Detailed Clinical Features of PTPRQ-Associated Hearing Loss Identified in a Large Japanese Hearing Loss Cohort.

The PTPRQ gene has been identified as one of the genes responsible for non-syndromic sensorineural hearing loss (SNHL), and assigned as DFNA73 and DFNB84. To date, about 30 causative PTPRQ variants have been reported to cause SNHL. However, the detailed clinical features of PTPRQ-associated hearing loss (HL) remain unclear. In this study, 15,684 patients with SNHL were enrolled and genetic analysis was performed using massively parallel DNA sequencing (MPS) for 63 target deafness genes. We identified 17 possibly disease-causing PTPRQ variants in 13 Japanese patients, with 15 of the 17 variants regarded as novel. The majority of variants identified in this study were loss of function. Patients with PTPRQ-associated HL mostly showed congenital or childhood onset. Their hearing levels at high frequency deteriorated earlier than that at low frequency. The severity of HL progressed from moderate to severe or profound HL. Five patients with profound or severe HL received cochlear implantation, and the postoperative sound field threshold levels and discrimination scores were favorable. These findings will contribute to a greater understanding of the clinical features of PTPRQ-associated HL and may be relevant in clinical practice.

Fibrinogen-to-lymphocyte Ratio Predicts the Outcomes of Hypopharyngeal Squamous Cell Carcinoma Treated With Definitive Radiotherapy.

BACKGROUND/AIM: Previous studies have identified several inflammatory biomarkers that are useful as prognostic biomarkers for various cancer types. However, the fibrinogen-to-lymphocyte ratio (FLR) has not been addressed in head and neck squamous cell carcinoma. Here, we aimed to examine the value of pretreatment FLR as a prognostic marker in patients who received definitive radiotherapy for hypopharyngeal squamous cell carcinoma (HpSCC). PATIENTS AND METHODS: This retrospective study included 95 patients treated with definitive radiotherapy for HpSCC between 2013 and 2020. The prognostic factors for progression-free (PFS) and overall (OS) survival were identified. RESULTS: The optimal cut-off value of pretreatment FLR for discriminating PFS was 2.46. Based on this value, 57 and 38 patients were classified into groups with high and low FLR, respectively. A high FLR was significantly associated with advanced local disease and overall stage, and with the development of synchronous second primary cancer compared with a low FLR. The high FLR group had significantly lower PFS and OS rates than the low FLR group. Multivariate analysis showed that having a high pretreatment FLR was an independent prognostic factor for poorer PFS and OS [PFS: hazard ratio (HR)=2.14, 95% confidence interval (CI)=1.09-4.19, p=0.026; OS: HR=2.86, 95% CI=1.14-7.20, p=0.024]. CONCLUSION: The FLR has a clinical effect on PFS and OS in patients with HpSCC, suggesting that it has potential application as a prognostic factor for patients with HpSCC.

Narrow-field supracricoid partial laryngectomy: Procedure development and initial clinical experiences.

OBJECTIVES: To evaluate the feasibility of narrow-field supracricoid partial laryngectomy with cricohyoidoepiglottopexy (NF-SCPL-CHEP). METHODS: Between 2019 and 2020, five patients with glottic cancers underwent NF-SCPL-CHEP. The mean durations of surgical drains, tracheostomy canula, and nasogastric tube use were evaluated. Length of stay following NF-SCPL-CHEP was compared with that of our open SCPL historical controls. A case summary is provided for the first patients, with detailed information about postoperative management and function. RESULTS: All five patients achieved uneventful postoperative recoveries without major complications. The average time for surgical drains, tracheostomy canula, and nasogastric tube use were 2, 15, and 46 days, respectively. The mean overall hospitalization period was 36 days for NF-SCPL-CHEP patients. The mean period of hospitalization based on our early experiences between 1997 and 2005 with classical open SCPL was 72 days. All patients were fully functional and local recurrences or distant metastases were not encountered during a mean observation period of 39 months. CONCLUSIONS: NF-SCPL-CHEP with 6 cm cervical access appeared technically feasible and oncologically sound in this initial clinical experience. An extra 2 cm incision, which enabled lateral neck dissection, was not felt to detract from the overall minimally invasive basis of NF-SCPL-CHEP. The clinical results were encouraging with limited complications and predictable postoperative recovery. The length of stay for patients undergoing NF-SCPL was half that of open SCPL historical controls. Less damages to local circulation may associate with the positive influences. Further study with a large patient sample across multiple institutions are needed to carefully evaluate long-term functional and oncological outcomes.

