by the Airway-Dysphonia-Voice-Swallowing (ADVS) staging system, Voice Handicap Index-30 (VHI-30), and Eating Assessment Tool-10 (EAT-10) questionnaires. RESULTS: Quality of life significantly improved as measured by the VHI-30 questionnaire with a median variation of - 31.0 (p = 0.003), the EAT-10 with a median variation of - 4.0 (p = 0.042), and the ADVS with a median variation of - 3.5 (p < 0.001). No significant changes were observed in swallowing scores. We were able to decannulate 7 of 9 patients (almost 80%) with previous tracheotomy. CONCLUSION: In conclusion, even if there is still no general agreement on an exact therapeutic algorithm to treat PGS, our results confirm that transoral surgery, in terms of scar tissue removal, combined in selected patients with posterior cordotomy and pedicled local flaps and/or placement of stents, represents a safe and effective surgical approach even for more severe PGS.

Surgical management and oncological outcome of non-squamous cell carcinoma of the larynx: a bicentric study.

PURPOSE: Non-squamous cell carcinoma (non-SCC) accounts for about 5% of laryngeal malignancies. Survival data are limited, and consensus on management principles is lacking. The present study reviews our experience in the surgical treatment of non-metastatic non-SCC of the larynx and compares oncological and functional outcomes in a cohort of patients affected by traditional SCC. METHODS: We collected data on 592 patients affected by laryngeal neoplasms. Univariate and multivariable survival analyses were performed using Cox proportional-hazards models; survival estimates were reported by hazard ratios (HR) with 95% confidence intervals (CI), and survival curves were established with the Kaplan-Meier method. RESULTS: We identified 326 patients affected by untreated SCC, while 21 had non-SCC histotypes. The non-SCC cohort was composed of 5 soft tissue sarcomas, 8 chondrosarcomas, 2 adenoid cystic carcinomas, 2 neuroendocrine carcinomas, 2 solitary fibrous tumors, 1 Kaposi's sarcoma, and 1 malignant peripheral nerve sheath tumor. Overall survival and disease-specific survival were not significantly different according to histology (p = 0.6 and p = 0.349, respectively). The non-SCC group showed an increased risk of recurrence (HR 5.87; CI95 2.15-16.06; p < 0.001). Nonetheless, no significant difference (p = 0.31) was found at multivariable analysis between the two groups in total laryngectomy-free survival with an organ preservation rate over 5 years of 81% for the non-SCC histologies. CONCLUSION: Non-SCC is a broad spectrum pathology, but generalized laryngeal surgical management principles are still feasible and it is possible to identify patients amenable to conservative surgical treatment without affecting survival.

Transoral laser microsurgery: feasibility of a new exoscopic HD-3D system coupled with free beam or fiber laser.

In the last decades, new technological devices and instruments have been developed to overcome the technical limits of transoral laser microsurgery. The recent introduction of 3D endoscopy seems to be a promising tool in the field of diagnostic and operative laryngology as an alternative to the traditional microlaryngoscopy. Our work aims to present a novel transoral microsurgical setting that expands the use of exoscopic systems (in this case the VITOM® 3D-HD) as an alternative to the standard operating microscope. A customized support arm and an adaptor to firmly connect the VITOM® 3D-HD camera to the laser micromanipulator were specially designed. This setup was used as an alternative to the standard operating microscope in a cohort of 17 patients affected by suspicious early to intermediate pharyngo-laryngeal neoplasms. A historical cohort of patients treated with the traditional setting and matching the same inclusion criteria was used as a reference for the duration of surgical procedures. The surgical procedures comprised 7 cordectomies, 2 endoscopic partial supraglottic laryngectomies, 4 tongue base resections, and 4 lateral oropharyngectomies or hypopharyngectomies. In 6 cases (35%), a simultaneous neck dissection was performed. The low rate of positive deep (6%) or superficial (12%) margins reinforced the safety of this platform, and the results obtained in terms of operating time were comparable to the control group (p > 0.05), which confirms the feasibility of the system. Our surgical setting setup is a convincing alternative to traditional transoral laser microsurgery for early to intermediate pharyngo-laryngeal neoplasms. The main advantages of this system are comfortable ergonomics for the first surgeon and a potential benefit in terms of teaching if applied in university hospitals, since the entire surgical team can view the same surgical 3D-HD view of the first operator. Further work is still needed to objectively compare the traditional and new technique, and to validate our preliminary clinical findings.

