Joseph C. K. Lee

Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus

We have established a cell line (C666‐1) from undifferentiated nasopharyngeal carcinoma (NPC). This cell line consistently carries the Epstein‐Barr virus (EBV) in long‐term cultures. C666‐1 is a subclone of its parental cell line, C666, derived from an NPC xenograft of southern Chinese origin. It grows as an adherent culture and lacks contact inhibition. In addition, it is tumorigenic in athymic nude mice. The cells consistently express EBV‐encoded RNAs and are positively stained for cytokeratin, an epithelial marker. In addition, they express EBNA1 protein, LMP1 and LMP2 transcripts and thus resemble the EBV latency II pattern. The virus genotype is EBV‐1 with the latent membrane protein 1 gene showing a 30‐bp deletion at the carboxyl terminus, both consistent with findings in southern Chinese NPC tumours. Cytogenetic analysis revealed a sub‐diploid status with a chromosomal modal number of 45. C666‐1 is unique among NPC cell lines in that it carries EBV. These cells may serve as a good investigative tool as the viral latency pattern and genotype are observed in the majority of primary NPC biopsies from Chinese patients. Int. J. Cancer 83:121–126, 1999.

High frequency of P16INK4A gene alterations in hepatocellular carcinoma

The tumor suppressor gene p16 (CDKN2/MTS-1/INK4A) is an important component of the cell cycle and inactivation of the gene has been found in a variety of human cancers. In order to investigate the role of p16 gene in the tumorigenesis of hepatocellular carcinoma (HCC), 48 cases of HCC were analysed for p16 alterations by: methylation-specific PCR (MSP) to determine the methylation status of the p16 promoter region; comparative multiplex PCR to detect homozygous deletion; PCR – SSCP and DNA sequencing analysis to identify mutation of the p16 gene. We found high frequency of hypermethylation of the 5′ CpG island of the p16 gene in 30 of 48 cases (62.5%) of HCC tumors. Moreover, homozygous deletion at p16 region were present in five of 48 cases (10.4%); and missense mutation were detected in three of 48 cases (6.3%). The overall frequency of p16 alterations, including homozygous deletion, mutation and hypermethylation, in HCC tumors was 70.8% (34 of 48 cases). These findings suggest that: (a) the inactivation of the p16 is a frequent event in HCC; (b) the p16 gene is inactivated by multiple mechanisms including homozygous deletion, promoter hypermethylation and point mutation; (c) the most common somatic alteration of the p16 gene in HCC is de novo hypermethylation of the 5′ CpG island; and (d) in contrast to other studies, high frequency of genomic alterations are not uncommon in the 9p21 of the p16 gene. Our results strongly suggest that the p16 gene plays an important role in the pathogenesis of HCC.

Detection of recurrent chromosomal gains and losses in primary nasopharyngeal carcinoma by comparative genomic hybridisation

Nasopharyngeal carcinoma (NPC) is a common cancer in Southern China but rare in Western countries. To search for genetic alterations in NPC, we examined a series of 20 primary tumours with comparative genomic hybridisation. The identified common chromosomal alterations included gain of chromosomes 1q, 8, 12, 19 and 20 as well as loss of chromosomes 1p, 3p, 9p, 9q, 11q, 13q, 14q and 16q. In concordance with our previous loss of heterozygosity studies in primary NPC, a high incidence of loss was detected on chromosomes 3p (75%), 11q (70%) and 14q (65%). Losses of 9q (60%), 13q (50%) and 16q (40%) were also identified. Novel chromosomal gains were observed on chromosome 12, with a high frequency (70%). Current analysis has revealed a comprehensive profile of the chromosomal regions showing losses and gains in primary NPC. Our findings may provide an entry point for conducting further investigations to locate the putative tumour‐suppresser genes and oncogenes that may be involved in the tumourigenesis of NPC. Int. J. Cancer 82:498–503, 1999.

Loss of heterozygosity on the short arm of chromosome 3 in nasopharyngeal carcinoma

A consistent loss of constitutional heterozygosity within a specific chromosome locus in a tumor type is suggestive of a tumor suppressor gene important in the genesis of that tumor. We studied whether such genetic alterations are involved, in the development of nasopharyngeal carcinoma (NPC). Tumor and matched blood leukocytes DNA from eleven Hong Kong Chinese patients with primary NPC stages I to IV were subjected to restriction fragment length polymorphism (RFLP) analysis using chromosome 3-specific polymorphic probes. Such probes are assigned to chromosomal region 3p25 (RAF-1), 3p24-22.1 (ERBA beta), 3p21 (DNF15S2), 3p14 (D3S3), and 3q12 (D3S1). The breakpoint varied among tumors, ranging in extent from 3p21-14. However, 100% frequency of complete loss of heterozygosity was observed at two chromosomal loci: RAF-1 locus (ten of ten cases at 3p25) and D3S3 locus (nine of nine cases at 3p14), in all evaluable NPC patients, suggesting the presence of putative tumor suppressor gene(s) within or close to these defined regions. The observed consistent deletion of alleles on the short arm of chromosome 3 in the NPC cases, which is in line with our previously reported and present cytogenetic findings, may represent a critical event in the multistep genesis of NPC. The present report also identifies defined loci for linkage studies on NPC families.

