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.

Association of Epstein-Barr virus with lymphoepithelioma-like carcinoma of the lung in southern China.

Having reviewed the data on 3,663 consecutive cases of primary lung carcinoma in southern China, we found that 32 cases could meet the criteria for lymphoepithelioma-like carcinoma (LELC) of the lung. To study the relationship between pulmonary LELC and Epstein-Barr virus (EBV) infection, we used in situ hybridization and immunohistochemistry techniques to detect the EBV-encoded small nonpolyadenylated RNA (EBER), latent membrane protein 1 (LMP1), and viral capsid antigen (VCA) in 32 cases of LELC and 19 cases of non-LELC lung carcinoma. Of the 32 cases, 30 (94%) showed EBER positivity. Of the 30 EBER-positive pulmonary LELC cases, 16 and 7 expressed LMP1 and VCA, respectively. In contrast with LELC, none of the 19 cases of non-LELC lung carcinoma showed EBER-, LMP1-, or VCA-positive signals in carcinoma cells. The results demonstrate that there is a close relationship between EBV infection and pulmonary LELC. EBV infection may have an essential role in the tumorigenesis of pulmonary LELC. EBV latent infection is the main status in pulmonary LELC except for individual EBV entering into a lytic cycle.

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 nonkeratinizing 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.

p53 mutational spectrum of esophageal carcinomas from five different geographical locales in China.

A mutational spectrum for exons 5-8 of the p53 tumor suppressor gene in esophageal carcinomas in mainland China and Hong Kong was established. This study involved 209 squamous cell carcinoma specimens obtained from five different geographical locales in China: Zhengzhou, Taiyuan, Shantou, Guangzhou, and Hong Kong. Zhengzhou and Shantou were high-incidence regions for esophageal cancer, whereas the other three regions had low or intermediate incidence of the disease. Analysis by single-strand conformation polymorphism and DNA sequencing showed that 87 specimens (41.6%) contained mutations in exons 5-8 of the p53 gene compared to 163 cases (78%) that had accumulation or aberrant expression of the protein, as detected by immunohistochemical staining. Point mutations accounted for 80.4% (87/107) of all genetic changes. The specimens from northern China exhibited fewer p53 gene aberrations and a more even distribution of mutations in exons 5-8 compared to those from southern China in which 60% of all mutations were found in exon 5. A major hot spot was found at codon 176 in exon 5, where 41 samples from Shantou, Guangzhou, and Hong Kong had a G-->T transversion. It is likely that among southern Chinese this codon is susceptible to mutagenesis by carcinogens. Codons 175, 203, 245, 250, 273, and 282 were also shown to be mutational hot spots, with three or more mutations observed at each site. The p53 mutational data obtained in this study showed that Chinese esophageal carcinomas are often associated with some unique genetic alterations, which may be attributed to specific dietary or environmental carcinogens that affect the Chinese but not Caucasians.

[Study on the role of EB virus in carcinogenesis of nasopharyngeal carcinoma by use of in situ hybridization].

Cryosections of 45 nasopharyngeal carcinomas and 9 non-tumorous biopsies were studied for EBV DNA by use of in situ hybridization. No EBV DNA was detected in all types of non-epithelial cells. One to three copies of EBV genome as shown by Namalwa cells could occasionally be found in a small number of normal, hyperplastic, squamously metaplastic and dysplastic epithelial cells. A considerable number of neoplastic cells in 6 para-tumourous carcinoma in situ foci contained numerous EBV genome copies as shown by Raji cells. The majority of infiltrating cancer cells of 42 non-keratinizing carcinomas and 3 keratinizing squamous cell carcinomas also exhibited quite a number of EBV genome copies. The above findings illustrate that EBV may have a promotive effect during the early stage just after the malignant conversion of epithelial cells to increase the proliferative rate of carcinoma cells.

Infiltrating lymphocytes and accessory cells in nasopharyngeal carcinoma.

The infiltrating lymphocytes (LCs) and accessory cells (ACs) including dendritic cells (DCs) and monocytes/macrophages in nasopharyngeal biopsies taken from 4 groups of nasopharyngeal carcinoma (NPC) patients were observed by using an immunostaining technique and the correlation of the results to the clinical manifestations and follow-up data was examined. The findings were as follows. (1) NPCs without lymph node metastasis always had marked infiltrating LCs and DCs as compared with those with lymph node(s) metastasis. (2) Advanced NPCs with lymph node(s) involvement (T1-4N1-3M0) and a rapid development of distant metastasis followed by death within 1 year after radiotherapy always showed fewer infiltrating LCs and DCs as compared with those with lymph node(s) metastasis (T1-4N1-3M0) and having longer than 5-year survival after radiotherapy. The amount of both LCs and ACs, especially DCs, infiltrating in NPC tissues appears to be an indicator of the activity of host immune defence mechanisms against cancer and influences the progression of the neoplasm as well as the prognosis.