A first case report of nasopharyngeal Mycobacterium abscessus subspecies massiliense infection.

BACKGROUND: Mycobacterium abscessus subspecies massiliense is a non-tuberculous mycobacteriosis and was subdivided from Mycobacterium abscessus in 2006. This article is the first report on nasopharyngitis caused by Mycobacterium abscessus subspecies massiliense. CASE PRESENTATION: A 45-year-old woman had an 18-month history of recurrent nasopharyngitis and presented with pain in the throat. Mycobacterial tissue culture and polymerase chain reaction testing revealed the presence of Mycobacterium abscessus subspecies massiliense in the nasopharyngeal tissue. This patient underwent surgery, followed by multiple rounds of chemotherapy with oral and intravenous antibiotic agents for 16 weeks. She has had no recurrence during the 56 weeks since treatment. CONCLUSION: It is difficult to detect the presence of Mycobacterium abscessus subspecies massiliense in a culture from the swabbing sample. The tissue culture from a biopsy specimen is mandatory for the identification of the species. Currently, no definite treatment policy is available and only empirical treatment is applied. This case is an important for the diagnosis and treatment of this bacterial infection on nasopharynx.

FosL1 Regulates Regional Metastasis of Head and Neck Squamous Cell Carcinoma by Promoting Cell Migration, Invasion, and Proliferation.

BACKGROUND/AIM: We evaluated the impact of FosL1, a member of the activated protein-1 family, on the pathways leading to regional metastasis of head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: We examined the influence of small interfering RNA (siRNA) and short heparin RNA (shRNA) mediated knockdown of FosL1 on cell migration, invasion, and proliferation in vitro as well as on regional metastasis in vivo. The prognostic significance of FosL1 was also analyzed using the Kaplan- Meier plotter using data from an HNSCC patient database. RESULTS: Down-regulation of FosL1 inhibited cell migration, invasion, and proliferation in vitro, decreased the incidence of regional metastases, and prolonged the survival of mice in vivo. We also determined that HNSCC patients with higher expression levels of FosL1 had a significantly shorter survival time than those with low expression of FosL1. CONCLUSION: FosL1 plays a crucial role in promoting cell migration, invasion, and proliferation in HNSCC.

Long-term treatment outcome of type 1 thyroplasty using novel titanium medialization laryngoplasty implant combined with arytenoid adduction for unilateral vocal cord paralysis: single-arm interventional study at a single institution.

OBJECTIVE: To evaluate the long-term treatment outcome of type 1 thyroplasty with novel rearrangeable titanium medialization laryngoplasty implant (TMLI) combined with arytenoid adduction (AA) for unilateral vocal cord paralysis (UVFP) in the authors' institution. METHODS: A total of 16 Japanese patients with UVFP who received type 1 thyroplasty using TMLI with arytenoid adduction was enrolled in this single-arm, non-randomized interventional study. The results of the auditory perceptual assessment, aerodynamic examination, acoustic measurement, and patient-based survey on these patients were evaluated preoperatively and at 3, 6, and 12 months postoperatively. RESULTS: Type 1 thyroplasty using TMLI with arytenoid adduction for our patient series presented significant improvements in maximum phonation time, mean flow rates, GRBAS scale, the Voice Handicap Index and the Voice-Related Quality of Life score over the 12-month postoperative period. CONCLUSION: Type 1 thyroplasty using TMLI with arytenoid adduction was quite effective for obtaining satisfactory postoperative voice improvement without any surgical complication over the long-term period.

