The efficacy and safety of dalpiciclib, a cyclin-dependent kinase 4/6 inhibitor, in patients with advanced head and neck mucosal melanoma harboring CDK4 amplification.

BACKGROUND: Mucosal melanoma (MM) is a rare but devastating subtype of melanoma. Our previous studies have demonstrated robust anti-tumor effects of cyclin-dependent kinase 4/6 (CDK 4/6) inhibitors in head and neck MM (HNMM) patient-derived xenograft models with CDK4 amplification. Herein, we aimed to investigate the efficacy and safety of dalpiciclib (SHR6390), a CDK4/6 inhibitor, in HNMM patients harboring CDK4 amplification. METHODS: The anti-tumor efficacy of dalpiciclib was assessed by HNMM patient-derived xenograft (PDX) models and patient-derived tumor cells (PDC) in vivo and in vitro. Immunohistochemical analyses and western blot were then performed to assess the markers of cell proliferation and CDK4/6 signaling pathway. For the clinical trial, advanced recurrent and/or metastatic HNMM patients with CDK4 amplification were treated with dalpiciclib 125 mg once daily for 21 consecutive days in 28-day cycles. The primary endpoint was disease control rate (DCR). Secondary endpoints included safety, objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). RESULTS: Dalpiciclib profoundly suppressed growth of HNMM-PDX and PDC with CDK4 amplification, whereas it showed relatively weak suppression in those with CDK4 wild type compared with vehicle. And dalpiciclib resulted in a remarkable reduction in the expression levels of Ki-67 and phosphorylated Rb compared with control group. In the clinical trial, a total of 17 patients were enrolled, and 16 patients were evaluable. The ORR was 6.3%, and the DCR was 81.3%. The estimated median PFS was 9.9 months (95% CI, 4.8-NA), and the median OS was not reached. The rate of OS at 12 months and 24 months was 68.8% (95% CI, 0.494-0.957) and 51.6% (95% CI, 0.307-0.866), respectively. The most frequent adverse events were neutrophil count decrease, white blood cell count decrease, and fatigue. CONCLUSIONS: Dalpiciclib was well-tolerated and displayed a durable benefit for HNMM patients with CDK4 amplification in this study. Further studies on CDK4 inhibitors and its combination strategy for MM are worth further exploration. TRIAL REGISTRATION: ChiCTR2000031608.

Fibroblast growth factor receptors 1 and 4 combined with lymph node metastasis predicts poor prognosis in oral cancer.

OBJECTIVES: The fibroblast growth factor receptor (FGFR) members including FGFR1-4 have been identified as promising novel therapeutic targets and prognostic markers in multiple solid tumors. However, the predictive role of the expression of FGFR proteins in oral squamous cell carcinoma (OSCC) requires further exploration. MATERIALS AND METHODS: Immunohistochemical evaluation of FGFR1-4 was performed on 161 paired OSCC samples. The associations of FGFRs with clinicopathologic and prognostic parameters were analyzed. To further assess the contribution of FGFRs to OSCC proliferation, cell lines, and one PDX model was utilized to examine the anti-tumor effect of the pan-FGFR inhibitor AZD4547. RESULTS: All FGFR members were found to be overexpressed in OSCC tumors when compared to normal tissues, and their expression was significantly associated with poor overall survival and disease-free survival. Multivariate Cox regression analysis revealed high expression of FGFR1 (p = 0.014) and FGFR4 (p = 0.009) were independent prognostic factors and co-overexpression of FGFR1 and FGFR4 with lymph node metastasis increased HR for death (p = 0.02). The pan-FGFR inhibitor AZD4547 showed anti-tumor activity in cell lines and in a patient-derived xenograft of OSCC. CONCLUSIONS: This study highlights the co-overexpression of FGFR1 and FGFR4 as a significantly poor prognosis indicator in OSCC when combined with lymph node metastasis.

Apatinib potentiates the therapeutic effect of anti-PD-1 in locally advanced head and neck cancers.