Microsurgical procedures during COVID-19 pandemic: the VITOM® 3D-HD exoscopic system as alternative to the operating microscope to properly use personal protective equipment (PPE).

PURPOSE: In the field of microsurgery, the use of conventional operating microscope, adopted in several disciplines, is not suitable with the full adoption of appropriate personal protective equipment (PPE), as goggles and face shields, needing the eyes to be at close contact with oculars. METHODS: Herein we present an exoscopic surgical setup, implemented for transoral laser microsurgery, by the VITOM® 3D-HD system. RESULTS: Our proposed exoscopic setting overcomes safety limits of the conventional operating microscope, being suitable with the adoption of full PPE necessary facing with suspected or confirmed positive SARS-CoV-2 patients needing urgent microsurgical procedures. CONCLUSION: The use of protocols to reduce the virus spreading is mandatory to safely treat also patients potentially SARS-CoV-2 infected. The described surgical setup is advisable to manage urgent microsurgical procedures along the duration of the COVID-19 pandemic, being applicable PPE necessary to treat potentially or confirmed SARS-CoV-2-infected patients.

Transoral management of adult benign laryngeal stenosis.

PURPOSE: Management of benign laryngeal stenosis (BLS) remains challenging even though transoral treatments in selected cases have shown satisfactory results, at least comparable to open-neck approaches, with reduced invasiveness. To date, no overall consensus has been reached on many issues. The aim of this study is to assess the effectiveness of a purely transoral treatment in a cohort of patients affected by BLS. METHODS: We evaluated 40 patients affected by BLS, treated by transoral surgery between 2013 and 2017. The European Laryngological Society classification for laryngotracheal stenosis was applied for the staging. Improvement in airway patency and quality of life was assessed by decannulation rate, Airway-Dyspnea-Voice-Swallowing (ADVS) score, Voice handicap index (VHI)-30, and Eating assessment tool (EAT)-10 questionnaires. RESULTS: Mean age was 61 years and M:F ratio was 1.4:1. Previous laryngeal surgery was the most common cause of stenosis (50%), followed by radiotherapy (20%), idiopathic etiology (12%), granulomatosis with polyangiitis (10%), and prolonged intubation (8%). Transoral treatment entailed an improvement in quality of life with a significant decrease in the VHI score (p < 0.0001) and improvement in Airway (p = 0.008), Dyspnea (p < 0.0001), and Voice (p < 0.0001) scores. No major perioperative complications were observed. The decannulation rate among patients with a tracheostomy in place (N = 16) was 63%. CONCLUSIONS: Transoral treatment of selected BLS managed by a team with high-level expertise in surgery and anesthesiology is associated with significant improvement of quality of life, especially with regard to voice and breathing functions.

Quality of life after transoral CO2 laser posterior cordotomy with or without partial arytenoidectomy for bilateral adductor vocal cord paralysis.

OBJECTIVES: Bilateral adductor vocal cord paralysis (BAVCP) is a rare and challenging condition whose main consequence is reduction of airway patency at the glottic level, often causing respiratory distress, while vocal function tends to remain almost normal. We investigated the effect of transoral glottal widening on quality of life and decannulation rates in patients affected by BAVCP. METHODS: We retrospectively evaluated patients affected by BAVCP and treated by transoral CO2 posterior cordotomy with or without medial partial arytenoidectomy (PC ± MPA) at two referral centers. The primary outcome was change in quality of life, evaluated pre- and post-operatively by the ADVS, VHI-30, and EAT-10 questionnaires. Secondary outcomes were the need for retreatments and, for patients with tracheotomy, the time to decannulation. RESULTS: Thirty-three patients met selection criteria. The etiology was post-surgical in 27 cases (81.8%), idiopathic in 4 (12.1%), a trauma-related in 1 (6.0%), and to other causes in 1 (3.0%). In 22 cases (66.7%), PC was combined with MPA. A significant improvement in responses for the ADVS (p < .0001) and EAT-10 (p < .0001) was observed, whereas the VHI-30 score did not change significantly post-operatively. All nine patients with a tracheostomy were successfully decannulated within 18 months after the surgical procedure. CONCLUSIONS: For patients affected by BAVCP, PC ± MPA by transoral CO2 laser microsurgery is a safe, customizable and minimally invasive treatment that can guarantee an affordable balance between quality of life in terms of phonation and swallowing and acceptable airway patency.

