Estrogen induces the expression of EBV lytic protein ZEBRA, a marker of poor prognosis in nasopharyngeal carcinoma.

Several epidemiological studies have suggested that Epstein-Barr virus (EBV) lytic infection is essential for the development of nasopharyngeal carcinoma (NPC), as the elevation of antibody titers against EBV lytic proteins is a common feature of NPC. Although ZEBRA protein is a key trigger for the initiation of lytic infection, whether its expression affects the prognosis and pathogenesis of NPC remains unclear. In this study, 64 NPC biopsy specimens were analyzed using immunohistochemistry. We found that ZEBRA was significantly associated with a worsening of progression-free survival in NPC (adjusted hazard ratio, 3.58; 95% confidence interval, 1.08-11.87; p = 0.037). Moreover, ZEBRA expression positively correlated with key endocrinological proteins, estrogen receptor α, and aromatase. The transcriptional level of ZEBRA is activated by estrogen in an estrogen receptor α-dependent manner, resulting in an increase in structural gene expression levels and extracellular virus DNA copy number in NPC cell lines, reminiscent of lytic infection. Interestingly, it did not suppress cellular proliferation or increase apoptosis, in contrast with cells treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate, indicating that viral production induced by estrogen is not a cell lytic phenomenon. Our results suggest that intratumoral estrogen overproduced by aromatase could induce ZEBRA expression and EBV reactivation, contributing to the progression of NPC.

Expression of estrogen receptor alpha is associated with pathogenesis and prognosis of human papillomavirus-positive oropharyngeal cancer.

Human papillomavirus (HPV) has been identified as a causative agent of cervical cancer and oropharyngeal cancer (OPC). Intriguingly, estrogen and HPV were shown to play synergistic roles in cervical carcinogenesis. We recently demonstrated that the apolipoprotein B mRNA-editing catalytic polypeptide 3 (APOBEC3, A3) family, which is inducible by estrogen, could lead to HPV DNA hypermutation and cause viral DNA integration. In the present study, we examined the relationships between estrogen-estrogen receptor α (ERα) and A3s in HPV-positive OPC. ERα expression was associated with HPV positivity in OPC biopsy samples using immunohistochemical analysis and reverse-transcription quantitative polymerase chain reaction. In addition, ERα was significantly associated with improved overall survival in HPV-positive OPC (hazard ratio, 0.26; p = 0.029). APOBEC3A (A3A) mRNA was induced by estrogen in HPV and ERα-positive OPC cells. Furthermore, A3A mRNA and protein expression were significantly higher in ERα-positive cases than in ERα-negative ones, among HPV-positive biopsy samples (p = 0.037 and 0.047). These findings suggest that A3A is associated with a good prognosis in ERα-positive OPC, and indicate the prognostic significance of ERα in HPV-positive OPC. This is the first study to demonstrate the prognostic role of ERα in HPV-positive OPC.

Progression of understanding for the role of Epstein-Barr virus and management of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is very common in southern China and Southeast Asia. In regions where NPC is endemic, undifferentiated subtypes constitute most cases and are invariably associated with Epstein-Barr virus (EBV) infection, whereas the differentiated subtype is more common in other parts of the world. Undifferentiated NPC is a unique malignancy with regard to its epidemiology, etiology, and clinical presentation. Clinically, NPC is highly invasive and metastatic, but sensitive to both chemotherapy and radiotherapy (RT). Overall prognosis has dramatically improved over the past three decades because of advances in management, including the improvement of RT technology, the broader application of chemotherapy, and more accurate disease staging. Despite the excellent local control with modern RT, distant failure remains a challenging problem. Advances in molecular technology have helped to elucidate the molecular pathogenesis of NPC. This article reviews the contribution of EBV gene products to NPC pathogenesis and the current management of NPC.

Modulation of the tumor microenvironment by Epstein-Barr virus latent membrane protein 1 in nasopharyngeal carcinoma.