OBJECTIVES: Antiangiogenic inhibitors have been shown to synergize with immune checkpoint blockade, but the underlying mechanisms of the synergistic response are not fully understood. PATIENTS AND METHODS: We investigate the impact of VEGFR2 inhibition on tumor-infiltrating immune cells in vivo and the activity of the combination of apatinib and anti-PD-1 in synergistic mouse model of HNSCC. A patient with squamous cell carcinoma of the left tongue with cervical lymph node were received with combined induction treatment of camrelizumab and apatinib to validate the efficacy of neoadjuvant immunotherapy before surgery. RESULTS: We found that apatinib increased the infiltration of CD8+ T cells and decreased the population of Tregs in a preclinical syngeneic mouse model. The proportions of CD8+ PD1+ T cells were significantly increased in apatinib-treated tumors. The combined treatment of apatinib and anti-PD-1 demonstrated better therapeutic benefit than each treatment alone. The patient with squamous cell carcinoma of the left tongue with cervical lymph node achieved major pathologic response (MPR) after two cycles of combined induction treatment. CONCLUSION: Our study demonstrated that apatinib therapy synergized with an anti-PD-1 antibody in preclinical cancer models and in patient with advanced HNSCC. These results provide a new rationale for advancing this neoadjuvant immunotherapy in large scale of clinical trials of HNSCC.

Low-dose arecoline regulates distinct core signaling pathways in oral submucous fibrosis and oral squamous cell carcinoma.

BACKGROUND: Betel nut chewing plays a role in the pathogenesis of oral submucous fibrosis (OSF) and oral squamous cell carcinoma (OSCC). As the major active ingredient of the betel nut, the effect of arecoline and its underlying mechanism to OSF and OSCC pathogenesis remain unclear. METHODS: Next-generation sequencing-based transcriptome and dRRBS analysis were performed on OSF and OSCC cells under low-dose arecoline exposure. Functional analyses were performed to compare the different roles of arecoline during OSF and OSCC pathogenesis, and key genes were identified. RESULTS: In this study, we identified that low-dose arecoline promoted cell proliferation of both NFs and OSCC cells via the acceleration of cell cycle progression, while high-dose arecoline was cytotoxic to both NFs and OSCC cells. We performed for the first time the transcriptome and methylome landscapes of NFs and OSCC cells under low-dose arecoline exposure. We found distinct transcriptome and methylome profiles mediated by low-dose arecoline in OSF and OSCC cells, as well as specific genes and signaling pathways associated with metabolic disorders induced by low-dose arecoline exposure. Additionally, low-dose arecoline displayed different functions at different stages, participating in the modulation of the extracellular matrix via Wnt signaling in NFs and epigenetic regulation in OSCC cells. After exposure to low-dose arecoline, the node roles of FMOD in NFs and histone gene clusters in OSCC cells were found. Meanwhile, some key methylated genes induced by arecoline were also identified, like PTPRM and FOXD3 in NFs, SALL3 and IRF8 in OSCC cells, indicating early molecular events mediated by arecoline during OSF and OSCC pathogenesis. CONCLUSIONS: This study elucidated the contribution of low-dose arecoline to OSF and OSCC pathogenesis and identified key molecular events that could be targeted for further functional studies and their potential as biomarkers.

Palbociclib-based high-throughput combination drug screening identifies synergistic therapeutic options in HPV-negative head and neck squamous cell carcinoma.

BACKGROUND: Deregulation of cell-cycle pathway is ubiquitously observed in human papillomavirus negative (HPVneg) head and neck squamous cell carcinoma (HNSCC). Despite being an attractive target, CDK4/6 inhibition using palbociclib showed modest or conflicting results as monotherapy or in combination with platinum-based chemotherapy or cetuximab in HPVneg HNSCC. Thus, innovative agents to augment the efficacy of palbociclib in HPVneg HNSCC would be welcomed. METHODS: A collection of 162 FDA-approved and investigational agents was screened in combinatorial matrix format, and top combinations were validated in a broader panel of HPVneg HNSCC cell lines. Transcriptional profiling was conducted to explore the molecular mechanisms of drug synergy. Finally, the most potent palbociclib-based drug combination was evaluated and compared with palbociclib plus cetuximab or cisplatin in a panel of genetically diverse HPVneg HNSCC cell lines and patient-derived xenograft models. RESULTS: Palbociclib displayed limited efficacy in HPVneg HNSCC as monotherapy. The high-throughput combination drug screening provided a comprehensive palbociclib-based drug-drug interaction dataset, whereas significant synergistic effects were observed when palbociclib was combined with multiple agents, including inhibitors of the PI3K, EGFR, and MEK pathways. PI3K pathway inhibitors significantly reduced cell proliferation and induced cell-cycle arrest in HPVneg HNSCC cell lines when combined with palbociclib, and alpelisib (a PI3Kα inhibitor) was demonstrated to show the most potent synergy with particularly higher efficacy in HNSCCs bearing PIK3CA alterations. Notably, when compared with cisplatin and cetuximab, alpelisib exerted stronger synergism in a broader panel of cell lines. Mechanistically, RRM2-dependent epithelial mesenchymal transition (EMT) induced by palbociclib, was attenuated by alpelisib and cetuximab rather than cisplatin. Subsequently, PDX models with distinct genetic background further validated that palbociclib plus alpelisib had significant synergistic effects in models harboring PIK3CA amplification. CONCLUSIONS: This study provides insights into the systematic combinatory effect associated with CDK4/6 inhibition and supports further initiation of clinical trials using the palbociclib plus alpelisib combination in HPVneg HNSCC with PIK3CA alterations.

