THE EPIDEMIOLOGY OF NASOPHARYNGEAL CARCINOMA
Chee Khoon Chan, M.S.
A Thesis Submitted to the Faculty of
The Harvard School of l'ublic Health ; -. ., . .~ !. . . in Partial Fulfillment of the Requirements
for the Degree of Doctor of Science
in the Field of Epidemiology
Boston, Massachusetts
December 1990 ~692~6 \2-C- 't((O r'~ ~lf ~v ii
This thesis has been read and approved by
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Preface
.... . Greatly mourned by his wife but without children, Law had died
in a private hospital of the nasopharyngeal cancer so prevalent on the
southern coast, caused by a lifetime's inordinate consumption of the dried
salt fish he had loved so much .....
Timothy Mo, in Sour Sweet
(p. 43, Abacus, 1983)
Novelists rush in where epidemiologists fear to tread.
The point of the above quote is not just to note that literary
license provides for a degree of freedom not available to
epidemiologists, but also that the salted fish hypothesis in
NPc·etiology was sufficiently known, if not among the general
populace of southern China, then at least among the literary
public. This may not have had much impact on the case-control
findings on salted fish consumption, but it does argue for
some caution in appraising that literature.
I wish to record my appreciation and gratitude to Dr.
Nancy Mueller, my thesis committee chairperson, and to Dr.
Walter Willett and Dr. Anastasios Tsiatis, who also served on iv the committee. Their support and counsel, together with that of Dr. Dimitrios Trichopoulos, Dr. Fran Cook, Dr. c.c. Hsieh, and other friends in the Epidemiology Department, were crucial in seeing me through the more difficult moments of this thesis.
I am grateful to the Science University of Malaysia for a staff training fellowship which supported me through 4 years of doctoral studies. The Insti tut Pasteur de Lyon also provided generous support for a year's research work in France.
Chee Khoon Chan
v
Table of contents
Title Page ...... i
Preface ...... iii
Table of Contents ...... •...... •...... v
List of Figures ...... vi
List of Tables ...... vii
Epstein-Barr virus antibody titers preceding the diagnosis of nasopharyngeal carcinoma...... 1
Dietary risk factors for nasopharyngeal carcinoma
A case-control study in Zangwu County, Guangxi
Autonomous Region, People's Republic of China... 26
The epidemiology of nasopharyngeal carcinoma
A review...... 66 vi
List of Figures
Epstein-Barr virus antibody patterns preceding the diagnosis of nasopharyngeal carcinoma
Figure 1 Anti-VCA/IgG antibody titers preceding diagnosis of nasopharyngeal carcinoma
Figure 2 Anti-VCA/IgA antibody titers preceding diagnosis of nasopharyngeal carcinoma vii
List of Tables
Epstein-Barr virus antibody patterns preceding the diagnosis of nasopharyngeal carcinoma
Table 1 Epstein-Barr virus antibody titers of NPC patients and matched controls
Table 2 Prevalence of elevated titers and geometric mean titers of antibodies against Epstein Barr virus among NPC patients and controls
Table 3 Summary of findings of previous studies on EBV serology in NPC patients prior to diagnosis of nasopharyngeal carcinoma
Dietary risk factors for nasopharyngeal carcinoma
A case-control study in Zangwu County, Guangxi
Autonomous Region, People's Republic of China
Table 1 Childhood living conditions, current income & risk for NPC
Table 2 Weaning foods & risk for NPC
Table 3 Childhood diet (age 2-10 yrs) & risk for NPC
Table 4 Childhood dietary risks for NPC -- Estimates for subjects with living parent(s) compared to all-subject estimates
Table 5 Adult diet (two decades ago) & risk for NPC
Table 6 Multivariate analysis of dietary risk factors for NPC viii
The epidemiology of nasopharyngeal carcinoma A review
Table 1 Age-standardised NPC incidence rates for selected countries
Table 2 HLA antigens associated with risk for NPC
Table 3 Epstein-Barr virus antigens detected in nasopharyngeal carcinoma
Table 4 Salted fish consumption & risk for NPC
Table 5 Environmental inhalants & risk for NPC
Table 6 EBV antibody titers preceding diagnosis of NPC Epstein-Barr Virus Antibody Patterns Preceding the Diagnosis of Nasopharyngeal Carcinoma
1 2 Chee Khoon Chan, Ms • 1 3 Nancy Mueller, ScD ' Alfred Evans, MD, MPH4 Nancy L. Harris, MD5 and members of the EBV-NPC collaboration (in alphabetical order) George W. Comstock, MD, DrPH6 Egil Jellum, MD7 Knut Magnus, PhD8 Norman Orentreich, MD9 B. Frank Polk, MD6 ·* Joseph Vogelman, PhD6
1. Department of Epidemiology, Harvard School of Public Health, 677 Huntington Ave., Boston, MA 02115. 2. Science University of Malaysia. 3. To whom reprints requests should be addressed. 4. Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT. 5 Department of Pathology, Massachusetts General Hospital, Boston, MA. 6 Department of Epidemiology, School of Hygiene & Public Health, Johns Hopkins University, Baltimore, MD. 7. University Institute for Clinical Biochemistry, Rikshospitalet, Oslo, Norway. 8. Cancer Registry of Norway, Oslo, Norway. 9. The Orentreich Foundation for the Advancement of Science, New York, NY.
* Deceased This study was supported by PHS grants CA31747, CA30433, BRSG RR05446, BRSG RR05443, National Institutes of Health, Department of Health & Human Services and by a staff training fellowship from the Science University of Malaysia to C.K. Chan. The follow-up of the HDFP study population is supported by PHS grant CA34937. 2
Acknowledgements
We are indebted to Dr. Diana Petitti of the Division of Research, Permanente Medical Group for her participation in this study. We also wish to thank Mrs. Rose Udin and Mrs. Sylvia Feinson at Harvard and Ms Linda Cenabra and Ms Betty Olson at Yale, and Dr. Robert Chambers, former Director of Laboratories, Washington County Hospital. 3
Abstract
Nasopharyngeal carcinoma (NPC) patients have elevated IgG and IgA antibody titers against the Epstein-Barr viral capsid antigen (VCA) and the diffuse component of the early antigen complex (EA-D) at diagnosis.