Intraoral ultrasonography in the assessment of DOI in oral cavity squamous cell carcinoma: a comparison with magnetic resonance and histopathology.

OBJECTIVE: The first-line therapeutic approach for oral cavity squamous cell carcinoma (OCSCC) is complete surgical resection. Preoperative assessment of depth of invasion (cDOI) is crucial to plan the surgery. Magnetic resonance (MR) and intraoral ultrasonography (IOUS) have been shown to be useful tools for assessment of DOI. The present analysis investigates the accuracy of MR and IOUS in evaluating DOI in OCSCC compared to histological evaluation (pDOI). MATERIALS AND METHODS: Forty-nine previously untreated patients with cT1-T3 OCSCC were reviewed. Nine patients were staged with MR alone, 10 with IOUS alone, and 30 with both MR and IOUS. RESULTS: Mean difference between cDOIMR and pDOI values of 0.2 mm (95% CI - 1.0-1.3 mm) and between cDOIIOUS and pDOI of 0.3 mm (95% CI - 1.0-1.6 mm). Spearman R between cDOIMR and pDOI was R = 0.83 and between cDOIIOUS and pDOI was R = 0.76. Both radiological techniques showed high performance for the correct identification, with the optimum cut-off of 5 mm, of patients with a pDOI ≥ 4 mm and amenable to a neck dissection, with an AUC of 0.92 and 0.82 for MR and IOUS, respectively. CONCLUSION: Both examinations were valid approaches for preoperative determination of DOI in OCSCC, although with different cost-effectiveness profiles and indications.

Validation of the European Laryngological Society classification of glottic vascular changes as seen by narrow band imaging in the optical biopsy setting.

PURPOSE: In 2016, the European Laryngological Society (ELS) proposed a classification for vascular changes occurring in glottic lesions as visible by narrow band imaging (NBI), based on the dichotomic distinction between longitudinal vessels (not suspicious) and perpendicular ones (suspicious). The aim of our study was to validate this classification assessing the interobserver agreement and diagnostic test performance in detecting the final histopathology. METHODS: A retrospective study was carried out by reviewing clinical charts, preoperative videos, and final pathologic diagnosis of patients submitted to transoral microsurgery for laryngeal lesions in two Italian referral centers. In each institution, two physicians, independently re-assessed each case applying the ELS classification. RESULTS: The cohort was composed of 707 patients. The pathologic report showed benign lesions in 208 (29.5%) cases, papillomatosis in 34 (4.8%), squamous intraepithelial neoplasia (SIN) up to carcinoma in situ in 200 (28.2%), and squamous cell carcinoma (SCC) in 265 (37.5%). The interobserver agreement was extremely high in both institutions (k = 0.954, p < 0.001 and k = 0.880, p < 0.001). Considering the diagnostic performance for identification of at least SIN or SCC, the sensitivity was 0.804 and 0.902, the specificity 0.793 and 0.581, the positive predictive value 0.882 and 0.564, and the negative predictive value 0.678 and 0.908, respectively. CONCLUSION: The ELS classification for NBI vascular changes of glottic lesions is a highly reliable tool whose systematic use allows a better diagnostic evaluation of suspicious laryngeal lesions, reliably distinguishing benign ones from those with a diagnosis of papillomatosis, SIN or SCC, thus paving the way towards confirmation of the optical biopsy concept.