Epithelioid sarcoma: A clinicopathological study of 12 head and neck cases.

OBJECTIVES: To determine the clinicopathological features of epithelioid sarcoma presenting in head and neck region (HNES) and elucidate diagnostic key points and treatment options for HNES. MATERIALS AND METHODS: A total of 12 HNES cases were collected in our department from 2010 to 2020. Their clinical information and pathological features were documented, and relevant follow-up was performed. Immunohistochemistry was carried to analyze the protein markers of HNES. RESULTS: Of the 12 HNES cases, 10 were primary tumors and 2 were metastasized from foot and shoulder, respectively. The patients with primary tumors were significantly younger than those with metastasized ones (22.7 vs 41.5, p = .0157), and male patients outnumbered female patients (3:1). Of all HNES cases, 9 were classic subtype, and 3 were proximal subtype. HNES patients had a poor prognosis, with 5-year overall survival of 41.5% and 5-year relapse-free survival of 22.5%. A loss of INI1 was identified as the hallmark of HNES with 83.3% (10/12) of HNES cases presenting as EZH2 positive. CONCLUSIONS: HNES is more prevalent at younger ages and in males, has a poor prognosis, and exhibits a greater proportion of classic subtype than proximal subtype. EZH2 inhibitor has therapeutic potential in HNES.

The crosstalk between AXL and YAP promotes tumor progression through STAT3 activation in head and neck squamous cell carcinoma.

Receptor tyrosine kinases (RTKs) and Yes-associated protein (YAP) are critical driving factors in tumors. Currently, the regulation of RTKs in the Hippo-YAP pathway has been recognized as an important issue. However, the relationship between AXL, one of the RTKs, and YAP in head and neck squamous cell carcinoma (HNSCC) remains unknown. In this study, the crosstalk between AXL and YAP was thoroughly investigated in vitro and in vivo. We determined that there was a positive correlation between AXL and YAP in the HNSCC tissue samples and the Cancer Genome Atlas (TCGA) dataset, and high co-expression was associated with poor prognosis. Inhibiting YAP decreased AXL expression in HNSCC cells, while YAP overexpression increased AXL. Moreover, ectopic expression of AXL reversed tumor suppressor phenotypes mediated by YAP silencing. This reversal effect was also confirmed in vivo. In addition, AXL overexpression and Gas6, a ligand of AXL, stimulated YAP dephosphorylation, nuclear translocation, and target gene transcription. AXL inhibition decreased YAP dephosphorylation and nuclear translocation. Mechanistically, Gas6 induced a competitive binding to phosphorylated signal transducers and activators of transcription 3 (STAT3) with large tumor suppressor kinase 1 (LATS1) and inhibited the Hippo pathway. This study revealed a novel non-transcriptional effect of STAT3 in Gas6/AXL-induced YAP activity, suggesting that STAT3 acted as a critical "molecular switch" during the mutual promotion between AXL and YAP, which might be a promising therapeutic target in HNSCC.

Elevated sodium chloride drives type I interferon signaling in macrophages and increases antiviral resistance.