Epstein-Barr virus carriage by nasopharyngeal carcinoma in situ.

We studied the distribution of the EBV genome in tumour biopsies obtained from 42 patients with poorly differentiated or undifferentiated nasopharyngeal carcinoma (NPC) and 3 patients with well-differentiated NPC. Six carcinoma in situ (CIS) foci were seen in 5 tumour specimens. By in-situ hybridization, multiple copies of the EBV genome were detected in some of the tumour cells in 3 CIS lesions involving the full thickness of the mucosal epithelium, but without microinvasion, while the viral genome was present in the majority of the tumour cells contained in another 3 CIS lesions with microinvasion. In agreement with previous findings, poorly differentiated and undifferentiated carcinomas regularly carried the viral genome, the number of copies of which was similar to that seen in CIS, while some, but not all, of the tumour cells of the well-differentiated histological type carried the virus. The viral genome was otherwise rarely detected in other areas of the mucosal epithelium and, where present, the viral carriage was confined to a few epithelial cells, in which the viral genome contents were markedly lower than in tumour cells. These results suggest that EBV may first become associated with NPC at an early stage of the disease shortly after the tumour has been initiated.

[Morphometric study on nasopharyngeal carcinoma (NPC)].

Morphometric parameters of carcinoma cells in 78 NPC and columnar epithelium basal cells in 21 of these 78 NPCs were measured by Quantimet 520 analyser. The results showed that: 1. Prominent enlargement of nuclei was a pathognomonic feature of carcinoma cells, except small spindle carcinoma cells, as compared with the normal basal cells, 2. vesicular nucleus cell carcinoma and small spindle poorly differentiated squamous carcinoma had their own characteristic morphometric parameters as compared with those of the ordinary poorly differentiated squamous carcinoma, and 3. there was no significant morphometric difference between vesicular nucleus cell carcinoma and poorly differentiated adenocarcinoma.

[Nasopharyngeal carcinoma (NPC) mortality rate in two districts of Guangdong Province and their relation to IgA antibody level against EB virus capsid antigen].

Based upon the retrospective investigation data, the NPC mortality rate in the Pearl River Delta was found to be twice as high as that of Chaoshan district. The positive rate of IgA/VCA antibody in the Pearl River Delta was also found to be significantly higher than that in the Chaoshan district by viro-serological screening. The IgA/VCA antibody positive rate and mortality rate were concomitantly increased with age in the bracket of 30-59. Accordingly, the authors assume that activation of infected EB virus might be an important factor in the process of etiology-pathogenesis of NPC.

Immunoglobulin A against viral capsid antigen of Epstein-Barr virus and indirect mirror examination of the nasopharynx in the detection of asymptomatic nasopharyngeal carcinoma.

To evaluate the efficacy of population screening for early stage nasopharyngeal carcinoma (NPC) in southern China, the authors recruited 42,048 and 10,402 apparently healthy subjects residing in a high incidence and a low incidence area, respectively; all subjects were between the ages of 30 and 59 years. The subjects' serum specimens were tested for immunoglobulin (Ig) A antibody against viral capsid antigen (IgA/VCA) of Epstein-Barr virus (EBV). Of the subjects from the high incidence area, 2823 were found to be seropositive. In follow-up, they had yearly examinations of the nasopharynx by indirect mirror with or without biopsy; 41 were found to have histologically confirmed asymptomatic NPC during the first 2 years of follow-up. The tumors in most of these cases were localized and were at earlier stages than tumors of symptomatic cases of NPC seen in the same region before the screening. The yearly indirect mirror examination of the nasopharynx seems to have effectively identified most of the tumors at the stage of asymptomatic disease. The risk of harboring NPC was found to be different among the different sex and age subgroups of seropositive individuals. By limiting such screening to those who are at exceedingly high risk, the cost of the screening can be kept within the spending of the public health authority, and the effectiveness of the screening also is improved.

The efficacy of fiberoptic endoscopic examination and biopsy in the detection of early nasopharyngeal carcinoma.

Fiberoptic endoscopic examination and biopsy of the nasopharynx was done in 130 patients as a prospective study. They all had elevated titers of antibodies against the viral capsid antigen of Epstein-Barr virus but no symptoms or signs of nasopharyngeal carcinoma. Each underwent a biopsy from six fixed sites in the nasopharynx. Of the 780 biopsy specimens taken from seven patients, 11 showed the presence of nasopharyngeal carcinoma. Techniques to improve the yields of such biopsies with fiberoptic endoscopy are discussed. The presence of tumor was unrelated to the macroscopic endoscopic findings. The highest incidence of subclinical tumor is in the pharyngeal recess.

Nasopharyngeal carcinoma and lymphoinfiltration.

