Cyclin-dependent kinase 1 and survivin as potential therapeutic targets against nasal natural killer/T-cell lymphoma.

Nasal natural killer/T-cell lymphoma (NNKTL) is closely associated with Epstein-Barr virus (EBV) and is characterized by poor prognosis, resulting from rapid progression of lesions in the affected organs. Recent data have shown that NNKTL is associated with the aberrant expression of cyclin-dependent kinase 1 (CDK1) and its downstream target survivin, but little is known about the functional roles of CDK1 and survivin in NNKTL. In the current study, we show that knockdown of the EBV-encoded oncoprotein latent membrane protein 1 (LMP1) induces downregulation of CDK1 and survivin in NNKTL cells. Immunohistochemistry detected CDK1 and survivin expression in LMP1-positive cells of NNKTL biopsy specimens. Inhibition of CDK1 and survivin in NNKTL cells with several inhibitors led to a dose-dependent decrease in cell proliferation. In addition, the Sp1 inhibitor mithramycin, which can downregulate both CDK1 and survivin, significantly suppressed the growth of established NNKTL in a murine xenograft model. Our results suggest that LMP1 upregulation of CDK1 and survivin may be essential for NNKTL progression. Furthermore, targeting CDK1 and survivin with Sp1 inhibitors such as mithramycin may be an effective approach to treat NNKTL, which is considered to be a treatment-refractory lymphoma.

Programmed death-ligand 1 and its soluble form are highly expressed in nasal natural killer/T-cell lymphoma: a potential rationale for immunotherapy.

Nasal natural killer/T-cell lymphoma (NNKTL) is an aggressive neoplasm with poor therapeutic responses and prognosis. The programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) pathway plays an important role in immune evasion of tumor cells through T-cell exhaustion. The aim of the present study was to examine the expression of PD-L1 and PD-1 molecules in NNKTL. We detected the expression of PD-L1 in biopsy samples from all of the NNKTL patients studied. PD-L1 was found on both malignant cells and tumor-infiltrating macrophages, while PD-1-positive mononuclear cells infiltrated the tumor tissues in 36% of patients. Most significantly, soluble PD-L1 (sPD-L1) was present in sera of NNKTL patients at higher levels as compared to healthy individuals and the levels of serum sPD-L1 in patients positively correlated with the expression of PD-L1 in lymphoma cells of tumor tissues. In addition, the high-sPD-L1 group of patients showed significantly worse prognosis than the low-sPD-L1 group. Furthermore, we confirmed that membrane and soluble PD-L1 was expressed on the surface and in the culture supernatant, respectively, of NNKTL cell lines. The expression of PD-L1 was observed in tumor tissues and sera from a murine xenograft model inoculated with an NNKTL cell line. Our results suggest that sPD-L1 could be a prognostic predictor for NNKTL and open up the possibility of immunotherapy of this lymphoma using PD-1/PD-L1 axis inhibitors.

Circulating Epstein-Barr virus-encoded micro-RNAs as potential biomarkers for nasal natural killer/T-cell lymphoma.

Nasal natural killer/T-cell lymphoma (NNKTL) is an Epstein-Barr virus (EBV)-associated malignancy and is characterized by local invasion and widespread dissemination, with a consequent poor prognosis. Micro-RNAs (miRNAs) play roles in the pathogenesis of several malignancies by regulating gene expression and have been recently identified as stable entities in serum. Here, we investigated the value of circulating EBV-miRNAs as biomarkers for NNKTL. Sera of patients with NNKTL were subjected to miRNA polymerase chain reaction (PCR)-array analysis, after which serum EBV-miRNA levels were verified using quantitative PCR. The latter analysis revealed high miR-BART2-5p, miR-BART7-3p, miR-BART13-3p, and miR-BART1-5p expression levels in sera of patients with NNKTL and indicated accurate values for discriminating patients with NNKTL from healthy controls. Levels of these 4 EBV-miRNAs, which were secreted from NNKTL cells, significantly decreased after treatment compared with those before treatment. Furthermore, a high circulating miR-BART2-5p level was associated with disease progression and poor prognosis in patients with NNKTL. Our findings demonstrate that circulating EBV-miRNAs, particularly miR-BART2-5p, may serve as potential diagnostic and prognostic biomarkers in patients with NNKTL.

