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Abstract Polyploidy is frequently enhanced under pathological conditions, such as tissue injury and cancer in humans. Polyploidization is critically involved in cancer evolution, including cancer initiation and the acquisition of drug resistance. However, the effect of polyploidy on cell fate remains unclear. In this study, we explored the effects of polyploidization on cellular responses to DNA damage and cell cycle progression. Through various comparisons based on ploidy stratifications of cultured cells, we found that polyploidization and the accumulation of genomic DNA damage mutually induce each other, resulting in polyploid cells consistently containing more genomic DNA damage than diploid cells under both physiological and stress conditions. Notably, despite substantial DNA damage, polyploid cells demonstrated a higher tolerance to its impact, exhibiting delayed cell cycle arrest and reduced secretion of inflammatory cytokines associated with DNA damage-induced senescence. Consistently, in mice with ploidy tracing, hepatocytes with high ploidy appeared to potentially persist in the damaged liver, while being susceptible to DNA damage. Polyploidy acts as a reservoir of genomic damage by mitigating the impact of DNA damage, while simultaneously enhancing its accumulation.

Summary: The cochlea contains two extracellular fluids, perilymph and endolymph. Endolymph exhibits high potential of approximately +80 to +110 mV (depending on species), which sensitizes sensory hair cells. Other properties of this unique fluid remain elusive, owing to its minuscule volume in rodent cochlea. We therefore developed a technique to collect high-purity endolymph from mouse cochleae. Comprehensive proteomic analysis of sampled endolymph using liquid chromatography with mass spectrometry identified 301 proteins, dominated by molecules engaged in immunity and proteostasis. Approximately 30% of these proteins were undetectable in our perilymph. A combination of mass spectrometry and different approaches revealed that, compared to perilymph, endolymph was enriched with α2-macroglobulin, osteopontin, apolipoprotein D, apolipoprotein E, and apolipoprotein J/clusterin. In other cells or tissues, α2-macroglobulin, apolipoprotein E, and apolipoprotein J contribute to the clearance of degraded proteins from extracellular fluid. Altogether, with the proteins described here, endolymph may play a protective role in stabilizing cochlear homeostasis.

Abstract Circulating tumor DNA (ctDNA), which circulates in the blood after being shed from cancer cells in the body, has recently gained attention as an excellent tumor marker. The purpose of this study was to evaluate whether ct human papillomavirus (HPV) 16 DNA (ctHPV16DNA) levels were associated with quantitative PET parameters in patients with HPV-positive head and neck (HN) squamous cell carcinoma (SCC). Fifty patients with oropharyngeal SCC (OPSCC) and 5 with SCC of unknown primary (SCCUP) before treatment were included. They all underwent blood sampling to test ctHPV16DNA levels and FDG PET-CT examinations. Quantitative PET parameters included SUVmax, metabolic tumor volume (MTV), MTV of whole-body lesions (wbMTV), and 56 texture features. ctHPV16DNA levels were compared to texture features of primary tumors in OPSCC patients (Group A) or the largest primary or metastatic lymph node lesions in OPSCC and SCCUP patients (Group B) and to other PET parameters. Spearman rank correlation test and multiple regression analysis were used to confirm the associations between ctHPV16DNA levels and PET parameters. ctHPV16DNA levels moderately correlated with wbMTV, but not with SUVmax or MTV in Groups A and B. ctHPV16DNA levels exhibited a weak negative correlation with low gray-level zone emphasis in Groups A and B. Multiple regression analysis revealed that wbMTV and high gray-level zone emphasis were the significant factors for ctHPV16DNA levels in Group B. These results were not observed in Group A. This study demonstrated that ctHPV16DNA levels correlated with the whole-body tumor burden and tumor heterogeneity visualized on FDG PET-CT in patients with HPV-positive HNSCC.

Abstract Cholesteatoma, which potentially results from tympanic membrane retraction, is characterized by intractable local bone erosion and subsequent hearing loss and brain abscess formation. However, the pathophysiological mechanisms underlying bone destruction remain elusive. Here, we performed a single-cell RNA sequencing analysis on human cholesteatoma samples and identify a pathogenic fibroblast subset characterized by abundant expression of inhibin βA. We demonstrate that activin A, a homodimer of inhibin βA, promotes osteoclast differentiation. Furthermore, the deletion of inhibin βA /activin A in these fibroblasts results in decreased osteoclast differentiation in a murine model of cholesteatoma. Moreover, follistatin, an antagonist of activin A, reduces osteoclastogenesis and resultant bone erosion in cholesteatoma. Collectively, these findings indicate that unique activin A-producing fibroblasts present in human cholesteatoma tissues are accountable for bone destruction via the induction of local osteoclastogenesis, suggesting a potential therapeutic target.

PurposeDiagnosis of Menière’s disease (MD) relies on subjective factors and the patients diagnosed with MD may have heterogeneous pathophysiologies. This study aims to stratify MD patients using two objective data, nystagmus videos and contrast-enhanced magnetic resonance imaging (CE-MRI).MethodsThis is a retrospective cross-sectional study. According to the Japan Society for Equilibrium Research criteria (c-JSER), adults diagnosed with definite MD and who obtained videos recorded by portable nystagmus recorder immediately following vertigo attacks and underwent CE-MRI of the inner ear were included (ss = 91). Patients who obtained no nystagmus videos, who had undergone sac surgery, and those with long examination intervals were excluded (n = 40).ResultsThe gender of the subjects was 22 males and 29 females. The age range was 20–82 y, with a median of 54 y. Endolymphatic hydrops (EH) were observed on CE-MRI in 84% (43 patients). Thirty-one patients had unilateral EH. All of them demonstrated EH on the side of the presence of cochlear symptoms. The number of patients who had both nystagmus and EH was 38. Five patients only showed EH and 5 patients only exhibited nystagmus, while 3 patients did not have either. Of the 43 nystagmus records, 32 showed irritative nystagmus immediately after the vertigo episode. The direction of nystagmus later reversed in 44% of cases over 24 h.ConclusionPatients were stratified into subgroups based on the presence or absence of EH and nystagmus. The side with cochlear symptoms was consistent with EH. The c-JSER allows for the diagnosis of early-stage MD patients, and it can be used to treat early MD and preserve hearing; however, this approach may also include patients with different pathologies.