Type I IFN production and signaling in macrophages play critical roles in innate immune responses. High salt (i.e. high concentrations of NaCl) has been proposed to be an important environmental factor that influences immune responses in multiple ways. However, it remains unknown whether high salt regulates type I IFN production and signaling in macrophages. Here, we demonstrated that high salt promoted IFNß production and its signaling in both human and mouse macrophages, and consequentially primed macrophages for strengthened immune sensing and signaling when challenged with viruses or viral nucleic acid analogues. Using both pharmacological inhibitors and RNA interference we showed that these effects of high salt on IFNß signaling were mediated by the p38 MAPK/ATF2/AP1 signaling pathway. Consistently, high salt increased resistance to vesicle stomatitis virus (VSV) infection in vitro. In vivo data indicated that a high-salt diet protected mice from lethal VSV infection. Taken together, these results identify high salt as a crucial regulator of type I IFN production and signaling, shedding important new light on the regulation of innate immune responses.

Yes-associated protein promotes cell migration via activating Wiskott-Aldrich syndrome protein family member 1 in oral squamous cell carcinoma.

BACKGROUND: Yes-associated protein (YAP) is a candidate oncogene in various cancers including oral squamous cell carcinoma (OSCC). Our previous study demonstrated that TNF-alpha could inhibit cell proliferation and invasion by YAP phosphorylation in OSCC. However, the role of YAP in OSCC is not yet clear. The objective of the present study was to elucidate the function of YAP in promoting migration in OSCC and to explore the possible mechanism with a novel YAP inhibitor CA3. METHODS: A total of 68 OSCC patients were enrolled, and the expression levels of YAP were investigated in tissue specimens by immunohistochemical staining. The inhibitory effects of CA3, a novel inhibitor of YAP, were demonstrated by immunofluorescence, Western blotting, and transwell assays. A human PCR motility array was performed to screen the changes in the gene expression profiles of the cells. In addition, shRNA interference, YAP re-expression, and WAVE1 overexpression plasmids were used to detect the regulatory mechanism of YAP and its relationship with cell migration. RESULTS: Yes-associated protein nuclear expression levels were associated with metastasis and 5-year overall survival rate. CA3 exhibited potent inhibitory effects on OSCC migration. YAP knockdown significantly suppressed tumor cell migration in OSCC. These effects were rescued when YAP was re-expressed and during WAVE1 overexpression in YAP-shRNA stable cells. CONCLUSIONS: The present study revealed that YAP was associated with cell migration and that this process was regulated by YAP/WAVE1. We also demonstrated that CA3 exhibited marked inhibitory effects on YAP expression and that it could be considered a potential therapeutic target for the treatment of OSCC.

Spatial and Single-Cell Transcriptomics Reveal a Cancer-Associated Fibroblast Subset in HNSCC That Restricts Infiltration and Antitumor Activity of CD8+ T Cells.

Although immunotherapy can prolong survival in some patients with head and neck squamous cell carcinoma (HNSCC), the response rate remains low. Clarification of the critical mechanisms regulating CD8+ T-cell infiltration and dysfunction in the tumor microenvironment could help maximize the benefit of immunotherapy for treating HNSCC. Here, we performed spatial transcriptomic analysis of HNSCC specimens with differing immune infiltration and single-cell RNA sequencing of five pairs of tumor and adjacent tissues, revealing specific cancer-associated fibroblast (CAF) subsets related to CD8+ T-cell infiltration restriction and dysfunction. These CAFs exhibited high expression of CXCLs (CXCL9, CXCL10, and CXCL12) and MHC-I and enrichment of galectin-9 (Gal9). The proportion of MHC-IhiGal9+ CAFs was inversely correlated with abundance of a TCF1+GZMK+ subset of CD8+ T cells. Gal9 on CAFs induced CD8+ T-cell dysfunction and decreased the proportion of tumor-infiltrating TCF1+CD8+ T cells. Collectively, the identification of MHC-IhiGal9+ CAFs advances the understanding of the precise role of CAFs in cancer immune evasion and paves the way for more effective immunotherapy for HNSCC. SIGNIFICANCE: Spatial analysis identifies IFN-induced MHC-IhiGal9+ CAFs that form a trap for CD8+ T cells, providing insights into the complex networks in the tumor microenvironment that regulate T-cell infiltration and function.

Mapping the landscape of HPV integration and characterising virus and host genome interactions in HPV-positive oropharyngeal squamous cell carcinoma.