Novel treatment for early-stage nasal natural killer/T-cell lymphoma: intra-maxillary arterial infusion chemotherapy with concomitant radiotherapy.

Nasal natural killer (NK)/T-cell lymphoma (NNKTL) displays unusual clinicopathological features, and the prognosis is very poor, even in the early stages of the disease. For early stage NNKTL, we have developed a novel chemoradiotherapy regimen incorporating arterial infusion chemotherapy, administered via the superficial temporal artery, in combination with radiotherapy. The novel arterial infusion regimen consists of ifosfamide, carboplatin, methotrexate, peplomycin, and etoposide (MPVIC-P). From 2003 to 2011, 12 patients with early stage NNKTL were treated with the MPVIC-P regimen via arterial infusion with concomitant radiotherapy (54 Gy). We have previously reported on the presence of Epstein-Barr virus (EBV) genetic DNA in NNKTL. Therefore, the effect of the treatment was evaluated by using both clinical findings and serum EBV DNA copy number. The observation period ranged from 39 months to 111 months post-treatment (median: 81 months). All 12 patients achieved and maintained complete remission and, to date, show no sign of relapse. Serum EBV DNA copy numbers decreased to below detectable levels in all 12 patients tested. Manageable mucositis was the most common grade 3-4 toxicity, and it was seen in 10 (83%) patients. However, grade 3-4 hematological toxicity was only seen in 4 (33%) patients. We conclude that our regimen of intra-maxillary arterial chemotherapy with concomitant radiotherapy is an effective treatment with minimal toxicity for early stage NNKTL. Copyright © 2015 John Wiley & Sons, Ltd.

CCL17 and CCL22/CCR4 signaling is a strong candidate for novel targeted therapy against nasal natural killer/T-cell lymphoma.

Nasal natural killer/T-cell lymphoma (NNKTL) is associated with Epstein-Barr virus and has a poor prognosis because of local invasion and/or multiple dissemination. Various chemokines play a role in tumor proliferation and invasion, and chemokine receptors including the C-C chemokine receptor 4 (CCR4) are recognized as potential targets for treating hematologic malignancies. The aim of the present study was to determine whether specific chemokines are produced by NNKTL. We compared chemokine expression patterns in culture supernatants of NNKTL cell lines with those of other lymphoma or leukemia cell lines using chemokine protein array and ELISA. Chemokine (C-C motif) ligand (CCL) 17 and CCL22 were highly produced by NNKTL cell lines as compared to the other cell lines. In addition, CCL17 and CCL22 were readily observed in the sera of NNKTL patients. The levels of these chemokines were significantly higher in patients than in healthy controls. Furthermore, we detected the expression of CCR4 (the receptor for CCL17 and CCL22) on the surface of NNKTL cell lines and in tissues of NNKTL patients. Anti-CCR4 monoclonal antibody (mAb) efficiently induced antibody-dependent cellular cytotoxicity mediated by natural killer cells against NNKTL cell lines. Our results suggest that CCL17 and CCL22 may be important factors in the development of NNKTL and open up the possibility of immunotherapy of this lymphoma using anti-CCR4 mAb.

Downregulation of miR-15a due to LMP1 promotes cell proliferation and predicts poor prognosis in nasal NK/T-cell lymphoma.

Nasal NK/T-cell lymphoma (NNKTL) is an Epstein-Barr virus (EBV)-associated malignancy and has distinct clinical and histological features. However, its genetic features are hitherto unclear. MicroRNAs (miRNAs) play a crucial role in the pathogenesis of several malignancies via regulating gene expression. In this study, we investigated whether the specific microRNAs were related to the tumor behaviors in NNKTL. MiRNA array and Quantitative RT-PCR analyses revealed that miR-15a was expressed at a much lower level in NNKTL cells (SNK-1, SNK-6, and SNT-8) than in normal peripheral NK cells and EBV-negative NK cell line KHYG-1. Quantitative PCR and western blot analyses showed that the expression of MYB and cyclin D1, which are validated targets of miR-15a, was higher in NNKTL cells. Transfection of NNKTL cells (SNK-6 and SNT-8) with a miR-15a precursor decreased MYB and cyclin D1 levels, thereby blocking G1/S transition and cell proliferation. Knockdown of EBV-encoded latent membrane protein 1 (LMP1) significantly increased miR-15a expression in SNK-6 cells. In NNKTL tissues, we found that reduced miR-15a expression, which correlated with MYB and cyclin D1 expression, was associated with poor prognosis of NNKTL patients. These data suggest that downregulation of miR-15a, possibly due to LMP1, implicates in the pathogenesis of NNKTL by inducing cell proliferation via MYB and cyclin D1. Thus, miR-15a could be a potential target for antitumor therapy and a prognostic predictor for NNKTL.

