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Ovarian carcinomas (OCs) are poorly immunogenic and immune checkpoint inhibitors (ICIs) have offered a modest benefit. In this study, high CD3 + T-cells and CD163 + tumor-associated macrophages (TAMs) densities identify a subgroup of immune infiltrated high-grade serous carcinomas (HGSCs) with better outcomes and superior response to platinum-based therapies. On the contrary, in most clear cell carcinomas (CCCs) showing poor prognosis and refractory to platinum, a high TAM density is associated with low T cell frequency. Immune infiltrated HGSC are characterized by the 30-genes signature (OC-IS 30 ) covering immune activation and IFNγ polarization and predicting good prognosis (n = 312, TCGA). Immune infiltrated HGSC contain CXCL10 producing M1-type TAM (IRF1 + pSTAT1Y701 + ) in close proximity to T-cells. A fraction of these M1-type TAM also co-expresses TREM2. M1-polarized TAM were barely detectable in T-cell poor CCC, but identifiable across various immunogenic human cancers. Single cell RNA sequencing data confirm the existence of a tumor-infiltrating CXCL10 + IRF1 + STAT1 + M1-type TAM overexpressing antigen processing and presentation gene programs. Overall, this study highlights the clinical relevance of the CXCL10 + IRF1 + STAT1 + macrophage subset as biomarker for intratumoral T-cell activation and therefore offers a new tool to select patients more likely to respond to T-cell or macrophage-targeted immunotherapies.

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.

Plasmacytoid dendritic cells (pDCs) infiltrate a large set of human cancers. Interferon alpha (IFN-α) produced by pDCs induces growth arrest and apoptosis in tumor cells and modulates innate and adaptive immune cells involved in anti-cancer immunity. Moreover, effector molecules exert tumor cell killing. However, the activation state and clinical relevance of pDCs infiltration in cancer is still largely controversial. In Primary Cutaneous Melanoma (PCM), pDCs density decreases over disease progression and collapses in metastatic melanoma (MM). Moreover, the residual circulating pDC compartment is defective in IFN-α production. The activation of tumor-associated pDCs was evaluated by and microscopic analysis. The expression of human myxovirus resistant protein 1 (MxA), as surrogate of IFN-α production, and proximity ligation assay (PLA) to test dsDNA-cGAS activation were performed on human melanoma biopsies. Moreover, IFN-α and CXCL10 production by stimulated (i.e. with R848, CpG-A, ADU-S100) pDCs exposed to melanoma cell lines supernatants (SN-mel) was tested by intracellular flow cytometry and ELISA. We also performed a bulk RNA-sequencing on SN-mel-exposed pDCs, resting or stimulated with R848. Glycolytic rate assay was performed on SN-mel-exposed pDCs using the Seahorse XFe24 Extracellular Flux Analyzer. Based on a set of microscopic, functional and analyses, we demonstrated that the melanoma milieu directly impairs IFN-α and CXCL10 production by pDCs TLR-7/9 and cGAS-STING signaling pathways. Melanoma-derived immunosuppressive cytokines and a metabolic drift represent relevant mechanisms enforcing pDC-mediated melanoma escape. These findings propose a new window of intervention for novel immunotherapy approaches to amplify the antitumor innate immune response in cutaneous melanoma (CM).

Extracellular signal-regulated kinase 5 (ERK5) is a unique kinase among MAPKs family members, given its large structure characterized by the presence of a unique C-terminal domain. Despite increasing data demonstrating the relevance of the ERK5 pathway in the growth, survival, and differentiation of normal cells, ERK5 has recently attracted the attention of several research groups given its relevance in inflammatory disorders and cancer. Accumulating evidence reported its role in tumor initiation and progression. In this review, we explore the gene expression profile of ERK5 among cancers correlated with its clinical impact, as well as the prognostic value of ERK5 and pERK5 expression levels in tumors. We also summarize the importance of ERK5 in the maintenance of a cancer stem-like phenotype and explore the major known contributions of ERK5 in the tumor-associated microenvironment. Moreover, although several questions are still open concerning ERK5 molecular regulation, different ERK5 isoforms derived from the alternative splicing process are also described, highlighting the potential clinical relevance of targeting ERK5 pathways.

