The role of frozen section biopsy for parotid gland tumour with benign fine-needle aspiration cytology.

OBJECTIVES: This study focused on parotid gland tumours diagnosed as benign by fine-needle aspiration cytology and investigated the necessity of frozen section biopsy. METHODS: There were 104 cases of parotid gland tumour where fine-needle aspiration cytology was benign and frozen section biopsy was subsequently performed, between April 2006 and June 2016. In this retrospective study, the results of frozen section biopsy were analysed and compared with the final histological diagnosis. RESULTS: Among the 104 cases diagnosed as benign by fine-needle aspiration cytology, 102 cases and 2 cases were diagnosed as benign and malignant, respectively, by frozen section biopsy. The final histological diagnoses showed that 98 cases were benign and 6 cases were malignant. The sensitivity and specificity values of frozen section biopsy in detecting malignant tumours were 33 per cent and 100 per cent, respectively. CONCLUSION: The necessity of frozen section biopsy in cases with benign fine-needle aspiration cytology may be low in parotid gland surgery.

APOBEC3A associates with human papillomavirus genome integration in oropharyngeal cancers.

The prevalence of human papillomavirus (HPV)-related oropharyngeal cancers has been increasing in developed countries. We recently demonstrated that members of the apolipoprotein B mRNA-editing catalytic polypeptide 3 (APOBEC3, A3) family, which are antiviral factors, can induce hypermutation of HPV DNA in vitro. In the present study, we found numerous C-to-T and G-to-A hypermutations in the HPV16 genome in oropharyngeal cancer (OPC) biopsy samples using differential DNA denaturation PCR and next-generation sequencing. A3s were more abundantly expressed in HPV16-positive OPCs than in HPV-negative, as assessed using immunohistochemistry and reverse transcription quantitative PCR. In addition, interferons upregulated A3s in an HPV16-positive OPC cell line. Furthermore, quantitative PCR analysis of HPV DNA suggests that APOBEC3A (A3A) expression is strongly correlated with the integration of HPV DNA. These results suggest that HPV16 infection may upregulate A3A expression, thereby increasing the chance of viral DNA integration. The role of A3A in HPV-induced carcinogenesis is discussed.

Hypopharyngeal presentation of cicatricial pemphigoid: videofluorographic and direct laryngoscopic findings.

OBJECTIVE: Cicatricial pemphigoid can affect all mucosa of the upper aerodigestive tract; however, hypopharyngeal involvement is less frequent. CASE REPORT: This paper presents a 69-year-old male diagnosed as having cicatricial pemphigoid who was experiencing difficulty swallowing. Videofluorography with barium swallow demonstrated narrow flow through the medial hypopharynx, but not through the lateral hypopharynx. Direct laryngoscopy revealed that the postcricoid hypopharyngeal lumen had become narrow due to circumferential scar formation. Interestingly, detached thin membranous webs were observed beyond the circumferential scar. CONCLUSION: This report describes important videofluorographic and direct laryngoscopic findings showing rare hypopharyngeal involvement in a case of cicatricial pemphigoid.

Case report. Intra-arterial chemotherapy for laryngeal cancer via a non-bifurcating carotid artery.

The common carotid artery (CCA) usually divides into the internal carotid artery (ICA) and the external carotid artery (ECA). We present an extremely rare case of a non-bifurcating carotid artery through which intra-arterial chemotherapy for laryngeal cancer was administered. The CCA angiogram, as well as ultrasonographic evaluation of the carotid arteries, demonstrated a non-bifurcating CCA that subsequently constituted the ICA. Furthermore, several branches normally given off by the ECA arose directly from the single carotid artery. Superselective intra-arterial infusion of cis-diamminedichloroplatinum (II) (CDDP) was subsequently performed.

Prevention and inhibition of nasopharyngeal carcinoma growth by antiviral phosphonated nucleoside analogs.