Several studies have implied that the presence of anti-VCA-lgA can be used as a screening marker for early NPC. To evaluate this further, we undertook a serologic case-control study based on four serum banks which together had specimens from over 240,000 persons. Seven cases of undifferentiated or poorly differentiated NPC were diagnosed in the period after serum collection ranging from 26 months to 154 months. Two controls per case matched on serum bank, age, sex, race, and date of serum collection were selected by a pre-determined random process. For anti-VCA-IgG, the geometric mean titer for cases (88.3) was significantly higher than that for controls (75.5) (p < 0.05). The difference was greatest among the Asian patients. No significant differences were found for anti-VCA-IgA, anti-EA-D, anti-EA-R or anti
EBNA. No time effects were evident when titers were plotted against time of blood collection preceding diagnosis. Our results do not suggest EBV activation in the period preceding NPC diagnosis, nor that detectable IgA antibody against VCA is a marker for early disease.
key words Nasopharyngeal carcinoma, Epstein-Barr virus, serological
screening 4
The Epstein-Barr virus (EBV) has been consistently
associated with the occurrence of undifferentiated and poorly differentiated nasopharyngeal carcinoma (NPC). NPC
patients from around the world, at or following diagnosis,
often have high IgG titers against the viral capsid antigen
(VCA) and the diffuse component of the EBV early antigen
complex (EA-D) but not the restricted (EA-R) (de Schryver et al., 1969, 1974; Henle, w. et al., 1970, 1973; Lin et al., 1971; Henderson et al., 1974). In contrast, patients with
carcinomas of the oropharynx or hypopharynx or tumors of the nasopharynx other than carcinomas usually have much lower titers. IgA titers against the VCA and the EA-D are also markedly elevated in the sera and saliva of NPC patients
(Wara et al., 1975; Henle, G. et al., 1977). Elevated titers against an EBV-specific DNase has also been reported
(Cheng et al., 1980). Nucleic acid hybridization consistently demonstrated the presence of EBV-DNA in NPC biopsies (zur Hausen et al., 1970, 1974; Nonoyama et al.,
1973), and specifically in the anaplastic or poorly differentiated carcinoma cells (Wolf et al., 1973, 1975;
Desgranges et al., 1975). Biopsies from carcinomas at other sites of the head and neck and from other types of tumors of the nasopharynx have been mostly negative for EBV-DNA
(Andersson-Anvret et al., 1977). The EBV-associated nuclear 5 antigen (EBNA) is also detected in anaplastic NPC cells, but not in the lymphoid elements of the tumor (Wolf et al.,
1973, 1975; Huang et al., 1974; Klein et al., 1974;
Desgranges et al., 1975).
These findings have prompted much interest in the potential of EBV serologic markers -- in particular IgA antibodies against VCA -- as screening aids in early detection of NPC (Zeng, 1985). The data on EBV serology prior to diagnosis is sparse, and the temporal relationship between altered EBV serological profile and emergence of disease remains obscure. One clinical report, and three other studies have yielded conflicting results (Ho et al.,
1978; Lanier et al., 1980; Zeng et al., 1985; Chen et al.,
1985). The purpose of this study is to evaluate the pattern of pre-diagnosis EBV antibodies in a series of NPC patients.
MATERIALS AND METHODS
The study population and selection process as well as details of the serologic assays have been previously described (Mueller et al., 1990). Two controls were identified for each case. Biopsy slides were requested for all NPC cases for review by one of us (NLH). The slides were reviewed without knowledge of the previous diagnosis or clinical findings. 6
statistical Analyses
The relative risk (RR) associated with elevated anti
EBV titers, for subsequent occurrence of nasopharyngeal
carcinoma, was estimated using conditional logistic
regression. The criteria for elevation were taken at the
80th (or closest) percentile for titer distributions of
controls. Logistic regressions were performed with the
EGRET statistical package which uses an iterative algorithm
to obtain maximum likelihood estimates. Statistical
significance at the 5% level (two-tailed) corresponds to the
95% confidence interval for the relative risk excluding the null value of 1.0. Geometric mean titers were calculated
only for subjects with detectable antibody at the lowest dilution (1:5), with tests of significance performed on
logarithmic transforms of titer values.
RESULTS
Nine cases of nasopharyngeal carcinoma were diagnosed
in the period subsequent to collection of blood specimens.
Biopsy specimens were available for review for all but one of the patients. Six cases were confirmed as undifferentiated or poorly differentiated NPC. For the one case not reviewed, the original diagnosis of partially differentiated squamous cell NPC was accepted. One case was re-designated a squamous cell NPC, and the remaining case 7
was re-classified as an undifferentiated B-cell non
Hodgkin's lymphoma. The seven cases of undifferentiated or
poorly differentiated NPC were diagnosed between 1970-1979.
All seven cases occurred among subjects in the Kaiser
Permanente multiphasic health examination program.
Ethnically, four of the cases were Asian, and three
were white. All but one were male. The mean age at
diagnosis for Asian cases was 52.3 years (range 39-74), and
for whites, 59.3 years (range 53-66). Of the five cases for
whom staging information was available, three were diagnosed
at stage III, and two at stage IV. Age-matching to within ±
12 months was attained for 4 matched sets. One stratum had a control matched to ± 18 months. The remaining 2 sets had controls 20 months and 31 months distant from the index
case. The interval of time from serum collection to
diagnosis ranged from 26 months to 154 months. The mean
interval was 129 months for Asian cases, and 91 months for
white cases.
Antibody titers against the EBV of cases and controls
are shown in Table 1. Except for two white controls
negative for all EBV antibodies, all subjects had detectable
levels of anti-nuclear antigen (EBNA) and anti-VCA-IgG,
indicating past infection with EBV. None of the subjects had detectable anti-VCA-IgM. For the other five antibodies, 8
cases had higher prevalences of detectable antibody than
controls (table 2). The geometric mean titer of cases for
anti-VCA-IgG (88.3) was significantly higher than that for
controls (75.5) (p < 0.05). All Asian cases had titers as
high or higher than their controls; the reverse was true
among non-Asian cases. Comparisons for the other four
antibodies showed no significant differences. The excluded
case of a squamous differentiated NPC, a 43 year old white
woman, had undetectable anti-VCA-IgA and anti-EA-D 49 months
before diagnosis; anti-VCA-IgG however was detectable at
1:320 dilution.
Table 2 also shows estimates of relative risks obtained from conditional logistic regressions with antibody
titers treated as dichotomous variables (elevated/non
elevated). Elevated pre-diagnosis anti-VCA-IgA titers do
not appear to be associated with increased risk for NPC.
The Asian cases have a higher prevalence of anti-VCA-IgA
seropositivity (100%) than among their controls (63%). The
relative risk associated with elevated anti-VCA-IgA, for the
Asian group, was 2.0 (95% confidence limits-- 0.1, 32.0).
Elevated anti-EA-D was associated with an RR of 2.7 (0.2,
33.0). The point estimates for anti-EBNA and anti-EA-R were
below unity, but were also not significant.
The antibody titers for the individual cases and their matched controls are plotted out by the time prior to 9 diagnosis at which the specimen was drawn (figures 1 and 2).
For the single case whose serum was obtained less than 4 years prior to diagnosis, a 53 year old white male, the anti-VCA-IgA titer was lower than that of both matched controls. In five of the six remaining matched sets, case anti-VCA-IgA titers fell within the range of control titers.
For anti-VCA-IgG, six of seven case titers fell within the range of matched control titers, or lower, and for anti-EA
D, five of seven. No time effects were evident.
DISCUSSION
The pre-diagnosis EBV serological profile of NPC patients is of interest from an etiological standpoint, as well as for purposes of screening and early detection of pre-clinical NPC. In NPC biopsies, viral gene expression is limited to the latent antigens EBNA-1, which is responsible for maintenance of the latent virus in episomal form, two different membrane proteins, LMP1 and LMP2 (Fahraeus et al.,
1988; Young et al., 1988; Frech et al., 1990; Longnecker &
Kieff, 1990; Rowe et al., 1990) and other as yet poorly characterized transcripts (Hitt et al., 1989).