Summary: Interaction between the gut microbiome and host plays a key role in human health. Here, we perform a metagenome shotgun-sequencing-based analysis of Japanese participants to reveal associations between the gut microbiome, host genetics, and plasma metabolome. A genome-wide association study (GWAS) for microbial species (n = 524) identifies associations between the PDE1C gene locus and Bacteroides intestinalis and between TGIF2 and TGIF2-RAB5IF gene loci and Bacteroides acidifiaciens. In a microbial gene ortholog GWAS, agaE and agaS, which are related to the metabolism of carbohydrates forming the blood group A antigen, are associated with blood group A in a manner depending on the secretor status determined by the East Asian-specific FUT2 variant. A microbiome-metabolome association analysis (n = 261) identifies associations between bile acids and microbial features such as bile acid metabolism gene orthologs including bai and 7β-hydroxysteroid dehydrogenase. Our publicly available data will be a useful resource for understanding gut microbiome-host interactions in an underrepresented population.

Background: Chronic rhinosinusitis is classified into eosinophilic chronic rhinosinusitis (ECRS) and non-eosinophilic chronic rhinosinusitis (NECRS). ECRS is a refractory allergic disease involving a variety of immune and epithelial cells. S100A8 is a damage-associated molecular pattern that is closely related to allergic inflammation. However, the pathological implications of S100A8 in ECRS have not been clarified. Methods: We evaluated the role of S100A8 in the pathogenesis of ECRS. Gene expression profiles of nasal polyps obtained from patients with ECRS or NECRS were evaluated using RNA sequencing. Results: S100A8 was identified as a significantly upregulated gene in nasal polyps associated with ECRS. Immunohistochemistry consistently revealed intense S100A8 staining in nasal polyps from patients with ECRS. Human nasal epithelial cells expressed the receptor for advanced glycation end products and Toll-like receptor 4. Recombinant S100A8 protein induced interleukin-1β secretion in human nasal epithelial cells. Conclusions: Our data demonstrate that S100A8 results in production of interleukin-1β in the nasal epithelium, which may be involved in the pathogenesis of ECRS.

Tomofuji et al. perform non-targeted metabolomics on serum samples from people with type 2 diabetes with or without complications. The authors identify metabolites associated with retinal and renal complications.

Abstract Ménière’s disease (MD) is an inner ear disorder in which the main pathological feature is endolymphatic hydrops (EH). Positive pressure therapy (PPT) using a portable device is now a second-line therapy for intractable MD when initial medical treatment fails. However, it remains unknown whether PPT causes the morphological and functional changes of inner ear in patients with active MD in accordance with reduction of vertigo attacks. In this nonrandomized controlled trial of 52 patients with MD, the volume of EH significantly decreased with reduction of vertigo attacks during 8 months of PPT combined with medications while the volume of that significantly increased with medications alone. There was no difference between Control group (n = 26) and PPT group (n = 26) regarding the vertigo control, however, PPT group achieved a significant functional improvement of vertical semicircular canals. The effect of volume reduction by PPT has been firstly demonstrated and the functional changes of all semicircular canals during PPT have been firstly examined. Morphological and functional changes in the inner ear by administrating local positive pressure are quite different from those caused by medications alone. Clinical trial registration: UMIN-CTR UMIN000041164 (registered on July 20, 2020).

Abstract RNA is spliced concomitantly with transcription and the process is organized by RNA splicing factors, transcriptional regulators, and chromatin regulators. RNA is spliced in close proximity to transcription machinery. Hence, some RNA splicing factors may play a role in transcription. Here, we show that the splicing factor SF3B2 binds to gene regulatory elements and mRNA to modulate transcription and RNA stability in head and neck squamous cell carcinoma cells. High SF3B2 expression leads to poor prognosis in patients with head and neck squamous cell carcinoma and to progression of tumor growth in mice. SF3B2 promotes tumor growth, owing to its involvement in activation of gene expression associated with mitochondrial electron transport and transcription regulatory region DNA binding. SF3B2 is enriched around the promoter element on chromatin and the transcription termination site on RNA. SF3B2 is involved in the regulation of RNA stability. According to the SF3B2-binding profile, SF3B2 regulates RNA polymerase II activity, in addition to regulating RNA splicing. Mechanistically, SF3B2 promotes the binding of structural maintenance of chromosomes 1A and CCCTC-binding factor (CTCF) to the SF3B2-binding genomic regions. SF3B2 also modulates CTCF transcriptional activity. Our findings indicate that SF3B2 has a dual function in both transcription and RNA stability, leading to head and neck squamous cell carcinoma progression.

We previously reported that L-glutamine reduces the severity of mucositis caused by chemoradiotherapy in patients with head and neck cancer. However, the impact of glutamine on the anti-tumor effect of chemoradiotherapy remains controversial. This study, which included 40 patients, investigated whether L-glutamine influences survival. Radiation therapy (total: 66 or 70 Gy), cisplatin, and docetaxel were co-administered for a period of 6 weeks. Patients were randomly assigned to receive either glutamine (glutamine group, n = 20) or placebo (placebo group, n = 20) during the entire course of chemoradiotherapy. We compared the overall survival and progression-free survival rates between the two groups. At 5-year follow-up, 16 (80%) and 13 (72%) patients in the glutamine and placebo groups, respectively, survived (with no significant difference in overall survival [glutamine group: 55.2 ± 12.7 months vs. placebo group: 48.3 ± 21.3 months]). A total of 14 (70%) and 12 (67%) patients in the glutamine and placebo groups, respectively, did not experience disease progression (with no significant difference in progression-free survival [glutamine group: 46.7 ± 19.5 months vs. placebo group: 43.6 ± 25.2 months]). These findings indicate that L-glutamine does not influence the survival of patients with locally advanced head and neck cancer receiving chemoradiotherapy.

Background and objectivesPatients with benign paroxysmal positional vertigo of the posterior canal (pc-BPPV) exhibit BPPV fatigue, where the positional nystagmus diminishes with the repeated performance of the Dix–Hallpike test (DHt). BPPV fatigue is thought to be caused by the disintegration of lumps of otoconial debris into smaller parts and can eliminate positional nystagmus within a few minutes [similar to the immediate effect of the Epley maneuver (EM)]. In this study, we aimed to show the non-inferiority of the repeated DHt to the EM for eliminating positional nystagmus after 1 week.MethodsThis multicenter, randomized controlled clinical trial was designed based on the CONSORT 2010 guidelines. Patients who had pc-BPPV were recruited and randomly allocated to Group A or Group B. Patients in Group A were treated using the EM, and patients in Group B were treated using repeated DHt. For both groups, head movements were repeated until the positional nystagmus had been eliminated (a maximum of three repetitions). After 1 week, the patients were examined to determine whether the positional nystagmus was still present. The groups were compared in terms of the percentage of patients whose positional nystagmus had been eliminated, with the non-inferiority margin set at 15%.ResultsData for a total of 180 patients were analyzed (90 patients per group). Positional nystagmus had been eliminated in 50.0% of the patients in Group A compared with 47.8% in Group B. The upper limit of the 95% confidence interval for the difference was 14.5%, which was lower than the non-inferiority margin.DiscussionThis study showed the non-inferiority of repeated DHt to the EM for eliminating positional nystagmus after 1 week in patients with pc-BPPV and that even the disintegration of otoconial debris alone has a therapeutic effect for pc-BPPV. Disintegrated otoconial debris disappears from the posterior canal because it can be dissolved in the endolymph or returned to the vestibule via activities of daily living.Classification of evidenceThis study provides Class II evidence of the non-inferiority of repeated DHt to the EM for eliminating positional nystagmus after 1 week.Registration numberUMIN000016421.