BACKGROUND: Human papillomavirus (HPV) integration into the host genome is an important factor in HPV(+)OPSCC carcinogenesis, in conjunction with HPV oncoproteins E6/E7. However, a well-studied investigation about virus-host interaction still needs to be completed. Our objective is to characterise HPV integration to investigate potential mechanisms of tumourigenesis independent of E6/E7 oncoproteins. MATERIALS AND METHODS: High-throughput viral integration detection was performed on 109 HPV(+)OPSCC tumours with relevant clinicopathological information. Of these tumours, 38 tumours underwent targeted gene sequencing, 29 underwent whole exome sequencing and 26 underwent RNA sequencing. RESULTS: HPV integration was detected in 94% of tumours (with a mean integration count of 337). Tumours occurring at the tonsil/oropharyngeal wall that exhibit higher PD-L1 expression demonstrated increased integration sites (p = .024). HPV exhibited a propensity for integration at genomic sites located within specific fragile sites (FRA19A) or genes associated with functional roles such as cell proliferation and differentiation (PTEN, AR), immune evasion (CD274) and glycoprotein biosynthesis process (FUT8). The viral oncogenes E2, E4, E6 and E7 tended to remain intact. HPV fragments displayed enrichment within host copy number variation (CNV) regions. However, insertions into genes related to altered homologous recombination repair were infrequent. Genes with integration had distinct expression levels. Fifty-nine genes whose expression level was affected by viral integration were identified, for example, EPHB1, which was reported to be involved in cellular protein metabolic process. CONCLUSIONS: HPV can promote oncogenesis through recurrent integration into functional host genome regions, leading to subsequent genomic aberrations and gene expression disruption. This study characterises viral integrations and virus-host interactions, enhancing our understanding of HPV-related carcinogenesis mechanisms.

[An improved paraffin embedding method for small core needle biopsy: technical introduction and evaluation].

PURPOSE: Modified agar pre-embedded paraffin embedding method was proposed to evaluate the effects on tissue integrity, histological morphology, protein and DNA detection in small specimens of core needle biopsies. METHODS: The core needle biopsy specimens of 10 patients with oral mucosal squamous cell carcinoma were subjected to modified agar pre-embedded paraffin embedding using molded embedding molds and conventional paraffin embedding respectively, the dehydration time of the former was 3.5 h and that of the latter was 12 h. After tissue treatment, H-E staining, histological morphology, immunohistochemistry (IHC), and DNA fluorescence in situ hybridization (FISH) were performed, respectively. The results were compared and analyzed using GraphPad Prism 9 software package. RESULTS: The modified agar pre-embedding method was less difficult to perform than the agar pre-embedding method, and easier to be promoted. Compared with conventional paraffin embedding method, the tissue dehydration time was significantly reduced(P＜0.001), and the results of microscopic histological morphology and subsequent IHC and FISH assays were reliable. CONCLUSIONS: The modified agar pre-embedded paraffin embedding method meets the requirements of clinical pathological diagnosis for tissue processing, and is worthy of clinical application for core needle biopsy specimens.

Fibular flap mandibular reconstruction for third-stage medication-related osteonecrosis of the jaw: A retrospective single-center study.

Background/purpose: The incidence and patient population of medication-related osteonecrosis of the jaw (MRONJ) has dramatically increased due to the use of drugs suppressing bone metastasis. However, its clinical treatment is still very difficult. The aim of this study was to evaluate the effectiveness and outcome of immediate fibular flap reconstruction for MRONJ in the mandible. Materials and methods: Patients who underwent immediate fibular flap reconstruction for MRONJ in the mandible in our institution from 1990 to 2022 were screened and identified. Their demographics, drug history, symptoms, surgical parameters and follow-up data were collected and analyzed. Results: In total, 25 patients with MRONJ stage 3 were included. The main cause of drug administration was osseous metastasis (88%), and zoledronate was the main drug. Pain, swelling (44%), pyorrhea (28%), extraoral fistulas (16%) and necrotic bone exposure (12%) were the main chief complaints. After segmental mandibulectomy, the fibular flap harvest was 9.73 ± 3.37 cm, and 18/25 (72%) were cut into two segments to reconstruct the mandible. Sixty-eight percent had an intraoral skin paddle placed. All of the flaps survived, and 21/25 (84%) of the soft tissue underwent primary healing. During follow-up, the alleviation of symptoms was effective, and there was no primary disease progression or death. Conclusion: This is the largest comprehensive investigation of fibular flap reconstruction for MRONJ in the mandible, which is proved to be an alternative and effective treatment for managing advanced patients with MRONJ.