In contrast, the serological profile of NPC patients at diagnosis is characterized by elevated IgG and IgA titers against VCA and EA-D, antigens which are expressed during the lytic, replicative phase of the viral life cycle but are 10 not detected in NPC biopsies. In particular, the presence of IgA antibodies against VCA and EA-D suggest viral replication in the epithelial cells of the nasopharynx
(Desgranges et al., 1977). The etiological significance of elevated replicative titers remains unclear.
The first report of pre-diagnosis anti-VCA-IgA status came from Hong Kong (Ho et al., 1978). One of three NPC patients had a titer of 1:80, at thirty-five months before diagnosis. The remaining patients had pre-diagnosis titers of 1:5 and 1:20 at fifty-five months and thirty months respectively.
Lanier et al. (1980) reported a study in which sera from seven Alaskan NPC patients had been collected two to ten years before diagnosis of disease. Three sets of controls were chosen for each case : the first set matched on age, race, sex, village of residence; the second set comprised Alaskan Natives aged 40 and above living in an area geographically similar to the case; and the third set were Alaskan Natives with other head and neck tumors, mixed tumors of the parotid, or persons without tumors from whom two separate sera or more were available for testing. With the exception of anti-EBNA, pre-diagnosis anti-EBV spectra and titers for six of the seven cases did not differ from those of controls. Anti-EBNA titer was as high as, or 11 higher, for every case than its closest matched control.
However, the validity of the comparison is unclear since the
control sera were collected 6 to 13 years after the index
case sera. For the seventh case, sera obtained at 17 and 21 months before diagnosis showed antibody patterns
characteristic of NPC patients. Since this patient was
admitted in stage IV and died soon thereafter, the authors
considered it likely that the tumor was already advanced when the sera were collected.
In 1980, Chinese researchers conducted a mass
screening in Wuzhou City of the Guangxi Autonomous Region.
Sera were obtained from 20,726 individuals aged 40 years and above. 1,136 IgA/VCA positive cases were followed up with clinical and histological examinations once a year. After
four years, 17 incident cases of NPC were diagnosed : 5 in stage I, 11 in stage II and 1 in stage III (Zeng et al.,
1985). The incidence rate among IgA/VCA positive subjects was 7.5 times the age-adjusted rate for the general Wuzhou population. This result however estimates the RR associated with pre-diagnosis IgA/VCA positivity, since follow-up subjects were subjected to close otolaryngological examinations while incident cases from the general population, for any particular year, would include fewer of the early stage clinically less evident cases. This bias could be attenuated in subsequent years of follow-up. 12
In another follow-up of 1,138 IgA/VCA positive subjects in Zangwu County, 15 of 81 individuals whose anti
VCA-IgA titers increased four-fold or more during the 3-year follow-up, developed NPC. The 81 individuals represented
7.1% of the IgA/VCA positive screens, but accounted for
71.5% of NPC patients diagnosed in this group (de The &
Zeng, 1986).
A fourth study, conducted in Taiwan, reported pre diagnosis anti-VCA-IgA titers for four NPC patients (Chen et al., 1985). Two of the subjects had undetectable titers at
9 months and 29 months prior to diagnosis. A third subject had detectable titer at 1:10 dilution (12 months pre diagnosis), and the remaining subject had a titer of 1:40
(28 months pre-diagnosis) which increased to 1:160 over the next 14 months and maintained at that level till diagnosis.
Our results for the 7 cases combined do not support elevated anti-VCA-IgA as a risk factor in the pre-diagnosis period. This result also holds when the exposure is defined as pre-diagnosis anti-VCA-IgA positivity, RR = 1.3 (0.2,
7.5). This is consistent with earlier reports that even in stage I of NPC, when the tumor is still limited to the postnasal space and is of small size, some patients do not yet show the characteristic EBV antibody pattern (Henle, w. et al., 1973, 1977; Ringborg et al., 1983). In the Chinese 13 follow-up studies too, there were no marked differences between the geometric mean titers of pre-diagnosis anti-VCA
IgA, and that at diagnosis of stage I NPC cases. The contrasts were stronger for diagnoses made at stages II-IV
(Zeng et al., 1983, 1985). The increased risk for subjects whose anti-VCA-IgA rise abruptly is difficult to interpret, since this happens quite close to diagnosis and seems to be primarily a phenomenon of cases diagnosed at stages II-IV.
Our results overall do not support a role in the pathogenesis of NPC for EBV activation in the period preceding diagnosis nor a consistent role of anti-VCA-IgA as a marker for screening. Of interest, our Asian patients did have a higher prevalence of anti-VCA-IgA than their controls which was not significant. Our small sample size precludes any conclusions as to whether elevated anti-VCA-IgA, extending beyond 3 years pre-diagnosis, is an NPC risk factor among this high-risk population. Further analysis of the Wuzhou cohort with its additional years of follow-up would be valuable. 14
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Table 1 Epstein-Barr Virus Antibody Titers of NPC Patients and Matched Controls
Subjects Months Prior (age in years) to Diagnosis VCA/IgG VCA/IgA EBNA EA-R EA-D
Asian subjects :
Case : male (39) 134 160 20 20 40 20 Control 1 160 80 20 80 5 Control 2 10 < 5 20 10 < 5
Case : male (42) 153 40 20 160 20 < 5 Control 1 40 20 160 10 < 5 Control 2 20 < 5 20 40 5
Case : male (54) 154 80 10 80 20 < 5 Control 1 80 20 160 20 10 Control 2 40 5 160 20 < 5
Case : male (74) 76 160 40 80 80 20 Control 1 80 20 40 80 20 Control 2 40 < 5 40 160 < 5
White subjects :
Case : male (59) 122 80 10 160 20 < 5 Control 1 640 80 160 160 5 Control 2 < 5 < 5 < 5 < 5 < 5
Case : male (53) 26 80 < 5 10 20 5 Control 1 160 5 160 40 5 Control 2 160 20 40 20 < 5
Case : female (66) 124 80 < 5 20 20 5 Control 1 < 5 < 5 < 5 < 5 < 5 Control 2 160 5 40 40 < 5 22
Table 2 Prevalence of Elevated Titers and Geometric Mean Titers of Antibodies Against Epstein-Barr Virus Among NPC Patients and Controls
Epstein-Barr Virus Elevated Titers Geometric Mean Titer Antigen (Elevated Titer) Patients Controls Relative Riskt Patients(*) Controls(*)
Capsid antigen IgG (~ 1:320) 0/7 1/14 0 88.3*(100%) 75.5 (86%) IgA (~ 1:40) 1/7 2/14 1.0 (0.1-11.0) 17.4 (71%) 17.1 (64%)
Epstein-Barr nuclear antigen (~ 1:160) 2/7 5/14 0.7 (0.1-5.3) 48.8 (100%) 59.9 (86%)
Early antigen Diffuse (~ 1:10) 2/7 2/14 2.7 (0.2-33.0) 10.0 (57%) 7.1 (43%) Restricted (~ 1:80) 1/7 4/14 0.5 (.0006-388.6) 26.9 (100%) 37.8 (86%)
* percent of subjects with detectable antibody at 1:5 dilution or higher
estimated from univariate conditional logistic regression with 95% confidence limits in parentheses
p < 0.05 23
Table 3 Previous Studies on Pre-Dx EBV Serology & NPC
study Subjects Findings
Lanier et al. 7 Alaskan cases case anti-EBNAs higher; (1980) case sera 2-10 yrs pre-Dx; all other antibodies similar control sera collected 6-13 yrs after index case sera
Zeng et al. 20,726 adults screened (Guangxi, PRC) 7.5 fold incidence among (1985) 1,136 IgA/VCA (+) followed 4 yrs sero(+) relative to age-adj. population rate
Chen et al. 4 Taiwanese subjects IgA/VCA titer Mo. Pre-Dx (1985) patient 1 10 12 patient 2 < 10 9 patient 3 160 14 patient 4 < 10 29
Ho et al. 3 Hong Kong subjects (1978) patient 1 5 55 patient 2 20 30 patient 3 80 35 'Fig. 1 : Anti-VCA/IgG Antibody Titers Preceding Diagnosis of Nasopharyngeal Carcinoma .-- Cases Controls I 24 0 • White ~ Chinese ~ I • I • ,640 320 I -~ -9 -cJI C1 11. • ~ •• 160 cJf < I I I n •6 0 0 0 80 )> • I ld' t-4 I \0 •! • 40 Gl I --i I I I -I 20 -· • -~-. CJ) • I I 1 10 5 .•- j 0 i60 140 120 100 80 60 40 20 0 Time (mo.) between last bleeding Fig. 2 : Anti-VCA/IgA Antibody Titers Preceding Diagnosis of Nasopharyngeal Carcinoma 25
Cases Controls 0 • White 6. • Chinese 160 d' d' < 80 () • • j l> I iJt t-t tO 6 40 l> fJiJ I I d' -t 6 20 -· •• • • co lA -~ (/) AI I 0 10 ~ 11 I • ~ 5 I •I • • o•• • 0 j 0 60 140 120 100 80 60 40 20 0
Time (mo.) between last bleeding and NPC diagnosis DIETARY RISK FACTORS FOR NASOPHARYNGEAL CARCINOMA
A Case-Control Study in Zangwu County, Guangxi Autonomous Region, People's Republic of China
Chee Khoon Chan, M.S.