Originally referred to as 'lymphoepithelioma', undifferentiated and poorly differentiated nasopharyngeal carcinoma (NPC) tissues showed intense lymphoinfiltration. In a study of cryosections from 15 NPC tissues, we found that infiltrating lymphoid elements were comprised predominantly of lymphocytes, but plasma cells, follicular dendritic cells, and eosinophils were also commonly seen. Subpopulations of lymphocytes having the same phenotypes tend to aggregate, forming clusters or secondary follicles in stromatous tissues. The tumor areas were mainly infiltrated by T cells. Tumor cells and/or apparently normal epithelium in the paratumorous areas frequently expressed CD21, CD23, CD40 and a B lymphocytes carcinoma cross-reacting antigen (BLCa), all of which are involved in B cell activation and proliferation. CD21 and BLCa were strongly expressed near the surface of both squamous and columnar epithelium by those epithelial cells which are at advanced stage of differentiation, while CD40 was expressed by epithelial cells at earlier stages of differentiation located at or near the basement membrane. CD23 was mainly expressed by columnar cells and basal cells underlying squamous epithelium, but not, or weakly so, by flattened squamous cells or reserve cells underlying columnar epithelium. The large majority of tumor cells expressed CD40 and BLCa. A substantial proportion of them also expressed CD23, but the tumor cells were not reactive for CD21. Despite eosinophilic infiltration, IL-6 was not detected in tumor tissues. IL-1 was, however, detected in abundance in the cytoplasm of follicular dendritic-like cells and in the intercellular spaces in tumor areas and surrounding stromatous tissues. The immunobiology of NPC is discussed in the light of these observations.

[DNA content and its distribution pattern of poorly differentiated nasopharyngeal carcinoma measured by Feulgen cytophotometry].

Nuclear area and DNA content of the normal columnar cells and cancer cells which were small, fusiform, irregular, vesicular-nuclear, giant malformed-nuclear and giant vesicular-nuclear were measured by Leitz MPV-III microscope photometer with HP-85 microcomputer in 32 poorly differentiated nasopharyngeal carcinoma (NPC) and 5 chronic nasopharyngitis sections. Based on different DNA contents and distribution patterns, it was divided into 4 cancer cell populations: small, fusiform and/or irregular, vesicular-nuclear and giant tumor cell populations. It should be noted that the nuclear DNA pattern of vesicular-nuclear cancer cell population had pathognomonic characteristics. The nuclear unit DNA content ratio of vesicular-nuclear cancer cells to lymphocytes was under 0.4. This cancer cell population was sensitive to irradiation. If the biological characteristics of these four cancer cell populations could be clarified not only in nuclear DNA pattern but also in biochemistry at molecular level, it will be possible to design the different treatment trials on the different cancer cell populations, then resulting in better prognosis of NPC patients.

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.

A morphologic and follow-up study on the nasopharyngeal lymphoid hyperplasia and its relation to the cancer.

Fourty-three cases of nasopharyngeal lymphoid hyperplasia (NPLH) were studied morphologically and by follow-up during a period of 8.5 years in a high-incidence city of nasopharyngeal carcinoma (NPC). The authors suggest that NPLH might be recognized as a disease entity in adults. Based on the sites and gross appearances of the hyperplastic lymphoid tissue, two types of NPLH, namely, adenoids and nodular, could be distinguished. The histopathological changes of NPLH were described in detail, and its relation to the development of NPC was discussed. According to this study, the authors disagree with the opinion that NPLH is a precancerous lesion.

[A morphologic and follow-up study on nasopharyngeal lymphoid hyperplasia and its relation to cancer].

Forty-three cases of nasopharyngeal lymphoid hyperplasia (NPLH) were studied morphologically and had been followed-up for a period of 7.5 years in a high-incidence city of nasopharyngeal carcinoma (NPC). The authors suggest that NPLH might be recognized as a disease entity in adults. Basing on the sites and gross appearances of the hyperplastic lymphoid tissue, two types of NPLH, namely, adenoids and nodular might be distinguished. The histopathological changes of NPLH are described in detail and its relation to the development of NPC is discussed. According to the data of this study, the authors' opinion is that NPLH is not a precancerous condition.

[In situ lesion of para-nasopharyngeal carcinoma--microspectrophotometer study].

Eighteen tissue sections of in situ cancer of para-nasopharyngeal carcinoma (NPCIS) were assayed for nuclear area, DNA content, nucleolar area and RNA content using MPV III microspectrophotometer. In general, the nuclear area, DNA content, nucleolar area and RNA content in NPCIS cells were significantly increased as compared with those in the non-cancerous epithelial cells. The DNA-histogram showed aneuploidy in the majority of NPCIS. In the invasive carcinoma foci, the above-mentioned characteristics were more marked. The authors believe that NPCIS is a critical stage in the pathogenesis of NPC. In addition, the comparison is described as to the similarities and differences between the columnar and squamous cell types of NPCIS.