Latent membrane protein 1 (LMP1) is a primary oncogene encoded by the Epstein-Barr virus, and various portions of LMP1 are detected in nasopharyngeal carcinoma (NPC) tumor cells. LMP1 has been extensively studied since the discovery of its transforming property in 1985. LMP1 promotes cancer cell growth during NPC development and facilitates the interaction of cancer cells with surrounding stromal cells for invasion, angiogenesis, and immune modulation. LMP1 is detected in 100% of pre-invasive NPC tumors and in approximately 50% of advanced NPC tumors. Moreover, a small population of LMP1-expressing cells in advanced NPC tumor tissue is proposed to orchestrate NPC tumor tissue maintenance and development through cancer stem cells and progenitor cells. Recent studies suggest that LMP1 activity shifts according to tumor development stage, but it still has a pivotal role during all stages of NPC development.

EBV infection is prevalent in the adenoid and palatine tonsils in adults.

Epstein-Barr virus (EBV) is associated with the pathogenesis of several diseases in both adults and children. However, there have been no reports on the prevalence and amount of EBV in the adenoids of adults; thus, it is important to investigate these in the adenoids and tonsils of adults and children. In this study, 67 patients who underwent tonsillectomy or adenotonsillectomy were included and divided into two groups: adults aged ≥ 16 years (n = 35) and children aged <16 years (n = 32). Patients' adenoid and tonsil tissues were analyzed using quantitative polymerase chain reaction for EBV DNA. EBV was detected in 26 (74%) adenoids and 25 (71%) tonsils among the adult group and was detected 21 (66%) adenoids and 20 (63%) tonsils in the child group. There was no significant difference in EBV DNA prevalence between the adenoids and tonsils for each group. However, there was a significant correlation between EBV DNA load in the adenoids and tonsils of the same individual in both groups (r = 0.579, P < 0.01, adult group; r = 0.919, P < 0.01, child group). In conclusion, EBV infection is prevalent in the adenoids and tonsils in adults and children. These results indicate that EBV continuously reside in the nasopharyngeal region after primal infection and may develop several diseases.

Endoscopic management of petrous apex cholesteatoma.

We describe a technique for approaching petrous apex cholesteatoma using combined lateral microscopic/endoscopic approaches, and discuss the utility of endoscopy in managing matrix inside the petrous apex. In our two cases, total view inside the petrous apex was achieved under endoscopy without mobilizing the internal carotid artery, and the matrix was successfully removed. Neither patient has presented with postoperative recurrence thanks to the wide-angle endoscopic view inside the petrous apex. Since the number of patients was small, comparisons with microscopic treatments are not yet valid, but endoscopes could offer a helpful tool for operating inside the petrous apex.

Cochlear implantation in deaf patients with eosinophilic otitis media using subtotal petrosectomy and mastoid obliteration.

We investigated the usefulness and safety of our cochlear implantation method for two deaf patients with eosinophilic otitis media. The surgical approach was a subtotal petrosectomy to remove the theater of eosinophilic infiltration and to prevent leaching of foreign substances and entry of stimuli that are the cause of eosinophilic inflammations. The operative cavity was obliterated with abdominal fat. There were no complications or recurrent inflammation following surgery in the cases of both patients. It was confirmed by CT scan that the eustachian tube was closed and the operative cavity remained obliterated with abdominal fat. Following the procedure, the hearing threshold results of the two patients were 30 and 34 dB. Cochlear implantation procedures in this report for deaf patients resulting from eosinophilic otitis media may be effective and safe. Using steroids before surgery may be the better option. To further confirm the efficacy and safety of our surgical concept, we need to administer this treatment concept for a large number of cases in a future study.

Progress and controversy for the role of chemotherapy in nasopharyngeal carcinoma.

Since the publication of Intergroup Study 0099, representing a superiority of concurrent chemoradiotherapy with cisplatin followed by adjuvant chemotherapy to radiotherapy alone for the treatment of locoregionally advanced nasopharyngeal carcinoma, an efficacy of concurrent setting of cisplatin-based chemotherapy with radiotherapy has been repeatedly validated. In meanwhile, the role of adjuvant part of the protocol has been controversial. There is an increasing evidence for the positive role of neoadjuvant chemotherapy with following concurrent chemoradiotherapy whereas favorable contribution was not proven in the last century. This article reviews the role of chemotherapy in the treatment of locoregionally advanced nasopharyngeal carcinoma.

Protein Farnesylation on Nasopharyngeal Carcinoma, Molecular Background and Its Potential as a Therapeutic Target.