PurposeNon-resective pharyngoplasty techniques have been shown to be effective to treat oropharyngeal collapse in patients affected by obstructive sleep apnea-hypopnea syndrome (OSAHS). The aim of our study is to evaluate outcome predictors in a cohort of patients affected by OSAHS and treated with non-resective pharyngoplasty, including variation of pharyngeal measures at the end of the surgical procedure.MethodsA cohort of patients affected by OSAHS, with palatal or lateral pharyngeal wall collapse, who underwent non-resective pharyngoplasty, were enrolled between 2014 and 2017. Surgical procedures encompassed non-resective pharyngoplasty by expansion sphincter pharyngoplasty (ESP) or barbed antero-lateral pharyngoplasty with barbed reposition pharyngoplasty (BRP) or barbed suspension pharyngoplasty (BSP) techniques, eventually associated with nasal surgery. Pharyngeal measures were recorded intraoperatively and their variation at the end of the procedure was considered. Surgical success was evaluated at least 6 months after surgery with respiratory polygraphy and ESS questionnaire. Outcome predictors were examined by multivariable logistic regression and ROC curve analysis.ResultsSeventy patients met the study inclusion criteria. ESP, BRP, and BSP in a uni-/multilevel setting led to significant improvement of all respiratory polygraphic parameters and daily sleepiness (p < 0.0001). Outcome analysis showed that greater variation of antero-posterior pharyngeal measure was associated with success (p = 0.01), with an optimal cutoff value of 8.5 mm; low AHIpre, high ESSpre, and antero-lateral pharyngoplasty with barbed sutures were associated with a higher rate of cure (p < 0.05).ConclusionsNon-resective pharyngoplasty is effective in treating OSAHS patients affected by palatal or lateral pharyngeal wall collapse, and intraoperative variation of antero-posterior width may be a useful tool to predict surgical success.

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.

The retinoic-acid-receptor-related orphan receptor gamma t (RORγt) isoform is required for the development of lymphoid organs, T-helper 17 cells (Th17), and innate lymphoid cells (ILC3s). Recent data in mouse and human have revealed non-T, non-ILC3 cell populations with antigen presenting features that express RORγt. This study maps the presence of RORγt cells with dendritic cell (DC) features in human adult and fetal lymphoid tissues. By combining multicolor flow cytometry, RNAseq of peripheral blood cells, analysis of lymph node scRNAseq datasets, and microscopic analysis on human tissue sections and single-cell suspensions, this study maps the presence of RORγt cells with DC features in human tissues. RORγt-DCs are found in human lymphoid organs, particularly in lymph nodes. Lymph node RORγt-DCs are located in the interfollicular area surrounding high endothelial venules and in the marginal sinuses. In terms of phenotype, RORγt-DCs are distinct from other nodal dendritic cells. They express PRDM16 and PIGR as well as transcripts supporting the antigen presentation machinery, while lacking stromal markers. A significant fraction of RORγt-DCs is proliferating, suggesting local self-renewal. Moreover, most of them lack autoimmune regulator (AIRE) expression. Comparison with mouse RORγt Thetis cells (TC) and Janus cells (JC) shows more similarity with group II TC than group I and III TC or JC, all of which are tolerogenic and express AIRE. Overall, this study identifies human lymph nodes as a relevant niche for RORγt-DCs and establishes tools for their microscopic mapping in human disease states.