Facial paralysis results in the decline in the generation of facial expressions and is attributed to several causes. Intractable facial paralysis has a poor prognosis, and new treatments are required. Facial paralysis results in the decline in the generation of facial expressions and is attributed to several causes. Reactive oxygen species can inhibit peripheral nerve regeneration after injury. Therefore, the administration of an appropriate antioxidant can promote nerve regeneration. Silicon (Si)-based agents can react with water to generate antioxidant hydrogen. Oral administration of Si-based agents can effectively alleviate symptoms of disease models associated with oxidative stress. Thus, we orally administered a Si-based agent to a facial paralysis model mice to investigate whether promotion of nerve regeneration occurred. The combined administration of methylcobalamin (MeCbl) with the Si-based agent was also investigated. The Si-based agent improved the clinical score evaluation of facial paralysis. Electroneuronography and immunostaining showed that the Si-based agent promoted myelination and recovery of facial nerve function. Furthermore, in the drug-administered group, oxidative stress associated with facial nerve injury was reduced more than that in the non-administered group. The clinical score evaluation, neuroregeneration effect, and reduction of oxidative stress were improved in the combination group compared to the single administration group. The Si-based agent could rapidly improve the disappearance of facial expressions by promoting myelin sheath formation and alleviating oxidative stress. Combination therapy with a Si-based agent and MeCbl should improve the prognosis and treatment of intractable facial paralysis.

Abstract Sarcopenia, which is characterized by a decrease in muscle quantity or quality, is commonly observed in patients with cancer. Recent research has reported contradictory results on the association between sarcopenia and the efficacy of immune checkpoint inhibitors (ICIs). We conducted a systematic review and meta‐analysis to investigate this discrepancy. We systematically searched three electronic databases to identify articles reporting on the association between sarcopenia and treatment outcomes in patients with solid cancers who received ICIs. The outcomes assessed were hazard ratios (HRs) for overall survival (OS) and progression‐free survival (PFS), and odds ratios (ORs) for objective response rate (ORR), disease control rate (DCR), and toxicity. Pooled estimates and their 95% confidence intervals (CIs) were calculated. A total of 2501 patients from 26 studies were analysed. Sarcopenia was observed in 44.7% (95% CI: 38.2–51.3) of the patients and was significantly associated with poor survival (HR = 1.55, 95% CI = 1.32–1.82 for OS and HR = 1.61, 95% CI = 1.35 to 1.93 for PFS). The HRs (95% CIs) for OS according to the diagnostic measures used were 1.97 (0.88–4.41) for psoas muscle index (PMI), 1.41 (0.87–2.28) for skeletal muscle density (SMD), and 1.43 (1.23–1.67) for skeletal mass index (SMI). The HRs (95% CIs) for PFS were 1.86 (1.08–3.21) for PMI, 1.27 (0.94–1.71) for SMD, and 1.38 (1.11–1.71) for SMI. Poor radiological response to ICI therapy was observed in patients with sarcopenia (OR = 0.52, 95% CI = 0.34–0.80 for ORR and OR = 0.45, 95% CI = 0.30–0.67 for DCR). The ORs for ORR (95% CIs) were 0.56 (0.15–2.05) for PMI and 0.78 (0.56–1.09) for SMI. The oncologic outcomes associated with melanoma and non‐small cell lung cancer (NSCLC) were comparable with those observed overall (HR for OS = 2.02, 95% CI = 1.26–3.24 for melanoma and HR for OS = 1.61, 95% CI = 1.19–2.18 for NSCLC). In contrast, the occurrence of severe toxicity was not associated with sarcopenia (OR = 1.13, 95% CI = 0.51–2.52). Poor survival and poor response in patients with sarcopenia indicate a negative association between sarcopenia and efficacy of ICIs. Sarcopenia's predictive ability is consistent across various tumour types. For the selection of patients who may respond to ICIs pre‐therapeutically, the presence of sarcopenia should be assessed in clinical practice.

Abstract The V-shaped arrangement of hair bundles on cochlear hair cells is critical for auditory sensing. However, regulation of hair bundle arrangements has not been fully understood. Recently, defects in hair bundle arrangement were reported in postnatal Dishevelled-associating protein (ccdc88c, alias Daple)-deficient mice. In the present study, we found that adult Daple −/− mice exhibited hearing disturbances over a broad frequency range through auditory brainstem response testing. Consistently, distorted patterns of hair bundles were detected in almost all regions, more typically in the basal region of the cochlear duct. In adult Daple −/− mice, apical microtubules were irregularly aggregated, and the number of microtubules attached to plasma membranes was decreased. Similar phenotypes were manifested upon nocodazole treatment in a wild type cochlea culture without affecting the microtubule structure of the kinocilium. These results indicate critical role of Daple in hair bundle arrangement through the orchestration of apical microtubule distribution, and thereby in hearing, especially at high frequencies.