Nasopharyngeal carcinoma (NPC) is one of the Epstein-Barr virus (EBV)-associated malignancies. NPC is highly metastatic compared to other head and neck carcinomas, and evidence has shown that the metastatic features of NPC are involved in EBV infection. The prognosis of advanced cases, especially those with distant metastasis, is still poor despite advancements in molecular research and its application to clinical settings. Thus, further advancement in basic and clinical research that may lead to novel therapeutic modalities is needed. Farnesylation is a lipid modification in the C-terminus of proteins. It enables proteins to attach to the lipid bilayer structure of cellular membranes. Farnesylation was initially identified as a key process of membrane association and activation of the RAS oncoprotein. Farnesylation is thus expected to be an ideal therapeutic target in anti-RAS therapy. Additionally, more and more molecular evidence has been reported, showing that proteins other than RAS are also farnesylated and have significant roles in cancer progression. However, although several clinical trials have been conducted in cancers with high rates of ras gene mutation, such as pancreatic carcinomas, the results were less favorable than anticipated. In contrast, favorable outcomes were reported in the results of a phase II trial on head and neck carcinoma. In this review, we provide an overview of the molecular pathogenesis of NPC in terms of the process of farnesylation and discuss the potential of anti-farnesylation therapy in the treatment of NPC.

Concurrent superselective intra-arterial chemotherapy and radiotherapy for late-stage squamous cell carcinoma of the temporal bone.

OBJECTIVES: This study evaluated the efficacy of concurrent superselective intra-arterial chemotherapy and radiotherapy without surgery for late-stage squamous cell carcinoma of the temporal bone, which typically has a poor prognosis. METHODS: The subjects were 5 patients treated at our hospital between 2007 and 2010 for primary cancer of the temporal bone. One patient had a stage T3 tumor, and 4 patients had T4 tumors, according to the Pittsburgh staging system. All patients received irradiation with a conventional once-daily fraction of 2 Gy, and the total dose ranged from 60 to 66 Gy. Intra-arterial cisplatin via transfemoral catheterization and intravenous sodium thiosulfate were administered. The contribution of each vessel in supplying blood to the primary tumor was determined by real-time computed tomographic angiography. RESULTS: Three patients obtained a complete response. The same 3 patients remain alive without local recurrence (mean survival, 28 months), 1 patient died of distant metastasis without local recurrence after 19 months, and 1 patient remains alive with local recurrence. CONCLUSIONS: Although the small number of patients prevents comparisons with other treatments, the present study obtained good results. This may become an effective treatment for patients with late-stage squamous cell carcinoma of the temporal bone.

Epstein-Barr Virus LMP1 Induces Soluble PD-L1 in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy. The principal oncogene of EBV, latent membrane protein 1 (LMP1), induces the expression of programmed death-ligand 1 (PD-L1), which is an immunosuppressive transmembrane protein and a promising therapeutic target for various malignancies. Recent studies have revealed an association between the level of soluble PD-L1 (sPD-L1) and disease progression. However, the role of sPD-L1 in NPC or its relevance to LMP1 has not been elucidated. This study aimed to examine whether LMP1 induces sPD-L1 in vitro and analyze the clinical relevance of LMP1, PD-L1, and sPD-L1 in NPC patients. Analysis of nasopharyngeal cell lines revealed that LMP1 induces both cellular PD-L1 and sPD-L1. Analysis of biopsy specimens from 32 NPC patients revealed that LMP1 expression was significantly correlated with PD-L1 expression. Finally, the serum sPD-L1 level in NPC patients was higher than that in the controls. Moreover, the sPD-L1 level in the advanced stage was higher than that in the early stage. However, LMP1 expression, PD-L1 expression, and sPD-L1 levels were not associated with prognosis. These results suggest that LMP1 induces both sPD-L1 and PD-L1, which are associated with NPC progression.

Modified meatotympanoplasty for external auditory canal stenosis and lateralized tympanic membrane: a preliminary study.