Extracellular matrix remodelling and stiffening contributes to tumorigenesis of salivary carcinoma ex pleomorphic adenoma--A study based on patient-derived organoids.

BACKGROUND: Salivary carcinoma ex pleomorphic adenoma (CXPA) is defined as a carcinoma that develops from benign pleomorphic adenoma (PA). Abnormally activated Androgen signaling pathway and amplification of HER-2/neu(ERBB-2) gene are known to be involved in CXPA tumorigenesis. Recent progress in tumour microenvironment research has led to identification that extracellular matrix (ECM) remodelling and increased stiffness act as critical contributing role in tumour carcinogenesis. This study examined ECM modifications to elucidate the mechanism underlying CXPA tumorigenesis. RESULTS: PA and CXPA organoids were successfully established. Histological observation, immunohistochemistry (IHC), and whole-exome sequencing demonstrated that organoids recapitulated phenotypic and molecular characteristics of their parental tumours. RNA-sequencing and bioinformatic analysis of organoids showed that differentially expressed genes are highly enriched in ECM-associated terms, implying that ECM alternations may be involved in carcinogenesis. Microscopical examination for surgical samples revealed that excessive hyalinized tissues were deposited in tumour during CXPA tumorigenesis. Transmission electron microscopy confirmed that these hyalinized tissues were tumour ECM in nature. Subsequently, examination by picrosirius red staining, liquid chromatography with tandem mass spectrometry, and cross-linking analysis indicated that tumour ECM was predominantly composed of type I collagen fibers, with dense collagen alignment and an increased level of collagen cross-linking. IHC revealed the overexpression of COL1A1 protein and collagen-synthesis-related genes, DCN and IGFBP5 (p < 0.05). Higher stiffness of CXPA than PA was demonstrated by atomic force microscopy and elastic imaging analysis. We utilized hydrogels to mimic ECM with varying stiffness degrees in vitro. Compared with softer matrices (5Kpa), CXPA cell line and PA primary cells exhibited more proliferative and invasive phenotypes in stiffer matrices (50Kpa, p < 0.01). Protein-protein interaction (PPI) analysis of RNA-sequencing data revealed that AR and ERBB-2 expression was associated with TWIST1. Moreover, surgical specimens demonstrated a higher TWIST1 expression in CXPA over PA. After knocking down TWIST1 in CXPA cells, cell proliferation, migration, and invasiveness were significantly inhibited (p < 0.01). CONCLUSION: Developing CXPA organoids provides a useful model for cancer biology research and drug screening. ECM remodelling, attributed to overproduction of collagen, alternation of collagen alignment, and increased cross-linking, leads to increased ECM stiffness. ECM modification is an important contributor in CXPA tumorigenesis.

Characteristics and Correlations of the Oral and Gut Fungal Microbiome with Hypertension.

The mycobiome is an essential constituent of the human microbiome and is associated with various diseases. However, the role of oral and gut fungi in hypertension (HTN) remains largely unexplored. In this study, saliva, subgingival plaques, and feces were collected from 36 participants with HTN and 24 healthy controls for metagenomic sequencing. The obtained sequences were analyzed using the Kraken2 taxonomic annotation pipeline to assess fungal composition and diversity. Correlations between oral and gut fungi and clinic parameters, between fungi within the same sample types, and between different sample types were identified by Spearman's correlation analysis. Overall, the subgingival fungal microbiome had substantially higher alpha diversity than the salivary and fecal fungal microbiomes. The fungal microbiomes of the three sample types displayed distinct beta diversity from each other. Oral fungi but not gut fungi in HTN had beta diversity significantly different from that of controls. Among the fungi shared in the oral cavity and gut, Exophiala was the genus with the most notable changes. Exophiala spinifera was the most abundant salivary species in HTN. Some fungal species directly correlated with blood pressure, including gut Exophiala xenobiotica and Exophiala mesophila. The markedly impaired ecological cocorrelation networks of oral and gut fungi in HTN suggested compromised association among fungal species. Most fungi were shared in the oral cavity and gut, and their correlations suggested the potential interplays between oral and gut fungi. In conclusion, the oral cavity and intestine have unique fungal ecological environments. The fungal enrichment and ecology in HTN, the correlations between oral and gut fungi, and the associations between oral and gut fungi and clinical parameters suggest an important role that the fungal microbiome may play in HTN. IMPORTANCE Our study fills the gap in human studies investigating the oral and gut fungal microbiota in association with blood pressure. It characterizes the diversity and composition of the oral and gut fungal microbiome in human subjects, elucidates the dysbiosis of fungal ecology in a hypertensive population, and establishes oral-gut fungal correlations and fungus-clinical parameter correlations. Targeting fungi in the oral cavity and/or gut may provide novel strategies for the prevention and treatment of hypertension.