Expression of CD70 in nasal natural killer/T cell lymphoma cell lines and patients; its role for cell proliferation through binding to soluble CD27.

Nasal natural killer (NK)/T cell lymphoma (NNKTL) is associated with Epstein-Barr virus (EBV). The present study analysed gene expression patterns of the NNKTL cell lines SNK6, SNK1 and SNT8, which are positive for EBV and latent membrane protein (LMP)-1, using a complementary DNA array analysis. We found that CD70 was specifically expressed in SNK6 and SNT8. Reverse transcription polymerase chain reaction and flow cytometric analyses confirmed that CD70 was expressed in all 3 NNKTL cell lines, but not in the other EBV-positive NK-cell lines. In vitro studies showed that NNKTL cell lines proliferated, in a dose-dependent fashion, in response to exogenous soluble CD27, which is the ligand for CD70. In NNKTL patients, we confirmed that the CD70 was expressed on the lymphoma cells in NNKTL tissues and that soluble CD27 was present in sera at higher levels as compared to healthy individuals. Finally, complement-dependent cytotoxicity assay showed that anti-CD70 antibody mediated effective complement-dependent killing of NNKTL cells and the affected target CD70 expression on the cells. These results suggest that CD70 acts as a functional receptor binding to soluble CD27, resulting in lymphoma progression and that immunotherapy using anti-CD70 antibody may be a potential candidate for treatment for NNKTL.

Clinical manifestations of granulomatosis with polyangiitis (Wegener's granulomatosis) in the upper respiratory tract seen by otolaryngologists in Japan.

We studied 91 patients with granulomatosis with polyangiitis (GPA; Wegener's granulomatosis) who were dealt with by otolaryngologists in Japan. The upper respiratory tract (URT) alone was involved in 56%. Regarding findings of PR3-ANCA, histology and initial diagnosis, of the 40 patients in whom sites other than the URT were involved, 64 and 73% were positive for PR3-ANCA and histology, respectively. On the other hand, of 51 patients in whom only the URT was involved, only 49 and 31% were positive for PR3-ANCA and histology, respectively. With regard to diagnosis, definitive and probable diagnoses were made in 78 and 20%, respectively, of patients involving sites other than the URT. On the other hand, definitive and probable diagnoses were made in only 22 and 37%, respectively, of patients involving the URT alone; the Japanese diagnostic criteria were inapplicable to 41%. Of the 21 patients to whom the diagnostic criteria were inapplicable, 13 (62%) developed additional symptoms and signs during the observation period, and they then fulfilled the Japanese diagnostic criteria. Among these patients, 8 were MPO-ANCA-positive and 3 patients had 2 or more sub-lesions in the URT. If the diagnostic criteria included cases with MPO-ANCA (±) and 2 lesions in the URT, the diagnostic rates increased from 59 to 86% even though they were of limited form. Thus, about 60% of the patients with URT symptoms alone did not satisfy the Japanese diagnostic criteria at the initial visit.

Endoscopic Resection of a Massive Pleomorphic Adenoma of the Nasal Septum.

Pleomorphic adenoma is a benign tumor that commonly arises from the major salivary glands, such as the parotid and submandibular glands. However, they rarely originate from the nasal cavity. Herein, we describe the case of a 49-year-old woman who presented with nasal obstruction. Preoperative evaluation revealed a giant pleomorphic adenoma attached to the nasal septum. Transnasal endoscopic removal was successfully performed using a specimen retrieval bag. We performed an endoscopic complete en-bloc resection of a large pleomorphic adenoma in the nasal cavity, using a specimen retrieval bag. There were no complications seen, no local recurrences after 2 years of follow-up, and the patient satisfaction was high. We therefore recommend that this technique of using specimen retrieval bags can be favorably utilized for endoscopic removal of large nasal tumors like pleomorphic adenoma.

