M. H. Ng

Sustained elevation of Epstein-Barr virus antibody levels preceding clinical onset of nasopharyngeal carcinoma

We have monitored Epstein–Barr virus (EBV) IgA antibody levels of 39 nasopharyngeal carcinoma (NPC) cases for up to 15 years before clinical onset of NPC, and assessed preclinical serologic status of another 68 cases. Our results identify a serologic window preceding diagnosis when antibody levels are raised and sustained. This window can persist for as long as 10 years, with a mean duration estimated to as 37±28 months. Ninety-seven of these 107 NPC cases exhibited such a window. Cases that did not may reflect individual antibody response to EBV. Serologic screening at enrollment identified those cases who had already entered the window and became clinically manifested earlier (median=28 months) than those who entered the window after enrollment (median=90 months). The former account for 19 of 21 cases diagnosed within 2 years of screening. Nasopharyngeal carcinoma risk levels among seropositive subjects were also highest during this period. Both prediction rates and risk levels declined thereafter; cases detected at later times were composed of increasing proportions of individuals who entered the serological window after screening. Our findings establish EBV antibody as an early marker of NPC and suggest that repeated screening to monitor cases as they enter this window has considerable predictive value, with practical consequences for cancer treatment.

Detection of subclinical nasopharyngeal carcinoma by fibreoptic endoscopy and multiple biopsy.

Of 6054 people who had high titres of antibodies against the viral capsid antigen of Epstein-Barr virus but no symptoms or signs of nasopharyngeal carcinoma as assessed by conventional methods, 130 were randomly recruited and examined by fibreoptic endoscopy and biopsy of several sites of the nasopharynx. 7 cases of nasopharyngeal carcinoma were detected. The tumours were largely confined to the pharyngeal recess, which suggests that it is the area of the nasopharynx most prone to the development of the tumour. Tumour was found in both recesses in 1 subject, who also had evidence of transition from severe epithelial dysplasia to carcinoma in a sample from the left roof, which suggests that the disease was multifocal in origin. This study showed that endoscopy and biopsy of several sites of the nasopharynx are more effective than the conventional approach in the detection of subclinical nasopharyngeal carcinoma among seropositive individuals at high risk of the disorder.

Transcription of BamHI-A region of the EBV genome in NPC tissues and B cells.

Analysis by Northern blotting and sequencing of cDNA clones from a transcription library of a tumor biopsy from a nasopharyngeal carcinoma (NPC) patient showed that the BamHI-A region of the Epstein-Barr virus genome is abundantly and regularly transcribed in tumor tissues from NPC patients. The transcription occurred in a rightward direction terminating between coordinates 160,965 and 160,995, where two polyadenylation sites are located. Rightward transcription of this region also occurred in B lymphoid cells harboring the viral genome, albeit at a lower level than in the tumor tissues. Differential splicing yields a family of related transcripts displaying at least four splicing patterns. Different promoters may be utilized, further contributing to the diversity of this family of transcripts. A 2.8-kb unspliced transcript present in B95-8 cells was probably initiated from a TATA box located in position 158,204, while the other transcripts may utilize other promoters localized to other regions. All the transcripts encompass a putative open reading frame, BARFO, which is predicted to encode a basic protein of about 20 kDa. It shares 40% colinear amino acid sequence homology with the DNA binding region of a transcription factor, ICP4, specified by herpes simplex virus.

