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Background: Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). The metastasis to cervical lymph nodes represents a frequent initial manifestation of NPC. The usefulness of EBV detection by polymerase chain reaction (PCR) in the diagnosis of occult NPC with cervical metastasis has been reported. Our previous study showed that EBER1 in-situ hybridization was somewhat more sensitive and specific than PCR in detecting EBV in the evaluation of specimens from a population at high risk for NPC., Methods: Fine-needle aspiration cytologic specimens of neck masses from 30 patients were investigated, including 10 NPC primary tumors, 19 squamous cell carcinomas from other sites of the head and neck (9 oral cavity, 2 paranasal sinuses, 2 oropharynx, 3 larynx, and 3 hypopharynx), and 1 diffuse large-cell lymphoma. EBER1 in-situ hybridization was performed on direct smears made from aspirates., Results: EBER1 signals were detected in all neck metastases from the nasopharynx but none of the specimens from other primary sites., Conclusions: This study suggests that EBER1 in-situ hybridization can be used as a supplemental tool for differential diagnosis whenever fine-needle aspiration cytologic examination is presented with a neck metastasis without knowing the primary site., (Copyright 2000 John Wiley & Sons, Inc.)

Epstein-Barr virus (EBV) has a well-established association with nasopharyngeal carcinoma (NPC), while its association with sinonasal carcinoma is still controversial. The role of EBV in sinonasal carcinoma and the potential value of EBER is situ hybridization in differentiating carcinomas originating from these anatomically neighboring regions were investigated. EBER in situ hybridization was applied to detect EBV in the specimens of surgically proved sinonasal carcinoma and the biopsies of carcinoma of the nasopharynx. EBER in situ hybridization was performed on paraffin embedded tissues by using PCR-derived, digoxigenin-labeled EBER-1 DNA probes. EBV was detected in 2 of 31 (6.5%) surgical specimens of sinonasal carcinoma, including 1 keratinizing squamous cell carcinoma and 1 adenocarcinoma. EBV was detected in all of 31 (100%) biopsies of carcinomas of the nasopharynx, including 1 keratining squamous cell carcinoma, 15 nonkeratinizing carcinomas, 14 undifferentiated carcinomas, and 1 adenocarcinoma. The present study does not support the role of EBV in the development of sinonasal carcinoma as does in carcinoma of the nasopharynx. EBER in situ hybridization is a useful adjunct for differentiating carcinomas originating from the sinonasal region and the nasopharynx.

The mainstay treatment of nasopharyngeal carcinoma (NPC) is radiotherapy. Although NPC is a radiosensitive tumor, 25% of NPC patients still suffered from local relapse in Taiwan. Whether the pathologic changes induced by radiation have impact on the interpretation of suspicious recurrent NPC specimens is seldom mentioned. We retrospectively analyzed 20 irradiated NPC patients who were suspected to have local recurrence and received serial nasopharyngeal biopsies. A total of 31 nasopharyngeal tissues including 15 local recurrent specimens and 16 negative specimens were entered into this study. Compared with non-irradiated nasopharyngeal tissue, the irradiated nasopharynx showed fibrosis and areas of necrosis mixed with acute or chronic inflammation. Stroma cells were enlarged, with prominent nuclei and cytoplasm, and some bizarre cells might be confused with tumor cells. These post-irradiation effects did have an impact on histological interpretation. In this study, 2 out of 16 nasopharyngeal tissues were misdiagnosed as having no recurrent tumor by using H and E stain only. However, scattered cancer cells can be clearly visualized by using EBER1 in situ hybridization. Therefore, it is recommended that EBER1 in situ hybridization is routinely used in irradiated nasopharyngeal tissues in helping the diagnosis of recurrent NPC.