Nasopharyngeal carcinoma (NPC) is universally associated with EBV infection. We have shown that the phosphonated nucleoside analog, (S)-1-[3-hydroxy-2-(phosphonylmethoxy)-propyl]cytosine (HPMPC) strongly inhibits growth of NPC xenografts in nude mice by causing apoptosis (J. Neyts et al., Cancer Res., 58, 384-388, 1998). We, therefore, tested two additional members of this drug family that have different degrees of antiviral activity, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) and 9-2-(R)-(phosphonomethoxy)propyladenine (PMPA). Intratumoral injection of PMEA (75 microl of 2% solution) in C15 NPC xenografts, which are latently infected with EBV, slowed tumor growth moderately, whereas PMPA (75 microl of 2% solution) slowed tumor growth only marginally. Compared with the previous results showing complete regression of tumor, PMEA had less antitumoral effect than HPMPC, and PMPA had the least. After 4 weeks of preventive treatment, tumors formed in 12.5, 50, and 100% of mice treated with HPMPC, PMEA, and PMPA, respectively, in contrast to the development of tumors in all of the PBS-treated control mice. We also investigated the effect of each drug on the EBV-positive epithelial cell line NPC-KT in vitro. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed inhibition of growth of NPC-KT cells by HPMPC and PMEA, but not by PMPA, which correlates with the results observed in tumor xenografts. Growth inhibition was attributable to induction of apoptosis in NPC-KT cells as indicated by a DNA fragmentation assay. Cleavage of poly(ADP-ribose) polymerase after treatment of NPC-KT cells with HPMPC was observed, which suggested that the apoptosis may be mediated by caspase(s). The apoptotic effects of the drugs are independent of any effects on EBV DNA polymerase, which is not expressed in these latently infected NPCs. These results suggest that HPMPC as well as PMEA could provide an adjunctive treatment for NPC.

Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells.

Cyclooxygenase-2 (COX-2) is an inducible form of COX and is overexpressed in diverse tumors, raising the possibility of a role for COX-2 in carcinogenesis. In addition, COX-2 contributes to angiogenesis. The Epstein-Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1), is detected in at least 70% of nasopharyngeal carcinoma (NPC) and all EBV-infected preinvasive nasopharyngeal lesions. We found that in specimens of LMP1-positive NPC, COX-2 is frequently expressed, whereas LMP1-negative NPC rarely express the enzyme. We next found that expression of LMP1 in EBV-negative nasopharyngeal epithelial cells induced COX-2 expression. Coexpression of IkappaBalpha(S32A/S36A), which is not phosphorylated and prevents NF-kappaB activation, with LMP1 showed that NF-kappaB is essential for induction of COX-2 by LMP1. We also demonstrate that NF-kappaB is involved in LMP1-induced cox-2 promoter activity with the use of reporter assays. Two major regions of LMP1, designated CTAR1 and CTAR2, are signal-transducing domains of LMP1. Constructs expressing either CTAR1 or CTAR2 induce COX-2 but to a lesser extent than wild-type LMP1, consistent with the ability of both regions to activate NF-kappaB. Furthermore, we demonstrate that LMP1-induced COX-2 is functional because LMP1 increased production of prostaglandin E(2) in a COX-2-dependent manner. Finally, we demonstrate that LMP1 increased production of vascular endothelial growth factor (VEGF). Treatment of LMP1-expressing cells with the COX-2-specific inhibitor (NS-398) dramatically decreased production of VEGF, suggesting that LMP1-induced VEGF production is mediated, at least in part, by COX-2. These results suggest that COX-2 induction by LMP1 may play a role in angiogenesis in NPC.

Aspirin inhibits tumor cell invasiveness induced by Epstein-Barr virus latent membrane protein 1 through suppression of matrix metalloproteinase-9 expression.

Matrix metalloproteinases (MMPs) are thought to play crucial roles in tumor invasion and metastasis. Because we have shown that EBV latent membrane protein 1 (LMP1) enhances MMP-9 expression by activation of nuclear factor (NF)-kappaB and activator protein (AP)-1 (T. Yoshizaki, et al., Proc. Natl. Acad. Sci. USA, 95: 3621-3626, 1998), we therefore tested whether up-regulation of MMP-9 by LMP1 could be correlated with enhanced invasiveness of tumor cells in vitro. Whether aspirin and sodium salicylate could reduce invasiveness and whether LMP1 could enhance MMP-9 expression in tumors grown in nude mice were also tested. C33A cells stably expressing LMP1 had increased expression of MMP-9 and showed greater invasion through reconstituted basement membrane compared with vector-transfected C33A cells (P < 0.02). Treatment with aspirin or sodium salicylate inhibited invasiveness of the LMP1-expressing C33A cells (P < 0.03) and suppressed both the LMP1-induced MMP-9 expression in zymographic analyses and LMP1-induced MMP-9 promoter activity in CAT reporter assays (P < 0.01). Endogenous MMP-2 levels were unaffected by either drug. Both drugs repressed the CAT activity of the truncated MMP-9 promoter construct, which only contained a binding site for AP-1, to the basal level (P < 0.05). Moreover, EMSA indicated that the effects of the salicylates were through the inhibition of not only NF-kappaB but also AP-1 binding activity. Inhibitory effect of salicylates could be reversed by p50/p65 subunits of NF-kappaB or c-Jun overexpression. The inhibitory effect of aspirin on NF-kappaB activity was attributable to the inhibition of IkappaB kinase activity. Finally, tumors derived from C33A cells stably expressing LMP1 grown in nude mice showed enhanced MMP-9 levels compared with tumors derived from vector-transfected C33A cells. This enhancement was inhibited by treatment of the mice with aspirin. These results suggest that aspirin may be able to suppress invasion and metastasis of EBV-associated tumors that express LMP1 by suppression of MMP-9.