FLCN alteration drives metabolic reprogramming towards nucleotide synthesis and cyst formation in salivary gland.

FLCN is a tumor suppressor gene which controls energy homeostasis through regulation of a variety of metabolic pathways including mitochondrial oxidative metabolism and autophagy. Birt-Hogg-Dubé (BHD) syndrome which is driven by germline alteration of the FLCN gene, predisposes patients to develop kidney cancer, cutaneous fibrofolliculomas, pulmonary cysts and less frequently, salivary gland tumors. Here, we report metabolic roles for FLCN in the salivary gland as well as their clinical relevance. Screening of salivary glands of BHD patients using ultrasonography demonstrated increased cyst formation in the salivary gland. Salivary gland tumors that developed in BHD patients exhibited an upregulated mTOR-S6R pathway as well as increased GPNMB expression, which are characteristics of FLCN-deficient cells. Salivary gland-targeted Flcn knockout mice developed cytoplasmic clear cell formation in ductal cells with increased mitochondrial biogenesis, upregulated mTOR-S6K pathway, upregulated TFE3-GPNMB axis and upregulated lipid metabolism. Proteomic and metabolite analysis using LC/MS and GC/MS revealed that Flcn inactivation in salivary gland triggers metabolic reprogramming towards the pentose phosphate pathway which consequently upregulates nucleotide synthesis and redox regulation, further supporting that Flcn controls metabolic homeostasis in salivary gland. These data uncover important roles for FLCN in salivary gland; metabolic reprogramming under FLCN deficiency might increase nucleotide production which may feed FLCN-deficient salivary gland cells to trigger tumor initiation and progression, providing mechanistic insight into salivary gland tumorigenesis as well as a foundation for development of novel therapeutics for salivary gland tumors.

BHD-associated kidney cancer exhibits unique molecular characteristics and a wide variety of variants in chromatin remodeling genes.

Birt-Hogg-Dubé (BHD) syndrome is a hereditary kidney cancer syndrome, which predisposes patients to develop kidney cancer, cutaneous fibrofolliculomas and pulmonary cysts. The responsible gene FLCN is a tumor suppressor for kidney cancer, which plays an important role in energy homeostasis through the regulation of mitochondrial oxidative metabolism. However, the process by which FLCN-deficiency leads to renal tumorigenesis is unclear. In order to clarify molecular pathogenesis of BHD-associated kidney cancer, we conducted whole-exome sequencing analysis using next-generation sequencing technology as well as metabolite analysis using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Whole-exome sequencing analysis of BHD-associated kidney cancer revealed that copy number variations of BHD-associated kidney cancer are considerably different from those already reported in sporadic cases. In somatic variant analysis, very few variants were commonly observed in BHD-associated kidney cancer; however, variants in chromatin remodeling genes were frequently observed in BHD-associated kidney cancer (17/29 tumors, 59%). Metabolite analysis of BHD-associated kidney cancer revealed metabolic reprogramming toward upregulated redox regulation which may neutralize reactive oxygen species potentially produced from mitochondria with increased respiratory capacity under FLCN-deficiency. BHD-associated kidney cancer displays unique molecular characteristics that are completely different from sporadic kidney cancer, providing mechanistic insight into tumorigenesis under FLCN-deficiency as well as a foundation for development of novel therapeutics for kidney cancer.

Establishment and characterization of BHD-F59RSVT, an immortalized cell line derived from a renal cell carcinoma in a patient with Birt-Hogg-Dubé syndrome.