Specific latent membrane protein 1 gene sequences in type 1 and type 2 Epstein‐Barr virus from nasopharyngeal carcinoma in Hong Kong

We reported previously that a characteristic Epstein‐Barr virus latent membrane protein 1 (EBV‐LMP1) gene was associated with nasopharyngeal carcinoma (NPC) in Hong Kong. It showed a 30 bp deletion at the carboxyl terminus with specific amino acid substitution Asp at codon 335 with reference to Gly in B95‐8 LMP1. This deletion variant Asp335 was present in over 90% of NPC biopsy specimens. The present study attempted to determine the whole encoding sequence of the LMP1 gene in different EBV isolates from NPC, and its relation with EBV types. We found that 92% (34/37) of primary NPC tumours harboured EBV‐1 and possessed the LMP1 deletion variant, of which 86% were Asp335 and 6% were Gly335. EBV‐2 was present in 8% (3/37) of tumours and all contained the retention variant of the LMP1 gene. Sequencing of the whole encoding region of the LMP1 gene revealed that the deletion variant Asp335 and deletion variant Gly335 carried similar sequences. They showed 43 common nucleotide substitutions in 41 codons with reference to B95‐8. The retention variant showed 52 base changes in 46 codons compared with B95‐8. The amino acid alterations in both the deletion and retention variants were mostly clustered at the transmembrane domain of the protein. Furthermore, half of the substitutions were common to both variants, suggesting a common evolutionary selection pressure. Nonetheless, the 2 LMP1 variants showed differences in nucleotide alterations and were associated with different EBV types, suggesting the presence of 2 distinct EBV strains in Hong Kong NPC. Int. J. Cancer 76:399–406, 1998.© 1998 Wiley‐Liss, Inc.

Plasma Cell-free Epstein-Barr Virus DNA Quantitation in Patients with Nasopharyngeal Carcinoma: Correlation with Clinical Staging

Nasopharyngeal carcinoma (NPC) is an important cancer in southern China and Southeast Asia.1 In Hong Kong, nearly all NPC cases are undifferentiated or poorly differentiated and harbor Epstein-Barr virus (EBV) in tumoral tissues.1 The recent interest in the presence of tumor-derived DNA in the plasma and serum of cancer patients has prompted our group2 and Mutirangura et al.3 to look for EBV DNA in the plasma and serum of NPC patients. Our preliminary data indicate that this approach is sensitive and specific for NPC.2 Our core technology for EBV DNA detection is real-time quantitative PCR,4 which is able to provide a quantitative measurement of the concentrations of EBV DNA in the plasma of NPC patients. In this communication, we report our latest results based on an expanded sample size, thus allowing a better understanding of the relationship between circulating EBV DNA and NPC staging.

Quality of Life of Palliative Care Patients in the Last Two Weeks of Life

Quality of life (QOL) is the main consideration in caring for advanced cancer patients, yet little is known about the QOL in the terminal phase. We profiled the QOL of 58 advanced cancer patients during their last 2 weeks of life using the McGill QOL questionnaire-Hong Kong version. The patients provided ratings of QOL an average of 5.6 (median 6) days pre-death. Palliative care services were successful in maintaining the total QOL score during the dying phase. The mean score was 7.0 of 10. Among the various domains, the physical and existential domains scored relatively poorly at 5.9 and 6 of 10, respectively. The worst physical symptom and meaning of life were the individual items with the poorest scores (4.8 and 5.4 of 10, respectively). Compared with admission, there was statistically significant improvement in the worst physical symptom (P = 0.02) and eating item (P = 0.002), but deterioration in physical well-being (P = 0.03), meaning of existence (P = 0.007), and satisfaction with oneself (P = 0.04). In conclusion, QOL evaluation during the terminal phase identifies important aspects requiring improvement during the last two weeks of life. Physical and existential domains of dying cancer patients needed more attention.

Frequent allelic loss on chromosome 9 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and highly associated with chronic virus‐B or ‐C infection and cirrhosis. Molecular studies have shown high frequency of loss of heterozygosity (LOH) in some specific chromosome regions, but LOH on chromosome 9 in HCC has not been thoroughly investigated. In our investigation of chromosome 9 with 19 polymerase‐chain‐reaction (PCR)‐based polymorphic microsatellite markers, 30 of 48 HCC tissue samples (63%) had LOH, and a distinct common deletion region and a region of loss were identified. The first region was located at the 9p21 region and the minimal deletion region was located between loci D9S1747 and D9S1748. This is a region of approximately 200 kb which includes the p16 tumor‐suppressor gene. A region of loss was located on 9p13 to 9q33. The putative tumor‐suppressor gene for nevoid‐basal‐cell‐carcinoma syndrome (NBCCS) at 9q22.3 resides within this region. In addition to LOH, 4 HCC cases showed possible homozygous deletions at 9p21 with markers D9S1748, D9S1752 and D9S171 by multiplex PCR analysis. In 3 cases, the minimal region of possible homozygous deletion was approximately 300 kb and was defined between markers D9S1747 and D9S1752. Since this deletion region includes both the p15 and the p16 tumor‐suppressor genes, these genes were possibly inactivated by homozygous deletion in HCC. In addition, a second region of possible homozygous deletion was present on the centromeric side of 9p21. However, these changes are not associated with age, gender, size or tumor‐cell differentiation. Our data also suggest that inactivation of the p16 and the p15 genes and the possibility of other unknown tumor‐suppressor genes located on these defined deleted regions of chromosome 9 may be involved in the pathogenesis of HCC. Int. J. Cancer 81:319–324, 1999.

Prevalence and prognostic significance of tumor-associated tissue eosinophilia in nasopharyngeal carcinoma

Tumor‐associated tissue eosinophilia (TATE) has been associated with an improved prognosis in a variety of neoplasms.

Telemedicine in China

Telemedicine has been shown to have a considerable impact in medical education, conferencing and consultation. As a result, the Peoples Republic of China has been keen to develop telemedicine. In her attempts to further the development of telemedicine, China has looked to the progress of medical services in Western countries such as Europe and North America. The United States of America, however, has exceeded the rest in exchange of health-care information and telemedicine technologies with China. Although China has been enthusiastic about the exchange, telemedicine in China requires development in the technical infrastructure and professional infrastructure.