Affiliations of authors: P. K. S. Chan, J. L. K. Cheung, A. F. Cheng (Department of Microbiology), C.-W. Lam (Department of Chemical Pathology), T.-H. Cheung, K. W. K. Lo (Department of Obstetrics and Gynaecology), The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong SAR, China; W. W. H. Li, M. Y. M. Chan, Department of Obstetrics and Gynaecology, Queen Elizabeth Hospital, Hong Kong SAR, China.
Correspondence to: Paul K. S. Chan, MRC Path, Department of Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China (e-mail: paulkschan{at}cuhk.edu.hk).
ABSTRACT
Human papillomavirus (HPV) type 58 has been found to be prevalent among Chinese patients with cervical cancer. This study examined the oncogenic risk of HPV58 variants in Hong Kong, a southern part of China. Altogether, 1924 women were studied: 42.8% with a normal cervix, 16.2% with cervical intraepithelial neoplasia (CIN) I, 12.7% with CIN II, 20.8% with CIN III, and 7.6% with invasive cervical cancer (ICC). The overall prevalence of HPV58 was 11.4% (220) and increased statistically significantly with the severity of neoplasia (Ptrend<.001, 2 test for trend). Among HPV58-positive women, the occurrence of E7 632C
T (T20I) and E7 760G
A (G63S) variants (T20I/G63S) showed a positive trend of association with the severity of neoplasia (Ptrend<.001,
2 test for trend). HPV58 variants carrying these two substitutions showed an odds ratio (OR) for ICC of 26.79 (95% confidence interval = 10.14 to 74.72), and this OR was 6.9-fold higher than the ORs of variants without these substitutions. Patients with CIN III or ICC who were also infected with T20I/G63S variants had a statistically significant younger age at diagnosis than those infected with other variants (median age = 37 years versus 48 years; P = .038, two-sided MannWhitney U test). Thus, HPV58 variants carrying E7 T20I/G63S substitutions may be associated with an increased risk for cervical cancer.
Subjects were recruited from a colposcopy clinic and were classified according to biopsy examination findings. All subjects gave their written informed consent. Women found to have a normal cervix by colposcopy, hence not receiving a biopsy examination, were regarded as normal, provided that their subsequent Papanicolaou (Pap) smear taken 46 months later did not reveal neoplasia. Those women with abnormal or missing follow-up smears were excluded. Histologic findings of inflammatory changes or HPV effects but without neoplasia were also classified as normal. The study was approved by the local Institutional Ethics Committee.
The study included 1924 Chinese women, most of whom were descendants of families from Canton province in the southern part of China. Their mean age was 40.2 years (range = 1688 years; standard deviation = 10.7 years). Diagnoses among these 1924 women were as follows: 823 (42.8%) with a normal cervix, 311 (16.2%) with cervical intraepithelial neoplasia (CIN) I, 244 (12.7%) with CIN II, 400 (20.8%) with CIN III, and 146 (7.6%) with invasive cervical cancer (ICC)including 137 with squamous cell carcinoma, seven with adenocarcinoma, and two with adenosquamous carcinoma. Overall, 220 (11.4%) samples were positive for HPV58 by a type-specific polymerase chain reaction (PCR) that used primers E7-P1/E7-P2 (5'- CTGTAA CAACGCCATGAGAG-3' and 5'-TCAGGGTCATCCATTGCAGA-3'). The positive rate for HPV58 showed a statistically significant trend of increase with the degree of neoplasia (5.1% for normal cervix, 12.2% for CIN I, 17.2% for CIN II, 13.0% for CIN III, and 31.5% for ICC; Ptrend<.001 by the 2 test for trend; Epi Info 2000; Centers for Disease Control and Prevention, Atlanta, GA).
Sequences of E7 and E6 regions were obtained by PCR-based cycle sequencing with E7-P1/E7-P2 primers and E6-P1/E6-P2 primers (5'-GACCGAA ACCGGTGCATATA-3' and 5'- TCT CATGGCGTTGTTACAGG-3'), respectively. Sequencing reactions were performed on the ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA), according to the manufacturer's protocol (BigDye Terminator Cycle sequencing kit; Applied Biosystems). All sequences were confirmed by sequencing from both directions.
The HPV58 prototype (GenBank accession no. NC_001443), isolated from a Japanese cervical cancer patient, was used as the reference (8). Six nucleotide sequence variations were found over the 450-base-pair (bp) E6 ORF, and 14 were found over the 297-bp E7 ORF. E6 variations were scattered, whereas E7 variations were mainly found within the carboxyl-terminal residues (amino acids 4189). Sequence variability was higher for E7 than for E6. Only 10 (4.5%) isolates showed one or more variations at E6, whereas 176 (80.0%) isolates showed one or more variations at E7. The maximum number of variations for any variant was four (0.9%) for E6 and six (2.0%) for E7. The average chance of having a nucleotide sequence deviation from prototype was two substitutions per 1000 bp for E6 and eight substitutions per 1000 bp for E7. Thus, three of six E6 substitutions and nine of 14 E7 substitutions were nonsynonymous (Fig. 1). Mutations generating a frameshift or a premature stop codon were not observed.