Hereditary renal cell carcinomas (RCCs) are life-threatening disorders not only for the patients but also for their relatives. Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder caused by germline mutations in the folliculin gene (FLCN). The protein product, FLCN, functions as a tumor suppressor, and the affected patients have high risks of developing multiple RCCs. The carcinogenic mechanisms stemming from FLCN dysfunction have been investigated using rodent models and human RCC tissues. However, very limited information has been available about in vitro signaling of human renal cells with genetically mutant FLCN. Herein, we established a new cell line, BHD-F59RSVT, from a BHD patient's chromophobe RCC by transfecting SV40 large T antigen. We investigated FLCN mutations, chromosome profiles, and cytopathologic characteristics of the cell line. BHD-F59RSVT reflected the patient's FLCN germline mutation, a 3-nt deletion in exon 13 (c.1528_1530delGAG). Neither somatic mutation nor loss of heterozygosity of FLCN was detectable. Chromosome 17p11.2 of the FLCN proximal region demonstrated a trimodal pattern. Genome-wide chromosomal analysis revealed a loss of chromosome 16 and mosaic segmental gains in chromosome 7. BHD-F59RSVT cells were positive when immunostained for cytokeratin 7, supporting their origin from distal convoluted tubules. Western blotting analysis demonstrated severely suppressed FLCN expression at the protein level. The collective findings indicate that the established cell line will be suitable for functional analysis of the typical phenotype of BHD-associated RCC with suppressed FLCN expression.

Identification of human papillomavirus (HPV) 16 DNA integration and the ensuing patterns of methylation in HPV-associated head and neck squamous cell carcinoma cell lines.

Recent studies showed that human papillomavirus (HPV) integration contributes to the genomic instability seen in HPV-associated head and neck squamous cell carcinoma (HPV-HNSCC). However, the epigenetic alterations induced after HPV integration remains unclear. To identify the molecular details of HPV16 DNA integration and the ensuing patterns of methylation in HNSCC, we performed next-generation sequencing using a target-enrichment method for the effective identification of HPV16 integration breakpoints as well as the characterization of genomic sequences adjacent to HPV16 integration breakpoints with three HPV16-related HNSCC cell lines. The DNA methylation levels of the integrated HPV16 genome and that of the adjacent human genome were also analyzed by bisulfite pyrosequencing. We found various integration loci, including novel integration sites. Integration loci were located predominantly in the intergenic region, with a significant enrichment of the microhomologous sequences between the human and HPV16 genomes at the integration breakpoints. Furthermore, various levels of methylation within both the human genome and the integrated HPV genome at the integration breakpoints in each integrant were observed. Allele-specific methylation analysis suggested that the HPV16 integrants remained hypomethylated when the flanking host genome was hypomethylated. After integration into highly methylated human genome regions, however, the HPV16 DNA became methylated. In conclusion, we found novel integration sites and methylation patterns in HPV-HNSCC using our unique method. These findings may provide insights into understanding of viral integration mechanism and virus-associated carcinogenesis of HPV-HNSCC.

H255Y and K508R missense mutations in tumour suppressor folliculin (FLCN) promote kidney cell proliferation.

Germline H255Y and K508R missense mutations in the folliculin (FLCN) gene have been identified in patients with bilateral multifocal (BMF) kidney tumours and clinical manifestations of Birt-Hogg-Dubé (BHD) syndrome, or with BMF kidney tumours as the only manifestation; however, their impact on FLCN function remains to be determined. In order to determine if FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation leading to pathogenicity, we generated mouse models expressing these mutants using BAC recombineering technology and investigated their ability to rescue the multi-cystic phenotype of Flcn-deficient mouse kidneys. Flcn H255Y mutant transgene expression in kidney-targeted Flcn knockout mice did not rescue the multi-cystic kidney phenotype. However, expression of the Flcn K508R mutant transgene partially, but not completely, abrogated the phenotype. Notably, expression of the Flcn K508R mutant transgene in heterozygous Flcn knockout mice resulted in development of multi-cystic kidneys and cardiac hypertrophy in some mice. These results demonstrate that both FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation, but to different degrees. Based on the phenotypes of our preclinical models, the FLCN H255Y mutant protein has lost it tumour suppressive function leading to the clinical manifestations of BHD, whereas the FLCN K508R mutant protein may have a dominant negative effect on the function of wild-type FLCN in regulating kidney cell proliferation and, therefore, act as an oncoprotein. These findings may provide mechanistic insight into the role of FLCN in regulating kidney cell proliferation and facilitate the development of novel therapeutics for FLCN-deficient kidney cancer.