Association of Epstein-Barr virus infection with p53 protein accumulation but not bcl-2 protein in nasopharyngeal carcinoma.

AIMS: The aim of this study was to investigate the association of Epstein-Barr virus (EBV) infection with status of p53 protein expression in nasopharyngeal carcinoma (NPC). The expression of EBV gene and gene product, p53 protein and bcl-2 protein in NPC was histopathologically studied. METHODS AND RESULTS: In-situ hybridization using oligonucleotide probe to EBV-encoded small RNAs (EBERs) and immunohistochemistry using monoclonal antibodies against EBV latent membrane protein 1 (LMP1), p53 protein and bcl-2 proteins were performed in 56 primary NPCs. EBERs were detected in 46 (82%) cases and LMP1 in 17 (30%) cases. While 30 of 32 (94%) cases in differentiated nonkeratinizing carcinoma (NKC, WHO type 2) and 16 of 17 (94%) cases in undifferentiated carcinoma (UC, WHO type 3) showed EBERs expression, neither five cases of keratinizing squamous cell carcinoma (KSCC, WHO type 1) nor two cases of adenocarcinoma showed EBERs. bcl-2 protein was detected in 50 (89%) cases, but its expression did not depend on expression of LMP1. p53 protein was detected in 31 (55%) cases, and there was a correlation between expression of EBERs and p53 protein (P < 0.05) but not between LMP1 and p53 protein. CONCLUSION: In this study, close association of NKC and UC but not KSCC with the latent infection with EBV was demonstrated. The induction of bcl-2 protein by LMP1, as shown in vitro, was not demonstrated. The association between overexpression of p53 protein and the presence of EBV suggests that some EBV-encoded protein, which may be different from LMP1, may play a role for nuclear accumulation of p53 protein.

Matrix metalloproteinase 9 is induced by the Epstein-Barr virus BZLF1 transactivator.

Type IV collagenases, matrix metalloproteinase (MMP) 2 and MMP9 are implicated in tumor invasion and metastasis. In patients with nasopharyngeal carcinoma (NPC), poor prognosis due to development of local and distant metastasis has been reported to be predicted by antibody titers against the Z protein which is an AP-1 family transcription factor encoded by the EBV BZLF1 immediate-early gene. Here we report that in patients with NPC, expression of Z in tumor cells correlates with advanced cervical lymph node metastasis which may suggest that Z affects tumor invasion and metastasis. We therefore tested if Z would induce expression of type IV collagenases. Transfection of Z expression plasmid into the C33A epithelial cell line increased expression of MMP9, but MMP2 expression was unaltered. Mutational analysis of the Z protein revealed that, in addition to all three functional domains of Z (dimerization domain, DNA binding domain, and activation domain), the carboxyl terminal 17 amino acids which stabilize the Z protein were necessary for induction of MMP9 expression. Analysis of the MMP9 promoter demonstrated that only AP-1 site close to the transcriptional start-site was essential for transactivation by Z. Previously we reported that Epstein-Barr virus latent membrane protein 1 (LMP1) stimulates MMP9 expression (Yoshizaki et al. Proc. Natl. Acad. Sci. 1998; 95: 3621-6). Thus, Z together with LMP1 may contribute to invasion and metastasis of NPC by inducing expression of MMP9.

Detection of Epstein-Barr virus in nasopharyngeal carcinoma by in situ hybridization and polymerase chain reaction.

The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma (NPC) has been shown by various methods. The purpose of this study is to identify the most useful method to detect EBV in NPC. Both polymerase chain reaction (PCR) for EBV-DNA and in situ hybridization for EBV-encoded small RNAs (EBERs) were examined in formalin-fixed, paraffin-embedded NPC specimens. In situ hybridization was performed in 56 cases, and PCR for EBV-DNA was performed in 42 cases. EBV-DNA was detected in 0 of 3 keratinizing squamous cell carcinomas (KSCC), 22 of 24 nonkeratinizing carcinomas (NKC), all 13 undifferentiated carcinomas (UNPC), and 0 of 2 adenocarcinomas (AC). EBERs were detected in 0 of 5 KSCC, 30 of 32 NKC, 16 of 17 UNPC, and 0 of 2 AC. Among them, EBERs was detected in 35 of 42 cases in which PCR was also performed, 0 of 3 KSCC, 22 of 24 NKC, all 13 UNPC, and 0 of 2 AC, respectively. Both results were consistent in 40 of 42 cases. We conclude that both PCR and in situ hybridization are useful to detect EBV in NPC. In situ hybridization has a particular advantage because it can demonstrate the localization of EBV in neoplastic cells. In addition, close association of NKC and UNPC but not KSCC and AC with EBV is suggested.