The early detection of head and neck squamous cell carcinoma (HNSCC) is essential to improve patient prognosis and enable organ and function preservation treatments. The objective of this study is to assess the feasibility of using electrical bioimpedance (EBI) sensing technology to detect HNSCC tissue. A prospective study was carried out analyzing tissue from 46 patients undergoing surgery for HNSCC. The goal was the correct identification of pathologic tissue using a novel needle-based EBI sensing device and AI-based classifiers. Considering the data from the overall patient cohort, the system achieved accuracies between 0.67 and 0.93 when tested on tissues from the mucosa, skin, muscle, lymph node, and cartilage. Furthermore, when considering a patient-specific setting, the accuracy range increased to values between 0.82 and 0.95. This indicates that more reliable results may be achieved when considering a tissue-specific and patient-specific tissue assessment approach. Overall, this study shows that EBI sensing may be a reliable technology to distinguish pathologic from healthy tissue in the head and neck region. This observation supports the continuation of this research on the clinical use of EBI-based devices for early detection and margin assessment of HNSCC.

Objectives The role of the immune system in head and neck squamous cell carcinoma is controversial. The aim of our study was to analyze full blood counts and distribution of T cell subsets in patients affected by laryngeal squamous cell cancer (LSCC) and their association with clinical variables and survival. Study design Retrospective study. Methods We analyzed the levels of platelets, lymphocytes, and neutrophils, as well as the CD4+, CD8+, and CD3+ T‐cell subpopulations by cytofluorometry in LSCC patients. A cohort of healthy patients was used as control group. The disease‐specific survival (DSS) was considered as survival outcome. Results Sixty‐five LSCC patients and 48 controls were enrolled. In LSCC patients, neutrophils were higher than in the healthy group (P < .0001). The neutrophil‐to‐lymphocyte ratio (NLR) and the platelet‐to‐lymphocyte ratio (PLR) were both higher in LSCC patients (P < .0001). In patients treated for recurrent disease, the CD8+/CD3+ ratio was increased (P = .02), while the CD4+/CD8+ (P = .03) and CD4+/CD3+ (P = .04) ratios were lower. In patients with lymph node metastases, leukocytes (P = .03), CD3+ (P = .04), and CD4+ (P = .0098) were all higher. Among Stages III‐IV patients, low lymphocyte and low leukocyte count were associated with worse DSS. Conclusion Our data demonstrate that NLR and PLR are significantly increased in LSCC. Lower CD4+/CD8+ and CD3+/CD8+ ratios are related to recurrent disease and a higher level of CD3+ and CD4+ is associated with nodal metastasis. Level of Evidence 4

The optimal treatment for T3 laryngeal carcinoma (LC) is still a matter of debate. Different therapeutic options are available: Transoral laser microsurgery (TLM), open partial horizontal laryngectomies (OPHLs), total laryngectomy (TL), and organ preservation protocols (radiation therapy (RT) or chemo-radiation (CRT)). This study aimed to retrospectively evaluate oncologic outcomes of 104 T3 LCs treated by surgery or non-surgical approaches from January 2011 to December 2016 at a single academic tertiary referral center. Each case was evaluated by a multidisciplinary team (MDT) devoted to the management of head and neck cancers. We divided the cohort into two subgroups: Group A, surgical treatment (TLM, OPHLs, TL) and Group B, non-surgical treatment (RT, CRT). For the entire cohort, two- and five-year overall survival (OS) rates were 83% and 56%, respectively. The two- and five-year disease-free survival (DFS) rates were 75% and 65%, and disease-specific survival rates were 93% and 70%, respectively. The N category was a significant independent prognosticator for OS (p = 0.02), whereas Group B was significantly and independently associated with DFS (HR 4.10, p = 0.006). Analyzing laryngo-esophageal dysfunction-free survival as an outcome, it was found that this was significantly lower in higher N categories (p = 0.04) and in cases that underwent non-surgical treatments (p = 0.002). Optimization of oncologic outcomes in T3 LCs may be obtained only by a comprehensive MDT approach, considering that different treatment options have heterogenous toxicity profiles and indications.