Background: The surgical treatment of external auditory canal stenosis (EACS) and lateralized tympanic membrane (LTM) is challenging because there is a high risk of postoperative complications.Objectives: The aim of this study was to evaluate the postoperative hearing levels and other outcomes of 10 cases with EACS and LTM.Materials and methods: This was a retrospective preliminary study of patients with EACS and LTM who underwent meatotympanoplasty from 2008 to 2018. Their mean age at surgery was 31.6 years. The surgeries were performed taking into consideration the following: (1) creating a large EAC and functioning tympanic membrane (TM) and (2) avoiding complete mastoidectomy to prevent postoperative cavity problems.Results: The mean follow-up period was 3 years 10 months. The average preoperative and postoperative air-bone gaps (ABGs) were 40.4 dB and 23.0 dB, respectively, and there was a significant difference. A postoperative ABG less than 30 dB was achieved in 80% (8/10) of patients. Four cases had postoperative complications (re-stenosis of the EAC and/or re-lateralization of the TM).Conclusions and significance: The postoperative results presented were comparable to those of previous surgical techniques; however, further development is needed to prevent postoperative complications and earn better hearing results.

Deletion of CD38 and supplementation of NAD+ attenuate axon degeneration in a mouse facial nerve axotomy model.

Following facial nerve axotomy, nerve function is not fully restored even after reconstruction. This may be attributed to axon degeneration/neuronal death and sustained neuroinflammation. CD38 is an enzyme that catalyses the hydrolysis of nicotinamide adenine dinucleotide (NAD+) and is a candidate molecule for regulating neurodegeneration and neuroinflammation. In this study, we analyzed the effect of CD38 deletion and NAD+ supplementation on neuronal death and glial activation in the facial nucleus in the brain stem, and on axon degeneration and immune cell infiltration in the distal portion of the facial nerve after axotomy in mice. Compared with wild-type mice, CD38 knockout (KO) mice showed reduced microglial activation in the facial nucleus, whereas the levels of neuronal death were not significantly different. In contrast, the axon degeneration and demyelination were delayed, and macrophage accumulation was reduced in the facial nerve of CD38 KO mice after axotomy. Supplementation of NAD+ with nicotinamide riboside slowed the axon degeneration and demyelination, although it did not alter the level of macrophage infiltration after axotomy. These results suggest that CD38 deletion and supplementation of NAD+ may protect transected axon cell-autonomously after facial nerve axotomy.

Influences of Semaphorin 3A Expression on Clinicopathological Features, Human Papillomavirus Status, and Prognosis in Oropharyngeal Carcinoma.

Human papillomavirus (HPV) infection is now identified as a major etiologic factor for oropharyngeal cancer (OPC), and HPV positivity is well established better prognostic marker in OPC. Now, predictable markers for the prognosis of the patients who are stratified by HPV has been investigated in. Semaphorin 3A (SEMA3A) is a well-known axon guidance molecule in the nervous system. It is also known as a tumor suppressor in various cancers. In the present study, we examined the relationships between SEMA3A and clinicopathologic features, especially HPV status, and neoangiogenesis, and its prognostic significance for OPC patients. Thirty-two OPC patients and 17 normal patients were analyzed for SEMA3A expression by immunohistochemical analysis. We also analyzed 22 OPC specimens for CD34 expression as a marker of neoangiogenesis. SEMA3A was significantly downregulated in OPC compared with chronic tonsillitis tissues (p = 0.005). SEMA3A expression was negatively correlated with CD34 expression (r = -0.466, p = 0.033). Moreover, the higher SEMA3A expression cohort showed better survival than the lower SEMA3A expression cohort regardless of HPV status (p = 0.035). These results suggest that SEMA3A expression is a prognostic marker for survival regardless of HPV status and is associated with anti-angiogenesis in OPC.

Overexpression of Semaphorin 3A is a Marker Associated with Poor Prognosis in Patients with Nasopharyngeal Carcinoma.

Semaphorins were discovered as guidance signals that mediate neural development. Recent studies suggest that semaphorin 3A (Sema3A), a member of the semaphorin family, is involved in the development of several cancers. This study aimed to analyze the association of Sema3A with the clinical features of nasopharyngeal carcinoma (NPC), an Epstein-Barr virus-associated carcinoma, and the Epstein-Barr virus primary oncogene latent membrane protein 1 (LMP1). The expression of Sema3A and LMP1 was immunohistochemically examined in the 35 NPC specimens. The mean expression scores for Sema3A and LMP1 were 20.8% ± 14.5% and 13.9% ± 14.8%, respectively. The expression of Sema3A significantly correlated with that of LMP1 (r = 0.41, p = 0.014). In addition, the Sema3A high cohort showed significantly poorer prognosis than the Sema3A low cohort. Sema3A expression was higher in the LMP1-positive KH-1 and KR-4 cell lines compared to the LMP1-negative HeLa cells. Overexpression of LMP1 in the LMP1-negative AdAH cell line upregulated Sema3A expression, both at the transcriptional and translational level. Finally, Sema3A expression was associated with poor prognosis in patients with NPC. Our data suggest that LMP1 induces the expression of Sema3A, which may promote tumor progression in NPC.