ObjectiveBasal information of head and neck small-cell carcinoma (HNSmCC) including epidemiology, primary site, treatment, and prognosis remains sparse due to its rarity. We report here a multicenter retrospective study on the diagnosis, treatment, and outcomes of patients with HNSmCC.Materials and methodsThis study involved 47 patients with HNSmCC from 10 participating institutions. Eight patients were excluded for whom no pathological specimens were available (n = 2) and for discrepant central pathological judgements (n = 6). The remaining 39 patients were processed for data analysis.ResultsAs pretreatment examinations, computed tomography (CT) was performed for the brain (n = 8), neck (n = 39), and chest (n = 32), magnetic resonance imaging (MRI) for the brain (n = 4) and neck (n = 23), positron emission tomography-CT (PET-CT) in 23 patients, bone scintigraphy in 4, neck ultrasonography in 9, and tumor markers in 25. Primary sites were oral cavity (n = 1), nasal cavity/paranasal sinuses (n = 16), nasopharynx (n = 2), oropharynx (n = 4), hypopharynx (n = 2), larynx (n = 6), salivary gland (n = 3), thyroid (n = 2), and others (n = 3). Stages were II/III/IV-A/IV-B/IV-C/Not determined = 3/5/16/6/5/4; stage IV comprised 69%. No patient had brain metastases. First-line treatments were divided into 3 groups: the chemoradiotherapy (CRT) group (n = 27), non-CRT group (n = 8), and best supportive care group (n = 4). The CRT group included concurrent CRT (CCRT) (n = 17), chemotherapy (Chemo) followed by radiotherapy (RT) (n = 5), and surgery (Surg) followed by CCRT (n = 5). The non-CRT group included Surg followed by RT (n = 2), Surg followed by Chemo (n = 1), RT alone (n = 2), and Chemo alone (n = 3). The 1-year/2-year overall survival (OS) of all 39 patients was 65.3/53.3%. The 1-year OS of the CRT group (77.6%) was significantly better compared with the non-CRT group (31.3%). There were no significant differences in adverse events between the CCRT group (n = 22) and the Chemo without concurrent RT group (n = 9).ConclusionNeck and chest CT, neck MRI, and PET-CT would be necessary and sufficient examinations in the diagnostic set up for HNSmCC. CCRT may be recommended as the first-line treatment. The 1-year/2-year OS was 65.3%/53.3%. This study would provide basal data for a proposing the diagnostic and treatment algorithms for HNSmCC.

Background: We examined the severity of epistaxis in patients with hereditary haemorrhagic telangiectasia (HHT) and its relationship with the performed activities of daily living. Methodology: This cross-sectional survey included 36 patients with HHT in Japan. An anonymous questionnaire survey was conducted regarding the severity of epistaxis, the measures adopted to prevent epistaxis, and the limitations in the performed activities of daily living. The latter was assessed using a visual analogue scale (VAS). The correlation between epistaxis severity and the VAS score was analysed using Spearman’s rank correlation coefficient. Results: Of the 36 participants surveyed, 94.4% had >1 episode of epistaxis/week. The mean epistaxis severity score (ESS) was 4.3 (range, 0.9–8.4). Limitations in daily life, going out (within a day), meeting with others, eating with others, and going on overnight trips were positively correlated with the ESS. To prevent nosebleeds, 44.4% and 41.7% of the participants used medications and avoided drying their nasal cavities, respectively. Conclusions: Epistaxis impacted the daily life of patients with HHT in proportion to its severity. Nonetheless, less than half of the patients used medications or took precautions. Hence, further educational activities should be considered for medical professionals and patients.

Abstract In the interaural direction, translational linear acceleration is loaded during lateral translational movement and gravitational acceleration is loaded during lateral tilting movement. These two types of acceleration induce eye movements via two kinds of otolith-ocular reflexes to compensate for movement and maintain clear vision: horizontal eye movement during translational movement, and torsional eye movement (torsion) during tilting movement. Although the two types of acceleration cannot be discriminated, the two otolith-ocular reflexes can distinguish them effectively. In the current study, we tested whether lateral-eyed mice exhibit both of these otolith-ocular reflexes. In addition, we propose a new index for assessing the otolith-ocular reflex in mice. During lateral translational movement, mice did not show appropriate horizontal eye movement, but exhibited unnecessary vertical torsion-like eye movement that compensated for the angle between the body axis and gravito-inertial acceleration (GIA; i.e., the sum of gravity and inertial force due to movement) by interpreting GIA as gravity. Using the new index (amplitude of vertical component of eye movement)/(angle between body axis and GIA), the mouse otolith-ocular reflex can be assessed without determining whether the otolith-ocular reflex is induced during translational movement or during tilting movement.

Abstract Isolated sphenoid sinus opacifications (ISSOs) are clinically important because they can lead to serious complications. However, some patients with ISSOs are asymptomatic, and not all patients are properly referred to the otolaryngology department. Because past studies of ISSOs focused only on patients who received treatment, in this study we selected ISSO cases based on radiology reports, then determined whether these patients had symptoms and were appropriately referred for specialty care. We conducted a retrospective analysis of data collected from patients who underwent computed tomography or magnetic resonance imaging from January 2007 to March 2017 at Osaka General Medical Center. We searched for the terms “sphenoid” or “sphenoidal” using F-REPORT to identify patients who had a sphenoid disease. We checked all selected images and diagnosed ISSOs. Examination of 1115 cases revealed 223 cases of ISSOs, of whom 167 (74.9%) were asymptomatic. We categorized patients with ISSOs into four groups: inflammation, mucocele, fungal diseases, and unclassifiable; the final category was used when edges were irregular or complete opacity was encountered. In the unclassifiable group, the majority of cases required otolaryngology consultation, but 37 of 47 unclassifiable patients did not have an otolaryngology visit. ISSOs are often identified by chance on imaging tests performed by non-otolaryngologists. However, our study revealed that many patients with ISSOs who should be treated by otolaryngologists were not referred to the otolaryngology department. Accordingly, it is important to promote awareness of the disease among other types of clinicians.

Abstract Human papillomavirus (HPV) 16 infection in the oropharynx is one of the major risk factors for oropharyngeal carcinoma. Although the HPV E6 and E7 proteins are known to have a role in head and neck carcinogenesis, whether their expression is maintained once the tumour has developed still remains unclear. We evaluated the expression of these proteins in HPV16-positive cancer cell lines and clinical oropharyngeal specimens. Two out of the four commercially available antibodies directed against the E7 protein could detect the E7 protein overexpressed in the 293FT cells, human embryonic kidney cells, although none of the four commercially available anti-E6 antibodies could detect the overexpressed E6 protein. Whereas HPV16-positive head and neck or cervical carcinoma cell lines expressed the E7 mRNA, the antibodies with an ability to detect the E7 protein could not detect it in western blotting in these HPV16-positive cell lines. In clinical specimens, E7 protein was partially detected in p16-positive area in p16-positive and HPV16 DNA-positive samples, but not in p16-negative and HPV DNA-negative or p16-positive and HPV DNA-negative samples. Consistent with these findings, the E7 protein was poorly translated from the endogenous structure of the E7 mRNA, although significant E7 mRNA expression was detected in these samples. Our findings indicate that E7 protein is partially expressed in p16-positive area in p16-positive and HPV16 DNA-positive clinical specimens.