Department of Epidemiology Harvard School of Public Health Boston, Massachusetts U.S.A.
C.K. Chan was supported by a fellowship from Institut Pasteur de Lyon, in addition to a staff training fellowship from the Science University of Malaysia. The analyses for this report were performed in the laboratory of Dr. Guy de The in Lyon (CNRS Laboratoire d'Epidemiologie et Immunovirologie des Tumeurs). 27
Abstract
88 incident cases of histologically confirmed nasopharyngeal carcinoma
(NPC) and two controls per case matched on age, sex, and neighborhood were interviewed in Zangwu County, Guangxi, China. A major objective of the study was to determine if dietary risk for NPC extended to a broader range of preserved foods beyond Cantonese salted fish. Weaning use of salted fish carried a relative risk of 2.6 (95% conf.limits 1.3, 4.9).
The susceptible period appears to extend into post-weaning childhood and early adulthood. None of the other salted or preserved foods queried
(including salted and pickled vegetables, fermented bean pastes and sauces, other soybean products) emerged as significant risk factors.
Consumption of herbal teas two decades ago was a significant risk factor
-- relative risk 4.0 (1.4, 11.3). Recent consumption of fresh fruits, chives, garlic and monosodium glutamate (MSG) were associated with reduced risk. In a multivariate analysis of the dietary factors, salted fish consumed in the three age periods, and recent consumption of fresh fruits and MSG remained significant. The mean age-at-diagnosis for female cases with family history of NPC (24.7 yrs) was significantly lower than that for male cases with NPC family history (41.3 yrs.) (p
0.02). The social profile of risk may include a rural childhood, and small household size and relatively less crowded sleeping arrangements in childhood. 28
INTRODUCTION
Nasopharyngeal carcinoma (NPC) occurs rarely in most parts of the world, typically at rates below 1 per 100,000 per year (Waterhouse et al., 1982). In the coastal provinces of south China however, NPC occurs at rates up to a hundred-fold the incidence observed in Europe and North
America. In Guangdong Province, NPC ranks third for cancer mortality among men (fourth among women), accounting for 15% of male cancer deaths and 11% of female cancer deaths (Nat'l
Cancer Control Office, 1979).
Incidence rates among people of southern Chinese descent in Hong Kong (Waterhouse et a1., 1982), Taiwan (Lin et al., 1971a), Singapore (Shanmugaratnam et al., 1982) and
Malaysia (Armstrong et al., 1979) are similarly high. In
California and Hawaii however, NPC mortality among second generation US-born Chinese is about half that of foreign born Chinese Americans, but still remains substantially above Caucasian rates (Zippin et al., 1962; King & Haenszel, 1973; Buell, 1974; Fraumeni & Mason, 1974; Yu et al., 1981).
Eskimo and other native communities in the Arctic are also at high risk (Lanier et al., 1980), and there is moderate incidence among Maghrebian Arabs in North Africa (Muir,
1972) .
Three main risk factors have been identified. The
Epstein-Barr virus (EBV) is consistently associated with 29 undifferentiated and poorly differentiated NPC. Patients have a distinctive anti-EBV serological profile (de Schryver et al., 1969, 1974; Henle, W. et al., 1970, 1973; Lin et al., 1971b; Henderson et al., 1974; Wara et al., 1975;
Henle, G. et al., 1977) and regardless of geographic origin,
EBV-DNA sequences have been consistently detected in NPC biopsies (zur Hausen et al., 1970, 1974; Nonoyama et al.,
1973), specifically in the anaplastic or poorly differentiated carcinoma cells (Wolf et al., 1973, 1975;
Desgranges et al., 1975). Biopsies from carcinomas at other sites of the head and neck and from other types of tumors of the nasopharynx have been mostly negative for EBV-DNA
(Andersson-Anvret et al., 1977). EBV however is ubiquitous, and the lack of evidence correlating strain differences
(Rymo et al., 1979; Bornkamm et al., 1980; Zimber et al.,
1986; Young et al., 1987) or age at primary infection (Chen, personal communication) with geographic variation in NPC rates suggest that additional risk factors are important.
A putative disease susceptibility locus closely linked to the HLA region has been identified (Lu et al.,
1990) .
Among lifestyle factors, weaning use of Cantonese salted fish has been the most consistent finding reported for southern Chinese communities (Geser et al., 1978;
Armstrong et al., 1983; Yu et al., 1986, 1988, 1989a, 1990). 30
Two recent studies in Guangxi and Guangzhou suggest that
weaning use of other salted and preserved foods (including
preserved vegetables and preserved soybean products) may
also be risk factors for NPC (Yu et al., 1988, 1989a).
The primary objective in this study was to
investigate further the suggestion that dietary risk for NPC may extend to a broader range of salted and preserved foods
among southern Chinese. Potential interactions between some
of the risk factors were also explored.