Background: Epstein-Barr virus (EBV) infection in nonsmall cell lung carcinoma (NSCLC) has been demonstrated in some ethnic groups. The pathobiology and the role of EBV and oncoprotein expression in these tumors have not been studied extensively. In this study, the authors investigated EBV-encoded RNA-1 (EBER1) transcripts by in situ hybridization and the expression of latent membrane protein-1 (LMP-1) and bcl-2 protein by immunohistochemistry in NSCLC patients from Taiwan, where nasopharyngeal carcinoma is endemic., Methods: A total of 127 cases of NSCLC (43 cases of squamous cell carcinoma [SCC], 67 cases of adenocarcinoma [AD], 12 cases of large cell carcinoma [LCC], and 5 cases of lymphoepithelioma-like carcinoma [LE]) were included. A sensitive polymerase chain reaction-derived, digoxigenin-labeled DNA probe for in situ detection of EBER1 transcripts was performed for the detection of EBV. Immunohistochemistry using the avidin-biotin-immunoperoxidase method was also performed to evaluate the expression of bcl-2 and LMP-1., Results: EBER1 was detected in 11 of the 127 NSCLC cases (8.7%; 6 SCC cases and 5 LE cases). All 5 LE cases were EBV-positive, whereas only 6 of the 43 SCC cases (14%), 0 of 67 AD cases, and 12 LCC cases were EBV-positive (P < 0.05). All five LE cases showed diffuse, strong, positive staining of tumor cells; five of the six SCC cases showed diffuse but weak staining. Among the nontumor epithelial cells, there was no EBER1 staining of any of the 11 EBER1-positive cases. The mean age of the LE patients was 10 years younger than that of the patients with other histological types. All 5 LE patients were nonsmokers, whereas 3 of the 6 patients with EBER1-positive SCC (50%) were smokers. EBER1 expression did not correlate with the 2-year survival rate of overall cases, but all 5 LE patients were alive without clinical evidence of disease at last follow-up. Gender, lymph node or distant metastasis, and clinical stage were not found to have any correlation with EBER1 expression (P > 0.05). All LE cases had bcl-2 oncoprotein expression (100%). This frequency was significantly different from other histologic types (P < 0.05). The LMP-1 detection rate was low and demonstrated no correlation with bcl-2 expression., Conclusions: In this study, the authors found that the primary LE of the lung is associated with young age, a history of not smoking, high bcl-2 immunoreactivity, and better survival rate. These characteristics demonstrate that EBV-associated LE of the lung is a unique entity. The findings of the current study suggest that EBV infection may play a different role in the tumorigenesis of primary LE of the lung than it does in other EBER1-positive NSCLCs.

Background: Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). Detection of EBV in biopsy specimens may serve as a tumor marker., Methods: To assess the sensitivity and specificity of the polymerase chain reaction (PCR) and in situ hybridization in the diagnosis of NPC, formalin fixed, paraffin embedded nasopharyngeal biopsies from patients in Taiwan suspected of having NPC were studied., Results: In specimens from 107 patients with NPC, EBV was detected by PCR in 97 cases (90.7%) and by EBER in situ hybridization in 105 cases (98.1%). In specimens from 61 patients without neoplasia, EBV was detected by PCR in 7 cases (11.5%) and by EBER in situ hybridization in 0 cases., Conclusions: These results suggest that although EBV DNA may occasionally be detected in nonneoplastic nasopharyngeal tissues, cells expressing EBER are not. EBER in situ hybridization may therefore prove to be a useful adjunct in the diagnosis of NPC.

Background: The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma (NPC) is well documented. Previous studies reported abundant expression of EBER1 in primary NPC and tumors metastatic to lymph nodes. However, a large series of case studies correlating World Health Organization (WHO) histologic subtypes with EBER1 is needed., Methods: The authors applied the EBER1 in situ hybridization to investigate the expression of EBER1 in 140 primary NPCs, 11 metastatic tumors to lymph nodes, 6 metastatic tumors to bone marrow, and 2 metastatic tumors to the liver. All 19 metastatic tumors had paired specimens from their primary NPCs for comparison. The in situ hybridization method was performed on paraffin embedded tissues by using polymerase chain reaction-derived, digoxigenin-labelled EBER1 DNA probes., Results: The EBER1 signal was identified in nuclei of malignant epithelial cells in 135 of 140 (96.4%) primary NPCs, including 4 of 5 (80%) WHO-I histologic subtypes, 71 of 73 (97.3%) WHO-II histologic subtypes, and 60 of 62 (96.8%) WHO-III histologic subtypes (P > 0.05). However, the positive hybridization signal in WHO-I NPC was less in proportion to malignant cells, usually limited to basal cells, than in other histologic types of NPC. In 10 of 11 specimens with metastases to the lymph nodes, hybridization was always limited to the malignant cells and not associated with lymphocytes. All 10 paired primary NPCs also demonstrated positive EBER1 hybridization. Only one paired specimen showed negative EBER1 in primary NPC and a metastases to a lymph node. Eight distant metastases, all EBER1-positive in their primary NPC, also demonstrated positive EBER1 hybridization signals in the malignant cells. The proportion of EBER1 positivity in metastatic NPC is higher than that in primary lesions as observed in paired specimens from the same patient., Conclusions: Because of abundant expression of EBER1 in primary NPC as well as in metastatic malignant cells, it is recommended that EBER1 in situ hybridization be performed on routinely processed specimens whenever NPC is suspected.

A PCR-derived digoxigenin-labeled DNA probe was used for for Epstein-Barr early RNA (EBER) in situ hybridization in formalin-fixed paraffin-embedded tissues. The results showed that the hybridization signal was morphologically distinct and the intensity of signal was comparable with those by RNA riboprobe. The advantages of using PCR-derived DNA probes for EBER in situ hybridization include: (1) the synthesis of digoxigenin-labeled DNA probes is easy and simple by PCR; (2) the labeled amplification product can be used as a probe without further purification; (3) DNA probes are potentially more stable than RNA probes; and (4) the preparation of DNA probes is relatively efficient and rapid. It is concluded that this technique is an ideal candidate for detection of EBER expression in clinical specimens.