[Detection of Epstein-Barr virus genomes in nasopharyngeal carcinoma].

In situ hybridization using EBV encoded small RNAs (EBERs) probe and polymerase chain reaction (PCR) were performed to detect the presence of Epstein-Barr virus (EBV) genomes in 56 primary nasopharyngeal carcinomas. EBERs was detected in 46 cases(82%); 30 of 32 cases (94%) in differentiated non-keratinizing carcinoma (NKC, WHO-2) and 16 of 17 cases (94%) in undifferentiated nasopharyngeal carcinoma (UNPC, WHO-3). But none of 5 cases of squamous cell carcinoma (SCC, WHO-1) nor 2 cases of adenocarcinoma showed EBERs. EBV-DNA was also detected in 22 of 24 cases of NKC and in all 13 cases of UNPC by PCR but not in 3 cases of SCC or 2 cases of adenocarcinoma. This findings shows that the examination of EBERs and EBV-DNA is very useful, sensitive method in the detection of EBV.

Increased expression of membrane type 1-matrix metalloproteinase in head and neck carcinoma.

BACKGROUND: Three types of membrane type-matrix metalloproteinases (MT-MMPs) have been identified as activators of pro-MMP2 (gelatinase A/72-kilodalton Type IV collagenase), which is believed to be crucial for tumor invasion and metastasis. MT1-MMP has been shown to be widely expressed in various tissues and to be overexpressed in lung and gastric carcinomas. METHODS: Activation of pro-MMP2 was examined in head and neck squamous cell carcinoma (HNSCC) by gelatin zymography. The expression of the MT1-MMP mRNA transcript was studied by Northern hybridization and that of MT1-MMP protein by immunohistochemical staining with a monoclonal antibody against MT1-MMP. RESULTS: Activation of pro-MMP2 was observed in ten of ten HNSCC tissues. Increased expression of the MT1-MMP mRNA transcript was also detected in eight of eight of these tissues. MT1-MMP positive cells were detected at the tumor cell lesions in the majority of these carcinoma tissues by immunohistochemical staining (24 of 27). MT1-MMP expression was more intense in moderately and well differentiated tumors than in poorly differentiated ones (P < 0.05). CONCLUSIONS: These results suggest that MT1-MMP is involved in the activation of pro-MMP2 in HNSCC. These findings suggest that MT1-MMP is significant not only as a tumor marker but as a new target for chemotherapy against HNSCC.

[Interaction between Epstein-Barr virus (EBV) gene expression and antibodies to EBV in nasopharyngeal carcinomas].

In situ hybridization using EBERs and BHLF oligonucleotide probes and immunohistochemistry using monoclonal antibodies against LMP1, EBNA2, BZLF1 protein, p53 protein and bcl-2 protein were performed on 56 primary nasopharyngeal carcinomas. EBERs was detected in 46 cases (82%), and LMP in 17 cases (30%), but EBNA2 was not detected. While 30 of 32 cases (94%) in differentiated non-keratinizing carcinoma (NKC) and 16 of 17 cases (94%) in undifferentiated carcinoma (UNPC) showed EBERs, neither 5 cases of squamous cell carcinoma (SCC) nor 2 cases of adenocarcinoma showed EBERs. This finding confirms latent infection of EBV, especially phenotypical latency II, in NKC and UNPC but not in SCC. Bcl-2 protein was positive in 50 cases (89%), but its expression did not depend on expression of LMP1, which did not demonstrate induction of bcl-2 by LMP1 as seen in vitro. Cytoplasmic BZLF1 expression was detected in 18 cases (32%) whereas BHLF was positive only in 6 cases (11%). This suggests dysfunction of BZLF1, which disrupts viral latency despite its expression. p53 protein was positive in 31 cases (55%), and there was a distinct correlation between expression of BZLF1 and p53 protein (p < 0.001). This suggests that the interaction between BZLF1 protein and p53 protein, which inactivate each other, is one of the tumorigenic factors in NPC.