METHODS
The Guangxi Autonomous Region is populated by a
diversity of ethnic groups. Han Chinese make up 40% of the
population and are found in a broad swathe to the east, a
region of high prevalence for NPC which borders on Guangdong
Province. As in Guangdong, the Han dialect most commonly
used here is Cantonese. The largest among the non-Han minorities are the Zhuang (36% pop.) who occupy much of the western half of the province. The Miao, Dong, Boyei, Sui,
Molao, Maonan and other minorities are mostly concentrated
in the northern region bordering on Guizhou Province. NPC prevalence among the minorities is much lower than among the
Han. The study locations, Wuzhou city and Zangwu County are
in the predominantly Han area. zangwu county covers an area 31 of 4260 sq. mi. and is situated at the border with Guangdong Province. The terrain is hilly and the rural population of 550,000 is largely engaged in agriculture with the family as the basic work unit. Wuzhou City is a town of about 170,000 people, more or less centrally located within Zangwu County. It is situated at the confluence of the Gui and Yong rivers about 25 km from the border with Guangdong Province. Nanning, the provincial capital for Guangxi is 480 km upstream to the west, and Guangzhou (Canton) is 380 km downstream to the east.
cases Cases were histologically confirmed incident cases of undifferentiated or poorly differentiated NPC diagnosed between Jan 1, 1986 - Dec 31, 1987 and resident in wuzhou City or Zangwu County. All Wuzhou cases were recruited from patients diagnosed at the Wuzhou cancer Institute and who were residents of Wuzhou city at the time of diagnosis. Records were abstracted from the files of the Wuzhou Cancer Registry. Zangwu cases were rural residents in Zangwu County at tte time of diagnosis. They were identified from records at the Zanqwu cancer Unit, a facility 15 km from Wuzhou set up specifically for NPC detection in the rest of Zangwu County. Zangwu rural residents who are diagnosed in Wuzhou or in Guangdong are reported back to the Zangwu 32
Cancer Unit. The Unit itself is equipped for
histopathology, EBV serology, and basic epidemiology.
Treatment is carried out at the Wuzhou Cancer Institute. No
other institutions in Wuzhou or Zangwu are involved in NPC
diagnosis or treatment.
Controls
Controls were selected by the interviewers from
households in the immediate neighbourhood of the index case.
The first two eligible controls who were sex- and age
matched (± 5 years) and who agreed to participate in the study were interviewed. No more than one control was
selected from each control household. As far as was
possible, the controls were interviewed on the same day as
the index case, and under similar circumstances. Cases and
their matched controls were interviewed by the same
interviewer for 29 of the 88 matched sets.
Interviews
Interviews were conducted at home in the presence of
immediate family members. Most of the interviews were
conducted in the local dialect, which uses the same written
form as the Mandarin script questionnaire. The primary
respondent was the case (or control), but family members were encouraged to provide supplementary information and to 33 participate in the corroboration of informational recall.
The listing of household members indicate that 53 of 88 cases (60%) listed one or both parents as household members, and 105 of 176 controls (60%). 48 cases (55%) had mothers living in the same household, and 78 controls (44%). The interviews in Wuzhou were conducted by 3 interviewers; a similar number of interviewers conducted the Zangwu interviews. Interviewers were junior staff physicians at the Wuzhou Cancer Institute who had been trained by a food anthropologist responsible for the pilot survey. Seventy percent of the interviews were conducted within 6 months of diagnosis, and the rest within a year. The interviewers used structured questionnaires which took approximately 2 hours to complete. Information was requested on date and place of birth, father's place of birth, marital status, educational level, residential history, occupational history, family medical history, household income, housing and amenities, types of fuel used, use of incense and mosquito repellent coils, childhood feeding practices (till age 10), food habits one year ago and twenty years ago, other personal habits (alcohol and tobacco use, medicaments). Information pertaining to NPC diagnoses was abstracted from records at the Wuzhou Cancer Institute and from the Zangwu Cancer Unit. 34
Analysis
Conditional logistic regression was used to estimate odds ratios and 95% confidence limits. Computations were performed using the EGRET statistical package (version
0.23.21). Estimates were adjusted for other covariates as indicated; otherwise the reported results are from univariate analyses. For comparison and as a check on the results for early childhood feeding, a separate analysis was also performed on the subset of subjects who had one or both parents listed as a household member. Separate analyses were also attempted for the two strata with positive and negative family history of NPC. Unconditional logistic regression was used in the latter instance, with dichotomous age variable (above and below 35 yrs.) and gender variable included in the regression models.
RESULTS
Eighty-eight cases of NPC diagnosed between Jan 1,
1986 - Dec 31, 1987 were recruited into the study. Of these, 29 were Wuzhou residents and 59 were Zangwu residents. All 88 cases (24 female, 64 male) were from the
Han ethnic community. Histologically, 86 cases were diagnosed with poorly differentiated carcinomas, and 2 with undifferentiated carcinomas. Age at diagnosis ranged from
17 years to 59 years; mean age was 41.3 years (38.3 for 35 females, and 42.5 for males; p = 0.07). Mean age for controls was 41.4 years. Age-matching to within ± 5 years was attained for 83 out of the 88 matched sets. In the remaining five sets, controls were 7, 12, 12, 16 and 16 years distant from their index cases.
Two of the 88 cases, and ten of the 176 controls reported their birthplace as outside Guangxi, mostly in
Guangdong. The relative risk associated with birthplace in
Guangdong was 0.2 (0.02, 1.7).
17 cases and 17 controls reported cancer diagnosed among family members. NPC among family members, in particular, was reported by 11 cases, and 6 controls. The relative risk (RR) for NPC, for a person with a family member previously diagnosed with this cancer was 6.4 (1.8 ,
23.3). The mean age at diagnosis for NPC cases with a family history of NPC (8 males and 3 females) was 36.7 years. Average age for the eight males was 41.3 years, for the three females 24.7 years (p = 0.02). Mean age at diagnosis for NPC cases without family history of NPC was
42.0 years. Mean age for these males was 42.7 years, and for females, 40.3 years.
Subject's level of schooling was not a significant risk factor (trend test p = 0.84). Mother's primary education was associated with a sixty percent risk reduction
(vs. no education), but this too was not significant. 36
None of the broad occupational categories
(agricultural, industrial, sales & administrative, military) were associated with risk. This remained so even when number of years worked was taken into account. The category
"other occupation" however had a significant positive trend with number of years worked (p = 0.02). The nine NPC patients included in this category were engaged in diverse occupations : carpenter, pharmacist, gold-mine worker, vehicular drivers, auto mechanic, security guards, dumpling vendor. Occupational exposures to smoke, fumes, pesticides, fertilisers, and other chemicals were not significant risk factors.
There was increasing risk for NPC as incomes declined
(linear trend for income per head p = 0.06} (Table 1}. The observed trend however is based on current income information, so the effect in part might be due to reduced income arising from the illness.
Larger sized households in childhood were associated with reduced risk (linear trend p = 0.02} (Table 1). Childhood spent in a rural-type house was also a significant risk factor-- relative risk 3.7 (1.0, 13.0}. Individual beds during childhood was associated with a twofold, significant risk-- RR 2.0 (1.1, 3.7).
Poor ventilation, absence of house windows or chimneys, and smoky interiors during childhood were not 37
significant risk factors. Nor were location of kitchen in
the living room area, use of incense and mosquito repellent
coils, and type of fuel used domestically associated with
risk.
weaning Diet
Breast feeding was a universal practice during
infancy of cases and controls. Use of powdered milk and
other forms of non-human milk was virtually nonexistent.
Anthropological observations had indicated that various herbal and cereal broths were fed by Cantonese mothers to
their newborn to make them nurse better. The use of such herbal mixtures did not appear as a significant risk factor
in this population. Mean age at introduction of solid foods was 19.7 months for cases and 19.2 months for controls.