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The 388AC (K93N) E6 nucleotide substitution showed a statistically significant negative trend of association with the severity of neoplasia (Ptrend<.001 by the exact test for trend; StatXact 4.0.1; Cytel Software Corp., Cambridge, MA), whereas 632C
T (T20I) and 760G
A (G63S) E7 nucleotide substitutions showed a statistically significant positive trend of association with the severity of neoplasia (Ptrend<.001 by the exact test for trend) (Table 1
, A). E7 T20I and E7 G63S amino acid substitutions were apparently linked, with both found in 66 isolates. Only four isolates from subjects with a normal cervix had the G63S substitution in the absence of T20I. No isolate carried the E7 T20I substitution in the absence of E7 G63S. The HPV58 E6/E7-HK-2 variant that carried the E7 T20I and G63S (T20I/G63S) substitutions was found to have a statistically significant positive trend of association with the severity of neoplasia (Ptrend<.001 by the exact test for trend; Table 1
, A).
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Among patients with CIN III or ICC, infection with E7 T20I/G63S variants was associated statistically significantly with a younger age at diagnosis than was infection with other variants (median age [interquartile range]: 37 years [30.559.3 years] versus 48 years [36.863.0 years], P = .038 by two-sided MannWhitney U test; SPSS 10.1.0; SPSS Inc., Chicago, IL). This observation is in line with a higher oncogenicity of E7 T20I/G63S.
Worldwide, HPV58 has been found in only 2% of cervical cancers (9). In contrast, one third of the patients with cancer in the current study were positive for HPV58. This unusually high prevalence has also been reported in Chinese populations living in Shanghai (10), Jiangxi (11), and Taiwan (12). HPV58 is closely related to HPV33 and is grouped with HPV16, -31, -33, -35, -52, and -67 under the same branch of an HPV phylogenetic tree (13). We found that sequence variability of HPV58 variants was fourfold higher for E7 than for E6, in contrast with HPV16, where E6 is reported to be more variable (25). The E7 protein has three domains: Conserved region (CR)-1 (amino-terminal 20 amino acids) and CR-2 (amino acids 2140) are similar to adenovirus E1A (14,15), and CR-3 (carboxyl-terminal amino acids 4198) contains two zinc-binding motifs (Cys-Xaa-Xaa-Cys) that are involved in dimerization and protein stability (16). We found two substitutions that were associated with a higher oncogenic risk. The first substitution (E7 T20I) is located at amino acid 20, close to the Leu-Xaa-Cys-Xaa-Glu domain that mediates association with the retinoblastoma protein and its related proteins, p107 and p130. The second substitution (E7 G63S) results in a change from glycine to serine. The E7 protein is phosphorylated on serine residues by casein kinase II (17), and a positive association between phosphorylation rate and oncogenic potential has been found (18). Thus, we suspected that the E7 G63S substitution might have created an additional phosphorylation site that confers increased transforming activity. However, our hypothesis was not supported by an artificial neural network-based computer prediction (NetPhos 2.0 [http://www.cbs.dtu.dk/services/NetPhos/]) (19). The NetPhos prediction gave a very low score for Ser-63 compared with Ser-31 and Ser-32, which are phosphorylated by casein kinase II (phosphorylation scores determined by NetPhos were 0.002 for Ser-63, 0.977 for Ser-31, and 0.992 for Ser-32). In the NetPhos prediction, a score closer to unity indicates a higher chance of phosphorylation.
In summary, our study provides epidemiologic evidence that HPV58 variants carrying E7 T20I/G63S substitutions are associated with an increased risk for cervical cancer. The exact mechanism for the increased oncogenicity needs further investigation. Cosegregating sequence variations in other parts of the virus genome may also play an important role. Finally, because this is a cross-sectional study, the magnitude of the risk should be interpreted cautiously.
NOTES
Supported by the Lee Hysan Foundation Research Grant and the Endowment Fund Research Grant (CA10830, CA10990) from United College, The Chinese University of Hong Kong. C. Lam is a Croucher Foundation Fellow.
Part of the results have been presented at the 18th International Papillomavirus Conference, Barcelona 2000 (abstract Nos. 095 and 096).
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Manuscript received February 5, 2002; revised May 21, 2002; accepted June 18, 2002.
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