EVIDENCE THAT RESPIRATORY TRACT IS MAJOR RESERVOIR FOR EPSTEIN-BARR VIRUS

Exfoliated cells harvested from bronchial washings of 53 patients with suspected bronchogenic carcinoma were tested by means of DNA dot hybridisation using the cloned large internal repeat (IR) sequence of Epstein-Barr virus (EBV) genome as a probe. 25 of these patients gave positive results. Since the patients had diseases that were not related to the virus, this finding suggests that the lower respiratory tract is a major reservoir for EBV. Attempts at cellular localisation of the virus revealed only an occasional cell which harboured the viral genome or expressed viral capsid antigens. These cells could not account for the quantity of the viral DNA detected in bronchial washings. Moreover, patients had similar profiles of serum EBV antibodies whether they were positive or negative for EBV DNA by dot hybridisation. These findings are compatible with a state of viral latency in which cells harbour a low copy number of the viral genome. Viral expression rarely occurs in these cells, which seem to elicit a minimum host immune response. If it is assumed that each latently infected cell harbours a maximum of approximately 30 EBV genomes (which is the lower limit of detection by the in-situ hybridisation method used in this study), the findings suggest that a considerable proportion of the exfoliative cells from the lower respiratory tract, of the order of 0.1-16%, harbour latent EBV.

Epstein-Barr virus expression within keratinizing nasopharyngeal carcinoma.

Three stages of maturation can be seen in keratinizing nasopharyngeal carcinomas. These stages are similar morphologically to basal cells, intermediate and superficial squamous cells seen in normal squamous epithelium. Taking advantage of such a diverse tumour cell population, 10 keratinizing nasopharyngeal carcinoma (NPC) were examined by in situ hybridization for the presence of latent Epstein‐Barr Virus (EBV) using EBV encoded RNAs (EBERs) and by immunohistology for the presence of EBV early antigen‐diffuse (EA‐D) and the 350/220 kd membrane glycoprotein of the EBV. The basal cell‐like tumour cells are mainly infected latently with the virus; viral replication was found in isolated intermediate squamous cells, whilst superficial squamous cells are largely depleted of all the viral markers. We used a control series of non‐keratinizing nasopharyngeal carcinomas composed of undifferentiated and poorly differentiated tumour cells and EBV latency was present in these tumours. Viral replication was detected by RT‐PCR, in the undifferentiated tumours but viral replication was not seen by immunohistology. The possible relationship between EBV life cycle in these tumours and tumour cell differentiation is discussed in the light of these findings. J. Med. Virol. 55:227–233, 1998.

Single-tube nested PCR in the diagnosis of tuberculosis.

AIMS: To evaluate the usefulness of a single-tube nested polymerase chain reaction (PCR) assay in the diagnosis of tuberculosis in 1497 pulmonary and 536 extrapulmonary specimens. METHODS: A single-tube nested PCR, utilising two sets of primers with different melting temperatures (88 degrees C for external primers; 70 degrees C for internal primers) to augment sensitivity and specificity without increasing the risk of amplicon contamination, was evaluated. Specimens were initially tested for the repetitive IS6110 sequences and if negative, retested for the universal 38 kilodalton sequence and for inhibitors. dUTP/Uracil-N-glycosylase and Instagene treatment were used to minimise contamination and the effect of inhibitors, respectively. RESULTS: Using culture as the gold standard, the overall sensitivity of the assay was 89% for pulmonary and 42% for extrapulmonary specimens. Sensitivity varied greatly with respect to sample type (92% for follow up specimens from a chest hospital and 70% for non-follow up specimens from a general hospital). The smear positivity rates were 15% for extrapulmonary specimens, and 69% and 45%, respectively, for follow up and non-follow up specimens from pulmonary sites. Specificity was 99.7%. Inhibitors were present more frequently in extrapulmonary than in pulmonary specimens (13.4% v 2.7%). CONCLUSION: Despite the high sensitivity of the PCR assay for the diagnosis of tuberculosis in pulmonary specimens, it was less effective in the extrapulmonary samples. This is probably because of the lower bacterial load in extrapulmonary specimens, the presence of more inhibitors adversely affecting the PCR assay and the higher volume of specimens used for culture.