(Solid foods in the questionnaire was interpreted to exclude
semi-solid weaning foods such as rice porridge, hence the discrepancy with weaning ages indicated below). Adult pre mastication of foods, which could favour EBV transmission at an early age, was not a risk factor (Table 3). Teething
foods which are occassionally used in other southern Chinese communities (such as fermented green onions) was not mentioned by any of the respondents.
Weaning age was not a significant risk factor -- the mean age at weaning for cases was 15.9 months, and for 38
controls, 17.1 months. A typical weaning staple would
consist of rice porridge to which might be added fish (fresh
or dried & salted), pork (fresh or salted & spiced),
vegetables (fresh or dried or salted/pickled). The relative
risk associated with weaning use of salted vegetables was
0.7 (0.4 , 1.3), and for pickled vegetables, 0.9 (0.5, 1.6)
(Table 2). Weaning use of dried salted fish however was a
significant risk factor RR 2.6 (1.3 , 4.9). No other weaning foods were significantly associated with risk.
Post-weaning Diet (age 2-10 years)
Between ages 2 and 10, salted fish consumption, in
soup, or cooked in rice porridge, was associated with
significant risk (Table 3). Salted fish as a side dish,
either steamed or fried in oil, did not appear to be a risk
factor. Weekly consumption of salted fish in soup carried a
threefold significant risk (linear trend p = 0.02). Monthly
consumption of salted fish in rice porridge appears to carry more risk -- RR 7.7 (2.5, 23.9) --than weekly consumption.
Only this last result remained significant when estimates were adjusted for weaning use of salted fish.
Pickled vegetable consumption in the post-weaning period was not a significant risk factor. Snack foods such
as preserved plums, melon seeds, fruit, or salted olives were also not associated with risk. 39
Table 4 compares the relative risks for selected food items in early childhood, with the results from a subset analysis in which one or both parents were listed as a household member. The point estimates are largely similar, except for weekly consumption of salted fish soup.
Adult Diets Two Decades Ago
For adult diets, respondents were queried about foods consumed a year ago and foods consumed two decades ago.
Consumption of salted fish two decades ago carried a relative risk of 2.1 (1.0, 4.1) (Table 5). This risk increased slightly after adjusting for weaning use of salted fish. Consumption of herbal teas twenty years ago also emerged as a significant risk factor-- RR 4.0 (1.4, 11.3).
Preserved vegetable consumption during this period was not a risk factor. Recent consumption of fresh fruits was associated with declining risk (linear trend p = 0.03); effects for leafy vegetables and fruit vegetables however were not estimable in this population (most cases and controls consumed these on at least weekly frequency).
Recent use of chives was associated with a marginally protective effect-- RR 0.4 (0.2, 1.0). Among households who made their own brine-preserved vegetables, frequency of changing the brine solution was not associated with risk.
None of the usual cooking oils and fats used (most 40 commonly peanut oil and other vegetable oils, pork fat) were associated with significant risk. Aside from recent use of monosodium glutamate and recent use of garlic, which were both associated with a fifty percent risk reduction, none of the other spices, sauces and other condiments (including fermented bean pastes) were associated with risk.
None of the beverages queried (unboiled water, tea, plant juices, aerated drinks, beer, other alcoholic drinks) were associated with risk.
There is a weakly suggestive dose response relationship for cigarettes smoked per day, in the recent past (linear trend p = 0.09), but not for total number of years smoked. Ever-smokers had 1.9 times the risk of never smokers, but this was not statistically significant.
Fathers' current smoking status was associated with a fourfold but not significant risk for the children.
In a multivariate analysis of the dietary factors identified above, 3 items remained significantly associated with risk : weaning use of salted fish, salted fish in rice porridge between ages 2-10, and salted fish two decades ago
(Table 6). Recent consumption of fresh fruits and MSG remained associated with reduced risk. 41
DISCUSSION
Family clustering of NPC cases has been reported on a number of occassions (Liang, 1964; Jung, 1965; Pang, 1965;
Ho, 1972a,b; Williams & de The, 1974; Lanier et al., 1979).
These observations are consistent with heritable susceptibility as well as with other exposures common to household family members. A recent HLA-linkage study has produced evidence for the existence of disease susceptibility gene(s) closely linked to the HLA region (Lu et al., 1990). The magnitude of the relative risk for the homozygous recessive -- 20.9 (5.1, infinity)-- and the possible range of the allelic frequency suggest that it might explain a large portion of the high risk among southern Chinese.
In the present study, the relative risk associated with NPC family history, a much less precise proxy for genotype, was 6.4. This relative risk was not appreciably changed by inclusion in the regression model other risk factors such as dietary exposures. If family history of NPC were a crude indicator of inherited susceptibility, one might expect a younger age-at-diagnosis for cases with family history. The observed difference between patients with and without family history of NPC is not large -- 36.7 vs. 42.0 years -- and could be partially accounted for by early detection bias since there was an active program of 42
early detection and treatment of NPC in the study area.
However, five of the eleven cases with family history were
diagnosed at stage II (45%), and the rest at stage III. On
the other hand, sixty of seventy six cases without family history were diagnosed at stage II or earlier (86%). The
age difference at diagnosis between the two subgroups is
primarily due to the young ages of female cases with family history of NPC (mean 24.7 years, versus 41.3 years for males with family history). This is intriguing given that males
have 2-3 times the risk of females.
It would be of interest to examine separately, for
the subjects with and without family history of NPC, the
relative risks associated with dietary and other exposures.
However, analyses stratified in this manner did not yield
estimable results. Analysis for interaction pointed towards
lower relative risks associated with salted fish and herbal tea consumption among those with family history of NPC, but the results were not significant. Epstein-Barr virus
serology at diagnosis, available only for the cases, showed
lower anti-VCA/IgA geometric mean titers for cases with NPC
family history (78.4), compared to cases without family history (124.0) (p = 0.43).
Among dietary exposures, weaning use of salted fish again emerges as a risk factor. Furthermore, the 43 susceptible period appears to persist into post-weaning childhood and even into early adulthood. The possibility of interviewer bias and recall bias cannot be excluded, since the salted fish hypothesis is widely known among Chinese health personnel in the study area, if not among the general populace. Nonetheless, the plausibility of salted fish as etiological agent gains support from the confirmation that malignant nasal cavity tumors can be induced in Wistar rats fed on salted fish at concentrations comparable to that in
Cantonese diets (Yu et al., 1989b, Huang et al., 1978).
These food items have also been analysed for volatile nitrosamines, before and after nitrosation, and corresponding genotoxicities assayed in s. t:yphimurium strains and by the SOS chromotest (Poirier et al., 1987, 1989).
Levels of N-nitrosodimethylamine (NDMA) in particular were higher (and for hard salted grouper, much higher) than levels in Western preserved (or fresh) fish products and in other foods. On the other hand, similarly high levels of
NDMA have been reported for dried and salted fish products in Japan, a low-incidence region for NPC (Kawabata et al.,
1979). Genotoxicity tests on samples of hard salted and dried grouper, and soft salted and dried Japanese mackerel, showed weak direct-acting genotoxicity in the sos chromotest for both fish varieties. Neither sample however was mutagenic in Salmonella TA 98. Chemical ni trosation increased 44 the genotoxic effect in the sos chromotest.