Infection with high-risk human papillomavirus (HPV) types is linked to the onset of several cancers. The mechanism of HPV infection, however, has not yet been fully elucidated. Here, using the newly developed HPV infectious pseudovirion (HPV PsV) and a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening system, we established an experimental system and searched for genes involved in HPV infection. The HPV PsV has the truncated herpes simplex virus thymidine kinase (dTK) to kill PsV-infected cells when combined with ganciclovir. The five rounds of selection of 293FT cells by infection with HPV PsVs identified two candidate genes involved in the HPV infection pathway. The validation experiments showed that SLC25A17, which encodes the peroxisomal membrane protein PMP34, was involved in the HPV infection pathway. The gRNAs against SLC25A17 attenuated the efficiency of HPV PsV infection in 293FT and HeLa cells. Although further experiments are required to determine whether PMP34 acts as the HPV infection pathway, these results indicate that our screening system is useful for identification of the genes involved in the HPV infection pathway.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background: Transnasal endoscopic surgery (TES) is an established procedure for the treatment of chronic rhinosinusitis, septal deviation, and paranasal benign tumours. Postoperative haemorrhage (POH) is a common complication following TES. Various studies have addressed the risk factors for intraoperative bleeding and the methods to improve the operative field by reducing bleeding. However, the factors affecting POH following TES have not been fully elucidated. Therefore, we aimed investigate the risk factors for POH following TES. Methodology: We studied 807 patients who underwent TES. The variables between cases with and without POH were compared using univariate and multivariate models. We also examined the site and timing of bleeding. Results: POH was observed in 21 cases (2.6%). Seven cases required treatment in the operation room (0.9%). The most common sites of bleeding were around sphenopalatine foramen, and the median time to POH was the 7th day after surgery. Multivariate analysis showed that anti-thrombotic medication, low platelet count, and surgical indication for sinonasal tumour were independent risk factors for POH. Conclusions: POH after TES is a relatively common complication. Careful intraoperative haemostatic procedures may be required in these cases.

Background Verrucous carcinoma (VC) is a rare variant of squamous cell carcinoma. Although VC is considered radioresistant, concrete evidence for this is absent. Methods We obtained data on VC treated with surgery or radiation from the Surveillance, Epidemiology, and End Results database. Treatment selection bias was reduced by propensity score matching. Overall survival (OS) and disease-specific survival (DSS) rates were estimated using the Kaplan-Meier method. Hazard ratios (HRs) were estimated using Cox proportional hazards models. Results Five-year OS rates in the radiation and surgery groups were 72.7% and 72.0%, respectively (P = 0.111); five-year DSS rates in the same were 86.7% and 88.4%, respectively (P = 0.234). HRs of radiation compared with surgery were 1.68 (95% confidence interval (CI), 0.96–2.95) for OS and 1.95 (95% CI, 0.69–5.53) for DSS. Conclusions Similar prognoses were observed in patients with VC treated with radiation and surgery. VC can be treated using radiation.

Objective: In benign paroxysmal positional vertigo (BPPV), positional nystagmus becomes generally weaker when the Dix–Hallpike test is repeated. This phenomenon is termed BPPV fatigue. We previously reported that the effect of BPPV fatigue deteriorates over time (i.e., the positional nystagmus is observed again after maintaining a sitting head position). The aim of this study was to investigate whether the effect of BPPV fatigue attenuates after maintaining a supine position with the head turned to the affected side.Methods: Twenty patients with posterior-canal-type BPPV were assigned to two groups. Group A received Dix–Hallpike test, were returned to the sitting position (reverse Dix–Hallpike test) with a sitting head position for 10 min, and then received a second Dix–Hallpike test. Group B received Dix–Hallpike test, were kept in the supine position with the head turned to the affected side for 10 min, and then received reverse Dix–Hallpike test followed by the second Dix–Hallpike test. The maximum slow phase eye velocity (MSPEV) of positional nystagmus induced by the first, reverse, and second Dix–Hallpike test were analyzed.Results: The ratio of MSPEV of the positional nystagmus induced by the second Dix–Hallpike test relative to the first Dix–Hallpike test was significantly smaller in group B than that in group A. There was no difference in the MSPEV of the positional nystagmus induced by the reverse Dix–Hallpike test between group A and B.Conclusions: The effect of BPPV fatigue is continued by maintaining a supine position with the head turned to the affected side, while the effect is weakened by maintaining a sitting head position. On the basis of the most widely accepted theory of the pathophysiology of BPPV fatigue, in which the particles become dispersed along the canal during head movement in the Dix–Hallpike test, we found an inconsistency whereby the dispersed otoconial debris return to a mass during the sitting position but do not return to a mass in the supine position with the head turned to the affected side. Future studies are required to determine the exact pathophysiology of BPPV fatigue.Classification of Evidence: 2b.

Boron neutron capture therapy (BNCT) is a radiotherapy utilizing the neutron capture and nuclear fission reaction of 10B taken up into tumor cells. The most commonly used boron agent in BNCT, p-borono-l-phenylalanine (BPA), is accumulated in tumors by amino acid transporters upregulated in tumor cells. Here, by using dipeptides of BPA and tyrosine (BPA-Tyr and Tyr-BPA), we propose a novel strategy of selective boron delivery into tumor cells via oligopeptide transporter PEPT1 upregulated in various cancers. Kinetic analyses indicated that BPA-Tyr and Tyr-BPA are transported by oligopeptide transporters, PEPT1 and PEPT2. The intrinsic oligopeptide transport activity in tumor cells clearly correlated with PEPT1 protein expression level but not with PEPT2, suggesting that PEPT1 is the predominant oligopeptide transporter at least in tumor cell lines. Furthermore, using BPA-Tyr and Tyr-BPA, boron was successfully delivered into PEPT1-expressing pancreatic cancer AsPC-1 cells via a PEPT1-mediated mechanism. Intravenous administration of BPA-Tyr into the mice bearing AsPC-1 xenograft tumors resulted in significant boron accumulation in the tumors. It is proposed that the oligopeptide transporters, especially PEPT1, are promising candidates for molecular targets of boron delivery in BNCT. The BPA-containing dipeptides would have a potential for the development of novel boron carriers targeting PEPT1. Keywords: Boron delivery, Boron neutron capture therapy, p-Borono-l-phenylalanine-containing dipeptide, Oligopeptide transporter, PEPT1

Positive pressure therapy (PPT) is applied for medically-intractable vertigo in Ménière's disease (MD); however, it remains unknown whether PPT affects in vivo endolymphatic hydrops (EH). In this case report, we describe a 5-year course of MD in a patient in which EH was repeatedly observed. As the patient experienced recurrent vertigo attacks after endolymphatic sac surgery, he began to use the PPT device additionally and vertiginous episodes decreased in accordance with a decrease in the EH volume. The mechanism of PPT is suggested that the pressure increase in the middle ear inhibits EH development. PPT, if added after surgery, might be more effective to reduce EH volume compared with surgery alone. A larger study group size is required to test these preliminary data concerning EH changes.