Area-dependent change of response in the rat's inferior colliculus to intracochlear electrical stimulation following neonatal cochlear damage.

To understand brain changes caused by auditory sensory deprivation, we recorded local-field potentials in the inferior colliculus of young adult rats with neonatal cochlear damage produced by systemic injections of amikacin. The responses were elicited by electrical stimulation of the entire cochlea and recorded at various locations along a dorsolateral-ventromedial axis of the inferior colliculus. We found that hair cells were completely destroyed and spiral ganglion neurons were severely damaged in the basal cochleae of amikacin-treated animals. Hair cells as well as spiral ganglion neurons were damaged also in the middle and apical areas of the cochlea, with the damage being greater in the middle than the apical area. Amplitudes of local-field potentials were reduced in the ventromedial inferior colliculus, but enhanced in the dorsolateral inferior colliculus. Latencies of responses were increased over the entire structure. The enhancement of responses in the dorsolateral inferior colliculus was in contrast with the damage of hair cells and spiral ganglion cells in the apical part of the cochlea. This contrast along with the overall increase of latencies suggests that early cochlear damage can alter neural mechanisms within the inferior colliculus and/or the inputs to this midbrain structure.

Microglial activation in the cochlear nucleus after early hearing loss in rats.

OBJECTIVE: Microglia are highly specialized tissue macrophages in the central nervous system. Their activation in the auditory system has been reported in adult hearing loss models, but their status in the developing auditory system is less understood. Therefore, we investigated microglial status in the cochlear nucleus (CN) during normal developing periods and after exposing rats to amikacin, a potent ototoxin, around the time of hearing onset. METHODS: To develop the deafness model, rats were administered with a daily intraperitoneal injection of amikacin (500 mg/kg) from postnatal day 7 (P7) to P15. To evaluate the expression of ionized calcium binding adaptor molecule 1 (Iba1), we performed immunohistochemical analysis using rat brains from P10-60. To compare the expression of microglia-related gene, reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis were performed. RESULTS: Immunohistochemical analysis revealed that, under normal conditions, microglia had relatively large cell bodies with several extended processes that surrounded other cells at P10, while the sizes and number of these cells gradually decreased afterward. In contrast, when amikacin was administered from P7 to P15, microglia maintained large cell bodies with relatively shorter processes at both P15 and P21. Furthermore, RT-qPCR analysis revealed upregulation of genes including phagocytotic and anti-inflammatory markers after amikacin administration. CONCLUSION: These results suggest that microglia are activated in the CN, and they may contribute to tissue remodeling after early hearing loss in the developing auditory system.

Inhibition of autophagy by chloroquine makes chemotherapy in nasopharyngeal carcinoma more efficient.

OBJECTIVES: A combination of platinum-based chemotherapy and radiotherapy is the standard treatment for nasopharyngeal carcinoma (NPC). However, the efficacy of chemotherapy has reached a plateau. Many autophagy studies suggest that autophagy can either promote or suppress to cancer progression. Thus, a role of autophagy in the acquisition of chemoradioresistance has recently been a notable event. Therefore, we examined the relationship between autophagy and chemotherapy in NPC. METHODS: The expression of Beclin 1 and microtubule-associated protein light chain 3 (LC3), a marker of autophagy, was determined by immunohistochemistry in the biopsy samples of patients with NPC before and after the first course of chemotherapy. Additionally, to investigate in the effect of autophagy suppression in chemotherapy, NPC cell line C666-1 cells were treated with cisplatin and/or chloroquine, an inhibitor of autophagy. RESULTS: The expression of Beclin 1 increased after chemotherapy in all patients. In NPC cell line C666-1, compared to cisplatin alone, combination therapy (cisplatin and chloroquine) reduced cell viability, and promoted cell apoptosis. CONCLUSIONS: These results suggest that autophagy, represented by Beclin 1, is upregulated after chemotherapy in both in vitro and in vivo NPC studies. Inhibition of autophagy could therefore be new strategy for NPC treatment.