Epstein-Barr Virus (EBV) DNA in Sera of Patients with Primary EBV Infection

ABSTRACT Detection of Epstein-Barr Virus (EBV) DNA by PCR in serum had a sensitivity of 80%, a specificity of 94%, and positive and negative predictive values of 95 and 79%, respectively, for the diagnosis of primary EBV infection. We suggest that this is a useful addition to the panel of tests used for this purpose.

LMP1 of Epstein-Barr virus induces proliferation of primary mouse embryonic fibroblasts and cooperatively transforms the cells with a p16-insensitive CDK4 oncogene

ABSTRACT The latent membrane protein LMP1 of Epstein-Barr virus (EBV) is often present in EBV-associated malignancies including nasopharyngeal carcinoma and Hodgkins lymphoma. Previous work demonstrates that the LMP1 gene of EBV is sufficient to transform certain established rodent fibroblast cell lines and to induce the tumorigenicity of some human epithelial cell lines. In addition, LMP1 plays pleiotropic roles in cell growth arrest, differentiation, and apoptosis, depending on the background of the target cells. To examine the roles of LMP1 in cell proliferation and growth regulation in primary culture cells, we constructed a recombinant retrovirus containing an LMP1 gene. With this retrovirus, LMP1 was shown to stimulate the proliferation of primary mouse embryonic fibroblasts (MEF cells). It has a mitogenic activity for MEF cells, as demonstrated by an immediate induction of cell doubling time. In addition, it significantly extends the passage number of MEF cells to more than 30 after retroviral infection, compared with less than 5 for uninfected MEF cells. Furthermore, LMP1 cooperates with a p16-insensitive CDK4 R24C oncogene in transforming MEF cells. Our results provide the first evidence of the abilities of the LMP1 gene, acting alone, to effectively induce the proliferation of primary MEF cells and of its cooperativity with another cellular oncogene in transforming primary cells.

Factors affecting serum IgA antibody to Epstein-Barr viral capsid antigens in nasopharyngeal carcinoma.

Irrespective of the ethnic origin of the patient, nasopharyngeal carcinoma (NPC), appears to stimulate the production of IgA antibodies against VCA. These antibodies are detected at high frequency and titres in sera from NPC patients but only rarely from control subjects. A majority of relapse-free survivors tested 1-12 years after radiotherapy (RT) sustain a detectable level of IgA anti-VCA. Serum titres of IgA anti-VCA remain relatively unchaged in individual NPC patients after RT, regardless of the disease evolution. These antibodies were detected in serum from one individual 9 months before NPC and the titre rose concomitantly with its clinical onset. Titres of IgA anti-VCA in multiple serum specimens from individual NPC patients, and in sera from different NPC patients, do not correlate with titres of IgG anti-VCA or with Serum IgA. It thus seems possible that the IgA anti-VCA in the sera of NPC patients might be largely derived near NPC. Apparently healthy individuals showing detectable IgA anti-VCA tend to aggregate in families of NPC patients. The distribution of siblings of these families who have the IgA anti-VCA reaction shows the binomial distribution expected for an autosomal recessive trait, implying the involvement of an autosomal recessive gene in the IgA anti-VCA response.

Immunohistochemistry of local immunoglobulin production in nasopharyngeal carcinoma.

Immunohistochemical investigations by the immunoperoxidase method have been carried out on sections of biopsy specimens obtained from the primary tumour sites of patients with nasopharyngeal carcinoma (NPC). It was found that, in many of the sections thus examined, there was an accumulation of plasma cells, particularly of the IgA type, in the connective tissues surrounding nests of NPC cells. Similar accumulation of plasma cells in the subepithelial connective tissues was likewise observed in a case of choanal polyp. Plasma cells were rarely observed in the section of a biopsy specimen of non-neoplastic oropharyngeal mucosa. These results indicated that the nasopharynx may be a site for the local production of IgA, but the antigenic specificity of these molecules is, however, not known. The possibility that the nasopharynx is a site for the local production of antibodies to the Epstein-Barr virus (EBV)-related antigens was discussed.