Background The objective of this study was to compare the prognostic impact of sarcopenia in patients with head and neck cancer (HNC) treated with surgery or radiation. Methods We systematically searched electronic databases to identify articles reporting the impact of sarcopenia on the prognosis of patients with HNC. Hazard ratios (HRs) and 95% confidence intervals (CIs) for overall survival (OS), disease-free survival (DFS), and disease-specific survival (DSS) were extracted and pooled. HR according to treatment modality were estimated using random-effects models. Statistical analyses were carried out using the Comprehensive Meta-Analysis software. Results In total, 18 studies enrolling 3,233 patients were included. Sarcopenia was associated with poor OS in both surgery and radiotherapy groups (hazard ratio [HR] 2.50, 95% confidence interval [CI] 1.95–3.21; HR 1.63, 95% CI 1.40–1.90, respectively). The HR was significantly higher in the surgery group than in the radiotherapy group (p = 0.004), with similar results obtained for DFS (HR 2.59, 95% CI 1.56–4.31; HR 1.56, 95% CI 1.24–1.97 for the surgery and radiotherapy groups, respectively) and DSS (HR 2.96, 95% CI 0.73–11.95; HR 2.67, 95% CI 1.51–4.73 for the surgery and RT groups, respectively). Conclusions Sarcopenia was a poor prognostic factor for HNC, regardless of the treatment modality. However, the adverse effects of sarcopenia on survival were more prominent in the surgery group than in the radiotherapy group. Sarcopenia assessment is required for appropriate treatment decision-making.

While microbiome plays key roles in the etiology of multiple sclerosis (MS), its mechanism remains elusive. Here, we conducted a comprehensive metagenome-wide association study (MWAS) of the relapsing-remitting MS gut microbiome (ncase = 26, ncontrol = 77) in the Japanese population, by using whole-genome shotgun sequencing. Our MWAS consisted of three major bioinformatic analytic pipelines (phylogenetic analysis, functional gene analysis, and pathway analysis). Phylogenetic case-control association tests showed discrepancies of eight clades, most of which were related to the immune system (false discovery rate [FDR] < 0.10; e.g., Erysipelatoclostridium_sp. and Gemella morbillorum). Gene association tests found an increased abundance of one putative dehydrogenase gene (Clo1100_2356) and one ABC transporter related gene (Mahau_1952) in the MS metagenome compared with controls (FDR < 0.1). Molecular pathway analysis of the microbiome gene case-control comparisons identified enrichment of multiple Gene Ontology terms, with the most significant enrichment on cell outer membrane (P = 1.5 × 10−7). Interaction between the metagenome and host genome was identified by comparing biological pathway enrichment between the MS MWAS and the MS genome-wide association study (GWAS) results (i.e., MWAS-GWAS interaction). No apparent discrepancies in alpha or beta diversities of metagenome were found between MS cases and controls. Our shotgun sequencing-based MWAS highlights novel characteristics of the MS gut microbiome and its interaction with host genome, which contributes to our understanding of the microbiome’s role in MS pathophysiology.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that shows progressive muscle weakness. A few treatments exist including symptomatic therapies, which can prolong survival or reduce a symptom; however, no fundamental therapies have been found. As a therapeutic strategy, enhancing muscle force is important for patients’ quality of life. In this study, we focused on skeletal muscle-specific myosin regulatory light chain kinase (skMLCK), which potentially enhances muscle contraction, as overexpression of skMLCK was thought to improve muscle function. The adeno-associated virus serotype 6 encoding skMLCK (AAV6/skMLCK) and eGFP (control) was produced and injected intramuscularly into the lower limbs of SOD1G37R mice, which are a familial ALS model. AAV6/skMLCK showed the successful expression of skMLCK in the muscle tissues. Although the control did not affect the muscle force in both of the WT and SOD1G37R mice, AAV6/skMLCK enhanced the twitch force of SOD1G37R mice and the tetanic force of WT and SOD1G37R mice. These results indicate that overexpression of skMLCK can enhance the tetanic force of healthy muscle as well as rescue weakened muscle function. In conclusion, the gene transfer of skMLCK has the potential to be a new therapy for ALS as well as for other neuromuscular diseases.

Reconstruction of complex three-dimensional defects or defects in separate locations after ablative head and neck surgery can be achieved with two simultaneous free flaps, a combination of a free flap and a pedicle flap, or a single multi-island free flap. The rectus abdominis flap is often used for reconstructions with a single multi-island free flap, but the flap is excessively bulky in some cases, leading to the unnecessary sacrifice of muscle. To overcome this issue and to preserve the muscle, we used a single muscle-sparing vertical rectus abdominis musculocutaneous flap with two completely separated skin islands based on two different skin perforators to simultaneously reconstruct separate locations (the orbit and parotid region) after orbital exenteration and total parotidectomy. Our findings highlight the feasibility of simultaneously reconstructing orbit and parotid contour deformities with a single double-island flap.

Abstract Due to habitual drinking and smoking and advanced age at diagnosis, patients with head and neck squamous cell carcinoma (HNSCC) frequently present with comorbidities. Several comorbidity indices have been developed and validated for HNSCC. However, none have become the standard method. In this study, we developed a new comorbidity index for Japanese patients with HNSCC, which was validated against an independent data set. A Cox proportional hazards analysis of 698 patients identified dementia, connective tissue diseases, and second primary malignancies in the oesophagus, head and neck, lungs, and stomach as prognostic comorbidities for overall survival. The Osaka head and neck comorbidity index (OHNCI) was generated from the weighted points of these comorbidities. In the independent data set, the 5-year overall survival rates for the low, moderate, and high scoring OHNCI groups were 62.1%, 64.3%, and 37.7%, respectively. In the multivariate analysis, the high scoring OHNCI group was an independent prognostic factor for overall survival (hazard ratio: 1.81, 95% confidence interval: 1.05–3.13; P = 0.031). The model including the OHNCI exhibited a higher prognostic capability compared to those including other commonly used comorbidity indices. The OHNCI could become the primary choice for comorbidity assessment in patients with HNSCC in Japan.

Endoscopic medial maxillectomy (EMM) was originally developed for surgery of maxillary sinus disease. This surgery was recently modified to preserve the inferior turbinate (IT) and the nasolacrimal duct (ND) and is commonly referred to endoscopic modified medial maxillectomy (EMMM). Here we present a case of endoscopic repair for a blowout fracture of the inferior orbital wall via access through the medial wall of the maxillary sinus, thereby preserving IT and ND, similar to EMMM. Two months postoperatively, a diplopia field test, computed tomography, and endoscopic observation were performed. Good recovery of diplopia was obtained, and empty nose syndrome, epiphora, and cheek numbness were not observed. Those complications such as empty nose syndrome, epiphora and cheek numbness can be avoided by the approach presented in this report; therefore, the maxillary medial wall approach, like EMMM, could become a preferred surgical method for blowout fractures of the inferior orbital wall.