Stapes Bar-Normal Stapedial Superstructure with a Mobile Footplate Fixed to the Promontory.

This report describes a case of stapedial superstructure fixation with a mobile footplate, which is a rare occurrence among the ossicular malformations that cause conductive hearing impairment. A 44-year-old man with symptoms of left-sided hearing impairment since childhood presented to our department. The eardrum was normal, and pure tone audiometry showed conductive hearing impairment with a three-frequency (500, 1000, and 2000 Hz) mean of 53.3 dB and a normal tympanogram result. An abnormal shadow was observed on the internal side of the left stapes during the three-dimensional reconstruction of computed tomography. We suspected a stapes malformation and performed an exploratory tympanotomy with transcanal endoscopic ear surgery. We found a bony bar between the superstructure of the stapes and the promontory. The mobility of the stapes was significantly improved by removing the bony bar. Findings of postoperative audiometry were normal after surgery. The treatment of such cases of stapedial fixation is simple, and the prognosis of hearing gain after surgery is quite good. Thus, regarding the treatment of patients with conductive hearing impairment, clinicians and surgeons should be aware of the possibility of single stapedial superstructure fixation.

Cervical Emphysema and Pneumomediastinum Caused by a Nasogastric Tube: A Case Report.

BACKGROUND The nasogastric tube (NGT) is a common medical device, and serious complications associated with NGT insertions are rare. The most common serious complication is tracheal insertion; cervical emphysema and pneumomediastinum are rare. There are several methods for confirming the location of the NGT, but a single method of confirmation is often inadequate. Confirmation by air insufflation into the NGT is currently not recommended and is highly invasive. Here, we report a case of cervical emphysema and pneumomediastinum caused by an NGT. CASE REPORT A 94-year-old woman experienced a stroke and was hospitalized for neurosurgery. The nurse inserted an NGT and performed insufflation, but air sounds were not detected. Chest radiography did not reveal the tip of the NGT. Computed tomography (CT) revealed cervical emphysema, pneumomediastinum, an NGT bent in the esophagus, and the distal end of the NGT in the nasopharynx. Nasopharyngeal endoscopy revealed damaged nasopharyngeal mucosa and the distal end of the NGT. The patient was diagnosed with insufflated air passing through the damaged nasopharynx, which had spread to the cervical area and mediastinum. The NGT was removed, and the patient was treated with antibiotics. CT showed cervical emphysema, and the pneumomediastinum resolved after 20 days. CONCLUSIONS It is important to recognize that there are numerous serious and unexpected complications associated with NGT. Different methods should be considered and used to confirm the location of an NGT. Further studies on the confirmation methods and dissemination of such knowledge are required to reduce NGT complications.

Hypopharyngeal Diffuse Large B-Cell Lymphoma in a 74-Year-Old Man Presenting with Dysphagia: A Case Report.

BACKGROUND Malignant lymphomas can occur at various sites. Hypopharyngeal tumors are at risk for airway obstruction and require rapid diagnosis and treatment. Most hypopharyngeal malignancies are squamous cell carcinomas; other tumors are rare. To date, only a few cases of malignant hypopharyngeal lymphoma have been reported, and its specific characteristics are unknown. Herein, we report a case of right hypopharyngeal diffuse large B-cell lymphoma (DLBCL) in a 74-year-old man with dysphagia. CASE REPORT A 74-year-old man presented to our hospital with dysphagia. He had no relevant medical history. Endoscopic examination revealed a right hypopharyngeal tumor. The surface of the tumor was smooth, with no evidence of hemorrhage. Computed tomography revealed a 40-mm mass located in the hypopharynx. We performed a tracheotomy and biopsy of the tumor. Histopathological examination revealed a diffuse proliferation of large atypical B cells with negative staining for Epstein-Barr virus by in situ hybridization. Immunohistochemical staining was positive for CD20 but negative for CD3 and CD10. The patient was administered chemotherapy. The tumor reduced in size, and the patient recovered completely. During the two-year follow up, no recurrence of cancer was observed. CONCLUSIONS Although most hypopharyngeal tumors are squamous cell carcinomas (SCCs), the possibility of other types of tumors should also be considered. Malignant lymphoma of the hypopharynx is rare, and more cases need to be studied and reported in the future.