Activation of EBV latent infection by preserved foods from the study area has also been investigated (Shao et al.,
1988). Three of eleven samples of preserved seafoods (two squid samples and one sample of dried salted Japanese mackerel) contained substances that activate EBV latency in
Raji cells. A serological follow-up study in Zangwu had indicated that abrupt rise in anti-VCA/IgA was a marker for risk of NPC (de The & Zeng, 1986), but the etiological significance of EBV reactivation is unclear since it appears to occur quite close to clinical diagnosis.
Salted vegetables (most commonly, mustard greens), and pickled vegetables (usually Chinese cabbage) did not appear as significant risk factors in this study even though volatile nitrosamine levels had been found elevated, but not to the levels observed for salted fish varieties (Poirier et al., 1987, 1989). We also failed to confirm earlier reports associating risk with other preserved foods such as salted duck eggs, fermented black bean and soybean pastes, fermented bean curd, fermented fish sauce, and preserved plums (Yu et al., 1988, 1989a). We did find however reduced risk associated with recent consumption of fresh fruits, chives, and MSG. 45
The social profile of risk may include low income and a rural childhood, in line with earlier observations associating risk with poorer, traditional lifestyle (Geser et al., 1978). It is also possible that the broader range of salted and preserved foods now suggested as NPC risk factors may be proxies for other exposures related to a poorer or traditional lifestyle. It is intriguing however that large household size and crowded sleeping arrangements in childhhood were associated with reduced risk. This parallels the finding of higher risk in smaller families for
Hodgkin's disease (Gutensohn & Cole, 1981), another illness for which there is some evidence of EBV involvement (Mueller et al., 1989). 46
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Table 1 Childhood Living Conditions, Current Income & Risk for NPC
Cases Controls Relative Risk*
Childhood living conditions
Type of House apt. building 13 38 1.0 level house (urban) 14 26 2.1 (0.7 , 6.6) rural house 59 111 3.7 (1. 0 , 13.0)
Number of household memberst 1-5 37 53 1.0 6-10 34 79 0.6 (0.3 , 1.1) > 10 5 22 0.3 (0.1 , 0.9)
Individual beds no 48 117 1.0 yes 40 59 2.0 (1.1 , 3.7)
Total household income (current) : < 400 yuan/mo. 58 108 1.0 400-800 yuanjmo. 25 51 0.9 (0.5 , 1. 7) > 800 yuan/mo. 5 16 0.6 (0.2 , 1. 7)
Income per head (current)* < 50 yuan/person/mo. 29 42 1.0 50-100 yuan/person/mo. 41 87 0.7 (0.3 , 1. 2) > 100 yuan/person/mo. 16 46 0.5 (0.2 , 1.1)
* relative risks from univariate conditional logistic regression; 95% confidence limits in parentheses trend test p = 0.02 trend test p = 0.06 58
Table 2 Weaning Foods & Risk for NPC
Cases Controls Relative Risk*
Age at weaning 0-12 months 51 99 1.0 13-24 months 33 59 1.1 (0.6 2.0) ' > 24 months 3 17 0.3 (0.1 1. 2) ' Weaning Foods
salted dried fish no 63 150 1.0 yes 24 21 2.6 (1. 3 4.9) ' salted pork no 78 162 1.0 yes 9 10 2.1 (0.7 5.7) ' fresh vegs. no 27 62 1.0 yes 60 110 1.5 (0.8 2.8) ' salted vegs. no 32 49 1.0 yes 55 119 0.7 (0.4 1. 3) ' pickled vegs. no 43 81 1.0 yes 41 83 0.9 (0.5 1. 6) ' dried vegs. no 54 104 1.0 yes 33 68 1.0 (0.5 1. 7) ' dried dates no 77 155 1.0 yes 10 17 1.1 (0.4 2.8) '
relative risks from univariate conditional logistic regression; 95% confidence limits in parentheses 59
Table 3 Childhood Diet (age 2-10 yrs) & Risk for NPC
Cases Controls Relative Risk*
Age began solid foods 0-6 months 12 19 1.0 7-12 months 22 so 0.6 (0.2 ' 1.8) 13-24 months 38 78 0.7 (0.2 ' 1.9) > 24 months 15 26 0.8 (0.2 ' 2.8)
Adult pre-mastication of food no 21 49 1.0 yes 66 123 1.3 (0.7 ' 2.6)
Post-Weaning Diet (age 2-10 years)
Salted fish (steamed or fried) rarely 31 75 1.0 monthly 34 57 1.4 (0.8 ' 2.6) weekly 22 44 1.2 (0.6 ' 2.5) Salt fish soupt rarely 71 160 1.0 monthly 7 9 1.8 (0.6 ' 5.0) weekly 9 7 3.2 (1.1 ' 10.0)
Salt fish in rice porridge rarely 62 157 1.0 monthly 15 6 7.7 (2.5 ' 23.9) weekly 10 13 2.0 (0.9 ' 4.8)
Pickled vegetables rarely 21 40 1.0 monthly 19 45 0.8 (0.4 ' 1. 7) weekly 48 90 1.0 (0.5 ' 1.9)
Snack Foods :
preserved plums no 84 166 1.0 yes 4 8 0.9 (0.3 ' 3.3)
melon seeds no 76 162 1.0 yes 12 12 2.0 (0.9 ' 4.6) 60 salted olives no 73 157 1.0 yes 15 17 1.9 (0.9 , 4.1)
* relative risks from univariate conditional logistic regression; 95% confidence limits in parentheses
trend test p = 0.02 61
Table 4 Childhood Dietary Risks for NPC -- Estimates for Subjects with Living Parent(s) Compared to All-Subject Estimates
Subjects with Living Parent(s) All Subjects (53 matched sets) (88 matched sets)
Rel. Risk (95% c.i.) Rel. Risk (95% c.i.)