Objective: The Epley maneuver (EM) has an immediate effect: rapid reduction of positional nystagmus. Benign paroxysmal positional vertigo (BPPV) causes BPPV fatigue, which constitutes fatigability of positional nystagmus and vertigo with repeated performance of the Dix-Hallpike test; notably, BPPV fatigability becomes ineffective over time. We hypothesized that the immediate effect of the EM is caused by BPPV fatigue. Therefore, we suspected that performance of the EM with intervals between head positions would worsen the immediate reduction of positional nystagmus in patients with BPPV, because BPPV fatigability would become ineffective during performance of this therapy.Methods: Forty patients with newly diagnosed BPPV were randomly assigned to the following two groups; one group performed the EM without intervals between positions (group A), and the other group performed the EM with 3 min intervals between positions (group B). The primary outcome measure was the ratio of maximum slow-phase eye velocity (MSPEV) of positional nystagmus soon after the EM, compared with that measured before the EM. Secondary outcome included whether a 30 min interval after the EM enabled recovery of MSPEV of positional nystagmus to the original value. This study followed the CONSORT 2010 reporting standards.Results: In both groups A and B, the immediate effect of the EM could be observed, because MSPEV during the second Dix-Hallpike test was significantly smaller than MSPEV during the first Dix-Hallpike test (p < 0.0001 in group A, p < 0.0001 in group B). The primary outcome measure was larger in group B than in group A (p = 0.0029). The immediate effect faded 30 min later (secondary outcome).Conclusions: This study showed that the EM had an immediate effect both with and without interval time in each head position of the EM. Because setting interval time in each head position of the EM reduced the immediate effect of the EM, interval time during the EM adds less benefit. This finding can reduce the effort exerted by doctors, as well as the discomfort experienced by patients with pc-BPPV, during EM. However, this immediate effect may be caused by BPPV fatigue, and may fade rapidly.Classification of Evidence: 1b

The aim of this study was to propose a new pathophysiological hypothesis for involuntary eye oscillation in infantile nystagmus (IN): patients with IN exhibit impaired gaze fixation, horizontal smooth pursuit and optokinetic nystagmus (OKN) and use saccadic eye movements for these underlying impairments. In order to induce saccades, they make enough angle between gaze and target by precedent exponential slow eye movements. IN consists of the alternate appearance of the saccade and the slow eye movements. Unlike most previous theories, IN is therefore considered a necessary strategy allowing for better vision and not an obstacle to clear vision. In five patients with IN, eye movements were analyzed during the smooth pursuit test, saccadic eye movement test, OKN test and vestibulo-ocular reflex (VOR) test. Their gaze fixation, horizontal smooth pursuit, OKN and the last half of the slow phase of VOR were impaired. The lines obtained by connection of the end eye positions of fast phase of nystagmus coincided with the trajectories of targets. The findings indicate that patients followed the target by the fast but not the slow phase of nystagmus, which supports our hypothesis. By setting the direction of slow phase of nystagmus opposite to the direction of the OKN stimulation, enough angle can be effectively made between the gaze and target for the induction of saccade. This is the mechanism of reversed OKN response. In darkness and when eyes are closed, IN weakens because there is no visual target and neither the saccade for catching up the target or slow phase for induction of the saccade is needed.

Light-gated ion channels and transporters have been applied to a broad array of excitable cells including neurons, cardiac myocytes, skeletal muscle cells and pancreatic β-cells in an organism to clarify their physiological and pathological roles. Nonetheless, among nonexcitable cells, only glial cells have been studied in vivo by this approach. Here, by optogenetic stimulation of a different nonexcitable cell type in the cochlea of the inner ear, we induce and control hearing loss. To our knowledge, deafness animal models using optogenetics have not yet been established. Analysis of transgenic mice expressing channelrhodopsin-2 (ChR2) induced by an oligodendrocyte-specific promoter identified this channel in nonglial cells—melanocytes—of an epithelial-like tissue in the cochlea. The membrane potential of these cells underlies a highly positive potential in a K+-rich extracellular solution, endolymph; this electrical property is essential for hearing. Illumination of the cochlea to activate ChR2 and depolarize the melanocytes significantly impaired hearing within a few minutes, accompanied by a reduction in the endolymphatic potential. After cessation of the illumination, the hearing thresholds and potential returned to baseline during several minutes. These responses were replicable multiple times. ChR2 was also expressed in cochlear glial cells surrounding the neuronal components, but slight neural activation caused by the optical stimulation was unlikely to be involved in the hearing impairment. The acute-onset, reversible and repeatable phenotype, which is inaccessible to conventional gene-targeting and pharmacological approaches, seems to at least partially resemble the symptom in a population of patients with sensorineural hearing loss. Taken together, this mouse line may not only broaden applications of optogenetics but also contribute to the progress of translational research on deafness.

Nicotinamide adenine dinucleotide (NAD) is an essential co-enzyme mediating various enzymatic reactions. Mitochondrial NAD particularly occupies a considerable amount of total NAD in cells, and serves as a co-enzyme in tricarboxylic acid cycle (TCA cycle), β-oxidation, and oxidative phosphorylation. Despite the importance of mitochondrial NAD, its synthesis pathway remains unknown. It has been proposed that NAD synthesis enzyme, Nmnat3, was localized in mitochondria, but its physiological relevance to the metabolism in mitochondria was not fully elucidated. Previously, we have reported that murine Nmnat3 protein was strongly expressed in the cytoplasm of mature erythrocytes, in which mitochondria were absent, and Nmnat3-deficient mice (Nmnat3-KO mice) exhibited splenomegaly and hemolytic anemia due to reduced NAD levels in mature erythrocytes. These results challenged the role of Nmnat3 in mitochondrial NAD synthesis. In this study, we demonstrated that mitochondrial NAD levels in various tissues, except for red blood cells, were unchanged in Nmnat3-KO mice. We also analyzed the metabolites in glycolysis and TCA cycle and found that there were no differences between Nmnat3-KO and WT mice. In addition, the aged Nmnat3-KO mice had comparable NAD levels to that observed in WT mice. Our results indicated that Nmnat3 is dispensable in the maintenance of mitochondrial NAD levels, and that other NAD regulatory pathways may exist in mitochondria.