Roles of oral microbiota and oral-gut microbial transmission in hypertension.

INTRODUCTION: Considerable evidence has linked periodontitis (PD) to hypertension (HTN), but the nature behind this connection is unclear. Dysbiosis of oral microbiota leading to PD is known to aggravate different systematic diseases, but the alteration of oral microbiota in HTN and their impacts on blood pressure (BP) remains to be discovered. OBJECTIVES: To characterize the alterations of oral and gut microbiota and their roles in HTN. METHODS: We performed a cross-sectional (95 HTN participants and 39 controls) and a 6-month follow-up study (52 HTN participants and 26 controls) to analyze the roles of oral and gut microbiota in HTN. Saliva, subgingival plaques, and feces were collected for 16S rRNA gene sequencing or metagenomic analysis. C57BL/6J mice were pretreated with antibiotics to deplete gut microbiota, and then transplanted with human saliva by gavage to test the impacts of abnormal oral-gut microbial transmission on HTN. RESULTS: BP in participants with PD was higher than no PD in both cross-sectional and follow-up cohort. Relative abundances of 14 salivary genera, 15 subgingival genera and 10 gut genera significantly altered in HTN and those of 7 salivary genera, 12 subgingival genera and 6 gut genera significantly correlated with BP. Sixteen species under 5 genera were identified as oral-gut transmitters, illustrating the presence of oral-gut microbial transmission in HTN. Veillonella was a frequent oral-gut transmitter stably enriched in HTN participants of both cross-sectional and follow-up cohorts. Saliva from HTN participants increased BP in hypertensive mice. Human saliva-derived Veillonella successfully colonized in mouse gut, more abundantly under HTN condition. CONCLUSIONS: PD and oral microbiota are strongly associated with HTN, likely through oral-gut transmission of microbes. Ectopic colonization of saliva-derived Veillonella in the gut may aggravate HTN. Therefore, precise manipulations of oral microbiota and/or oral-gut microbial transmission may be useful strategies for better prevention and treatment of HTN.

Candidate therapeutic agents in a newly established triple wild-type mucosal melanoma cell line.

BACKGROUND: Mucosal melanoma has characteristically distinct genetic features and typically poor prognosis. The lack of representative mucosal melanoma models, especially cell lines, has hindered translational research on this melanoma subtype. In this study, we aimed to establish and provide the biological properties, genomic features and the pharmacological profiles of a mucosal melanoma cell line that would contribute to the understanding and treatment optimization of molecularly-defined mucosal melanoma subtype. METHODS: The sample was collected from a 67-year-old mucosal melanoma patient and processed into pieces for the establishment of cell line and patient-derived xenograft (PDX) model. The proliferation and tumorigenic property of cancer cells from different passages were evaluated, and whole-genome sequencing (WGS) was performed on the original tumor, PDX, established cell line, and the matched blood to confirm the establishment and define the genomic features of this cell line. AmpliconArchitect was conducted to depict the architecture of amplified regions detected by WGS. High-throughput drug screening (HTDS) assay including a total of 103 therapeutic agents was implemented on the established cell line, and selected candidate agents were validated in the corresponding PDX model. RESULTS: A mucosal melanoma cell line, MM9H-1, was established which exhibited robust proliferation and tumorigenicity after more than 100 serial passages. Genomic analysis of MM9H-1, corresponding PDX, and the original tumor showed genetic fidelity across genomes, and MM9H-1 was defined as a triple wild-type (TWT) melanoma subtype lacking well-characterized "driver mutations". Instead, the amplification of several oncogenes, telomerase reverse transcriptase (TERT), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), melanocyte Inducing transcription factor (MITF) and INO80 complex ATPase subunit (INO80), via large-scale genomic rearrangement potentially contributed to oncogenesis of MM9H-1. Moreover, HTDS identified proteasome inhibitors, especially bortezomib, as promising therapeutic candidates for MM9H-1, which was verified in the corresponding PDX model in vivo. CONCLUSIONS: We established and characterized a new mucosal melanoma cell line, MM9H-1, and defined this cell line as a TWT melanoma subtype lacking well-characterized "driver mutations". The MM9H-1 cell line could be adopted as a unique model for the preclinical investigation of mucosal melanoma.