JunB promotes cell invasion, migration and distant metastasis of head and neck squamous cell carcinoma.

BACKGROUND: While treatment failure in cases of head and neck squamous cell carcinoma (HNSCC) frequently takes the form of locoregional recurrences and distant metastasis, our understanding of the mechanisms of metastasis in HNSCC is limited. We initially performed the upstream and key nodes analysis together with whole gene microarray analysis characterized by distant metastatic potential in vivo with HNSCC cell lines and identified JunB, a member of the activator protein-1 (AP-1) family, as a key molecule in the regulation of the pathways related to distant metastasis in HNSCC. We have therefore tested the hypothesis that JunB plays a crucial role in distant metastasis in HNSCC. METHODS: To study the role of JunB on metastatic potential of HNSCC, small interfering RNA (siRNA)-mediated knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (cas9) system (CRISPR/Cas9)-mediated knockout of JunB in HNSCC cells were established and the abilities of cell invasion and migration in vitro were examined. The efficacy of knockout of JunB was also examined using an experimental lung metastatic mouse model of HNSCC. In addition, to study if the role of JunB in HNSCC cell migration and invasiveness is related to epithelial-to-mesenchymal transition (EMT), cell morphology and expression of mesenchymal or epithelial marker on siRNA mediated JunB knockdown in HNSCC cells were examined with or without TGF-ß stimulation. RESULTS: siRNA knockdown and sgRNA knockout of JunB in metastatic HNSCC cells significantly suppressed both cell invasion and migration in vitro. In addition, the knockout of JunB in metastatic HNSCC cells significantly repressed the incidence of lung metastases and prolonged the survival in vivo. However, we did not observe any change in cell morphology with the down-regulation of mesenchymal markers and up-regulation of epithelial markers in response to siRNA-mediated JunB knockdown in HNSCC cells. CONCLUSION: These results suggested that JunB could play an important role in promoting cell invasion, migration and distant metastasis in HNSCC via pathways other than EMT and that the down-regulation of JunB may become an effective strategy for patients with invasive HNSCC.

Efficacy and toxicity of concurrent chemoradiotherapy in patients with advanced oropharyngeal squamous cell carcinoma.

PURPOSE: The aim of this study was to evaluate the feasibility and toxicity of concurrent chemoradiotherapy (CCRT) with docetaxel, cisplatin (CDDP) and 5-fluorouracil (5-FU) (TPF regimen) or with CDDP, 5-FU, methotrexate and leucovorin (PFML regimen) in previously untreated patients with advanced oropharyngeal squamous cell carcinoma (SCC). METHODS: Fifty-six eligible patients with stage III or IV oropharyngeal SCC were treated with CCRT. Forty-four patients were men and 12 were women, and the average age of the patients was 58.8 years (range, 37-72 years). In the TPF group, patients received CCRT with the TPF regimen [docetaxel (50 mg/m(2), day 1), CDDP (60 mg/m(2), day 4) and a continuous 5-FU infusion (600 mg/m(2)/day, days 1-5)]. In the PFML group, patients received CCRT with the PFML regimen [CDDP (60 mg/m(2), day 4), a continuous 5-FU infusion (600 mg/m(2)/day, days 1-5), methotrexate (30 mg/m(2), day 1) and leucovorin (10 mg/m(2)/day, days 1-5)]. The total radiation dose was between 66.6 and 70.2 Gy. RESULTS: The overall 5-year survival rate was 64.6% in all patients, 68.6% in the resectable group and 47.4% in the unresectable group. The 5-year disease-specific survival rate was 72.2% in all patients, 78.1% in the resectable group and 47.7% in the unresectable group. Regarding clinical stage, the 5-year disease-specific survival rates were 91% in stage III, 72% in stage IVa and 44% in stage IVb. CONCLUSION: CCRT with TPF or PFML regimen for advanced oropharyngeal SCC is tolerable and effective, especially in patients with resectable disease.