Various serum biomarkers have been developed for predicting head and neck squamous cell carcinoma (HNSCC) prognosis. However, none of them have been proven to be clinically significant. A recent study reported that the ratio of aspartate aminotransaminase (AST) to alanine aminotransaminase (ALT) had a prognostic effect on non-metastatic cancers. This study aimed to examine the effect of the AST/ALT ratio on the survival of patients with HNSCC. Clinical data of 356 patients with locoregionally advanced HNSCC were collected. The effect of the AST/ALT ratio on overall survival was analyzed using a Cox proportional hazard model. Moreover, recursive partitioning analysis (RPA) was used to divide the patients into groups on the basis of the clinical stage and AST/ALT ratio. The prognostic ability of this grouping was validated using an independent data set (N = 167). The AST/ALT ratio ranged from 0.42 to 4.30 (median, 1.42) and was a prognostic factor for overall survival that was independent of age, primary sites, and tumor stage (hazard ratio: 1.36, confidence interval: 1.08-1.68, P = 0.010). RPA divided patients with stage IVA into the following two subgroups: high AST/ALT (≥2.3) and low AST/ALT (

The mouse is the most commonly used animal model in biomedical research because of recent advances in molecular genetic techniques. Studies related to eye movement in mice are common in fields such as ophthalmology relating to vision, neuro-otology relating to the vestibulo-ocular reflex (VOR), neurology relating to the cerebellum's role in movement, and psychology relating to attention. Recording eye movements in mice, however, is technically difficult.We developed a new algorithm for analyzing the three-dimensional (3D) rotation vector of eye movement in mice using high-speed video-oculography (VOG). The algorithm made it possible to analyze the gain and phase of VOR using the eye's angular velocity around the axis of eye rotation.When mice were rotated at 0.5 Hz and 2.5 Hz around the earth's vertical axis with their heads in a 30° nose-down position, the vertical components of their left eye movements were in phase with the horizontal components. The VOR gain was 0.42 at 0.5 Hz and 0.74 at 2.5 Hz, and the phase lead of the eye movement against the turntable was 16.1° at 0.5 Hz and 4.88° at 2.5 Hz.To the best of our knowledge, this is the first report of this algorithm being used to calculate a 3D rotation vector of eye movement in mice using high-speed VOG. We developed a technique for analyzing the 3D rotation vector of eye movements in mice with a high-speed infrared CCD camera. We concluded that the technique is suitable for analyzing eye movements in mice. We also include a C++ source code that can calculate the 3D rotation vectors of the eye position from two-dimensional coordinates of the pupil and the iris freckle in the image to this article.

BackgroundImmunoglobulin (Ig)A nephropathy (IgAN) is the most common form of primary glomerulonephritis in the world. Some bacteria were reported to be the candidate of the antigen or the pathogenesis of IgAN, but systematic analysis of bacterial flora in tonsil with IgAN has not been reported. Moreover, these bacteria specific to IgAN might be candidate for the indicator which can predict the remission of IgAN treated by the combination of tonsillectomy and steroid pulse.Methods and findingsWe made a comprehensive analysis of tonsil flora in 68 IgAN patients and 28 control patients using Denaturing gradient gel electrophoresis methods. We also analyzed the relationship between several bacteria specific to the IgAN and the prognosis of the IgAN. Treponema sp. were identified in 24% IgAN patients, while in 7% control patients (P = 0.062). Haemophilus segnis were detected in 53% IgAN patients, while in 25% control patients (P = 0.012). Campylobacter rectus were identified in 49% IgAN patients, while in 14% control patients (P = 0.002). Multiple Cox proportional-hazards model revealed that Treponema sp. or Campylobactor rectus are significant for the remission of proteinuria (Hazard ratio 2.35, p = 0.019). There was significant difference in remission rates between IgAN patients with Treponema sp. and those without the bacterium (p = 0.046), and in remission rates between IgAN patients with Campylobacter rectus and those without the bacterium (p = 0.037) by Kaplan-Meier analysis. Those bacteria are well known to be related with the periodontal disease. Periodontal bacteria has known to cause immune reaction and many diseases, and also might cause IgA nephropathy.ConclusionThis insight into IgAN might be useful for diagnosis of the IgAN patients and the decision of treatment of IgAN.

The clinical significance of human papillomavirus (HPV) in neck node metastasis from cancer of unknown primary (CUP) is not well established. We aimed to address the relationship of HPV status between node metastasis and the primary tumor, and also the relevance of HPV status regarding radiographically detected cystic node metastasis in head and neck squamous cell carcinoma (HNSCC) and CUP. HPV DNA was examined in 68 matched pairs of node metastasis and primary tumor, and in node metastasis from 27 CUPs. In surgically treated CUPs, p16 was examined immunohistochemically. When tonsillectomy proved occult tonsillar cancer in CUP, HPV DNA and p16 were also examined in the occult primary. Cystic node metastasis on contrast-enhanced computed tomography scans was correlated with the primary site and HPV status in another series of 255 HNSCCs and CUPs with known HPV status. Node metastasis was HPV-positive in 19/37 (51%) oropharyngeal SCCs (OPSCCs) and 10/27 (37%) CUPs, but not in non-OPSCCs. Fluid was collected from cystic node metastasis using fine needle aspiration in two OPSCCs and one CUP, and all fluid collections were HPV-positive. HPV status, including the presence of HPV DNA, genotype, and physical status, as well as the expression pattern of p16 were consistent between node metastasis and primary or occult primary tumor. Occult tonsillar cancer was found more frequently in p16-positive CUP than in p16-negative CUP (odds ratio (OR), 39.0; 95% confidence interval (CI), 1.4-377.8; P = 0.02). Radiographically, cystic node metastasis was specific to OPSCC and CUP, and was associated with HPV positivity relative to necrotic or solid node metastasis (OR, 6.2; 95% CI, 1.2-45.7; P = 0.03). In conclusion, HPV status remains unchanged after metastasis. The occult primary of HPV-positive CUP is most probably localized in the oropharynx. HPV status determined from fine needle aspirates facilitates the diagnosis of cystic node metastasis.

Small cell carcinoma (SmCC) generally occurs in the lung and extrapulmonary SmCC is a relatively rare entity. Here, we present a case of a 42-year-old male with SmCC of the larynx diagnosed as cT3N0M0. Concurrent chemoradiotherapy using cisplatin (CDDP) and etoposide (VP-16) was given, which achieved complete response (CR). Lung metastasis became evident in 16 months whereas the locoregional control remained good. In spite of intensive chemotherapy using CDDP and irinotecan (CPT-11), the patient died of disease in 34 months after the first interview. Since the prognosis of laryngeal SmCC is extremely poor, organ preservation therapy should be considered instead of radical laryngectomy.