Anti-Swelling, Robust, and Adhesive Extracellular Matrix-Mimicking Hydrogel Used as Intraoral Dressing.

Due to the wet and dynamic environment of the oral cavity, the healing of intraoral wounds, such as tooth extraction wounds, requires stable and firm wound dressings. In clinical practice, cotton balls and gauzes, sponge plugs, or sutures are used to treat extraction wounds, but none of these means can continuously isolate the wound from the intraoral environment and facilitate ideal healing conditions. Herein, inspired by the natural extracellular matrix, a family of wound dressings is developed for intraoral wound repair. Infiltrating a ductile long-chain hydrogel network into a prefabricated, sturdy macromolecular meshwork and in situ crosslinking endowed the composite hydrogel with controllable swelling behaviors and robust mechanical properties. The macromolecular meshwork functioned as the backbone to support the composite and restricts the swelling of the long-chain hydrogel network. In vitro tests verified that this wound dressing can provide durable protection for intraoral wounds against complex irritations. Furthermore, accelerated wound healing occurred when the wound dressing is applied in vivo on a canine tooth extraction model, due to the effective reduction of acute inflammation. These results suggest that this family of bioinspired hydrogels has great potential for application as intraoral wound dressing.

Targeting cyclin-dependent kinase 4/6 as a therapeutic approach for mucosal melanoma.

Mucosal melanoma is a rare but devastating subtype of melanoma which typically has a worse prognosis than other melanoma subtypes. Large-scale next-generation sequencing studies, including our recent research, have also proved that the molecular landscape and potential oncogenic drivers of mucosal melanoma remain distinct from that of cutaneous melanoma. Recently, a number of selective cyclin-dependent kinase 4 (CDK4)/6 inhibitors have been approved for clinical application in breast cancer or entered phase III clinical trial in other solid tumors. Additionally, we have revealed that the dysregulation of cell cycle progression, caused by CDK4 amplification, is a key genetic feature in half of mucosal melanoma and targeting of CDK4 in selected mucosal melanoma patients is a potentially promising direction for precision cancer treatment by using molecular-characterized mucosal melanoma patient-derived-xenograft models. This review summarizes the current literature regarding CDK4/6 dysregulation in mucosal melanoma, preclinical and clinical studies of CDK4/6 inhibitors and potential combinational strategies in treating mucosal melanoma.

[Investigation and analysis on the status of conducting clinical research among oral health professionals].

PURPOSE: To understand oral health professionals' knowledge, attitude, behavior and training needs related to clinical research, and explore relevant factors affecting clinical research knowledge level. METHODS: An online self-designed questionnaire was conducted among oral health professionals from the collaborative innovation network member units of the National Clinical Research Center for Oral Diseases (Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine). The data were processed with SAS 9.4 software package. RESULTS: A total of 281 oral health professionals were enrolled in the study. Most of them had a positive attitude towards clinical research, 80% of them had an idea of carrying out clinical research, while only 22.8% of them implemented finally. The main causes restricting oral health professionals from conducting clinical research were lack of time (68.3%), insufficient teams (63.7%), and short of financial support (60.9%). Participants' mean score of clinical research knowledge was (13.72±7.20) points. Multiple linear regression model showed the type of hospital, clinical research participation in the past five years and epidemiologists' or statisticians' involvement in the latest project were related to participants' knowledge level of clinical research. CONCLUSIONS: Oral health professionals have a positive attitude towards clinical research, while their behavior and knowledge about clinical research were weak. Strengthening the top design of clinical research is inseparable from the cultivation of professionals' clinical research ability. The national clinical research center should give full play to the mission of the "national team", accelerate the establishment of a specialized, normalized and large-scale clinical research training pattern, and provide training opportunities to network member units.