Weaning Foods :
salted dried fish 2.3 (0.8 ' 6.6) 2.6 (1.3 ' 4.9)
salted vegetables 0.5 (0.2 ' 1.1) 0.7 (0.4' 1.3)
pickled vegetables 1.0 (0.5 ' 2.1) 0.9 (0.5 ' 1.6)
Post-Weaning Diet (age 2-10 years)
Salted fish (steamed or fried) rarely 1.0 1.0 monthly 1.1 (0.5 2.6) 1.4 (0.8 2.6) ' weekly 0.7 (0.2 ' 2.0) 1.2 (0.6 2.5) ' ' Salt fish soup rarely 1.0 1.0 monthly 1.4 (0.3 7.2) 1.8 (0.6 5.0) weekly 1.0 (0.2 ' 6.1) 3.2 (1.1 ' 10.0) ' ' Salt fish in rice porridge rarely 1.0 1.0 monthly 4.1 (0.8 22.3) 7.7 (2.5 23.9) weekly 1.5 (0.4 ' 6.1) 2.0 (0.9 ' 4.8) ' ' Pickled vegetables rarely 1.0 1.0 monthly 1.0 (0.3 2.9) 0.8 (0.4 1. 7) ' weekly 1.1 (0.4 ' 2.7) 1.0 (0.5 1. 9) ' ' 62
Table 5 Adult Diet (Two Decades Ago) & Risk for NPC
Cases Controls Relative Risk*
Preserved foods
salted fish no 18 56 1.0 yes 68 116 2.1 (1. 03 4.1) ' preserved pork no 69 144 1.0 yes 17 28 1.3 (0.6 2.7) ' salted eggs no 76 159 1.0 yes 12 17 1.6 (0.7 3.8) ' preserved vegs no 19 44 1.0 yes 67 127 1.3 (0.6 2.7) ' preserved tubers no 62 130 1.0 yes 24 42 1.2 (0.6 2.3) ' Spices & condiments :
tao si (black bean paste) no 56 114 1.0 yes 32 62 1.1 (0.6 1. 9) ' ferm. fish/shrimp paste no 88 175 yes 0 1 *** NO CONVERGENCE ***
fish sauce no 88 176 yes 0 0 *** NO CONVERGENCE ***
spice mixture no 86 169 1.0 yes 2 7 0.5 (0.1 2.8) ' 63
Other soyabean products
tao fu ~ l-2x /mo. 68 133 1.0 ~ l-2x /wk. 20 43 0.9 (0.4 , 1. 8) ferm. soya curd ~ l-2x /mo. 85 165 1.0 ~ l-2x /wk. 3 11 0.5 (0.2 , 2.0)
soyabean paste ~ l-2x /mo. 88 175 ~ l-2x jwk. 0 1 *** NO CONVERGENCE ***
soyabean sheet ~ l-2x /mo. 85 167 1.0 ~ l-2x /wk. 3 9 0.6 (0.2 , 2.5)
soyabean milk ~ l-2x /mo. 82 168 1.0 ~ l-2x /wk. 6 8 1.6 (0.5 , 4. 7)
Fruits and vegetables
leafy vegetables rarely 1 1 monthly 0 0 *** NO CONVERGENCE *** weekly 87 175
fruit vegetables rarely 2 0 monthly 9 12 *** NO CONVERGENCE *** weekly 77 164
root vegetables rarely 1 0 monthly 15 21 *** NO CONVERGENCE *** weekly 72 155
green onions rarely 29 64 1.0 monthly 31 49 1.5 (0.7 , 2.9) weekly 28 63 1.0 (0.5 , 2.0)
fresh fruitst rarely 16 21 1.0 monthly 41 85 0.6 (0.3 , 1. 3) weekly 31 70 0.5 (0.2 , 1. 2) 64
dried fruits rarely 79 151 1.0 monthly 7 21 0.6 (0.2 1. 6) weekly 2 4 0.9 (0.1 ' 5.8) ' Herbal teas no 56 130 1.0 yes 32 46 4.0 (1.4 11.3) '
* relative risks from univariate conditional logistic regression; 95% confidence limits in parentheses trend test p = 0.23 (trend test for recent consump. fresh fruits, p = 0.03) 65
Table 6 Multivariate Analysis of Dietary Risk Factors for NPC
Relative Risk (95% c.i.)
Weaning use of salted fish 2. 8* (1.1 7.0) ' Salt fish soup (age 2-10 years) monthly 0. 2t (0.03 1. 9) weekly 2.4 (0.4 ' 14.0) ' Salt fish rice porridge (age 2-10 years) monthly 15. 6t (2.4 100.6) weekly 1.1 (0.3 ' 3.9) ' Salt fish (2 decades ago) 3. 5* (1.4 8.8) ' Herbal tea (2 decades ago) 2.4* (0.7 8.5) ' Fresh fruits (1 year ago) monthly 0. 4t (0.1 1. 5) weekly 0.3 (0.1 ' 0.9) ' Chives (1 year ago) 0.4* (0.1 1. 2) ' MSG (1 year ago) 0. 3* (0.1 0.7) ' Garlic (1 year ago) o. 5* (0.2 1. 8) '
* baseline category no consumption
baseline category rarely consumed 66
THE EPIDEMIOLOGY OF NASOPHARYNGEAL CARCINOMA
A Review
Chee Khoon Chan, M.S.
Department of Epidemiology Harvard School of Public Health Boston, Massachusetts U.S.A.
C.K. Chan was supported by a staff training fellowship from the Science University of Malaysia. 67
1. Introduction
The outstanding feature of the epidemiology of nasopharyngeal carcinoma (NPC) is its high incidence among a
few communities around the world -- ethnic Chinese from the coastal provinces of southern China, Eskimo and other Arctic natives, and Maghrebian Arabs from north Africa. This markedly uneven distribution, ethnically and geographically, has been the rationale for studies looking into heritable susceptibility, distinctive cultural practices, and ecologic exposures co-extensive with high incidence regions.
Three main risk factors have been identified. The Epstein-Barr virus (EBV) is consistently associated with undifferentiated and poorly differentiated NPC. Patients have a distinctive anti-EBV serological profile and regardless of geographic origin, EBV-DNA sequences have been consistently detected in NPC biopsies, specifically in the anaplastic or poorly differentiated carcinoma cells.
Biopsies from carcinomas at other sites of the head and neck and from other types of tumors of the nasopharynx have been mostly negative for EBV-DNA. EBV however is ubiquitous, and the lack of evidence correlating strain differences or age at primary infection with geographic variation in NPC rates suggests that additional risk factors are important. A disease susceptibility locus closely linked to the HLA region has been identified from HLA linkage studies. Among 68 lifestyle factors, weaning use of salted fish has been the most consistent finding reported for southern Chinese communities. Two recent studies in Guangxi and Guangzhou suggest that weaning use of other salted and preserved foods may also be risk factors.
2. Histopathology
Nasopharyngeal carcinoma is a malignant epithelial tumour arising from the epithelium lining the surface and crypts of the nasopharynx. This epithelium is a mosaic of patches of stratified squamous epithelium, pseudo-stratified columnar ciliated epithelium, and epithelium intermediate in features between the two. NPC may arise from any of these types of epithelia (Shanmugaratnam & Muir, 1967).
Histologically, three subtypes of NPC are recognised by the
World Health Organisation classification scheme : squamous cell carcinoma, nonkeratinizing carcinoma, and undifferentiated carcinoma (Shanmugaratnam & Sobin, 1978).
There is continuing debate on the epidemiologic and biological significance of these histopathologic variants.
One view that NPC is a histologically homogeneous neoplasm derives from the observation that all histologic subtypes, including undifferentiated NPC with no evidence of squamous differentiation by light microscopy, consistently show ultra-structural evidence of squamous differentiation 69 by electron-microscopy (Svoboda et al., 1965). The five year survival rate for squamous cell carcinoma is poorer than that for nonkeratinizing carcinoma and undifferentiated carcinoma combined, but there are no other significant correlations between histologic subtype and distribution of
NPC patients by age, sex, or HLA antigen profiles, or cell mediated immune status (Shanmugaratnam et al., 1979). There are however conflicting reports on whether the association of NPC with the EBV is confined to undifferentiated and poorly differentiated NPC (Andersson-Anvret et al., 1977) or whether it extends to differentiated NPC as well (de
Schryver et al., 1969; Shanmugaratnam et al., 1979: Raab
Traub et al., 1987).
In moderate- and high-incidence regions such as
Singapore, Taiwan, Tunisia, and Uganda, nonkeratinizing NPC together with undifferentiated NPC constitute 80% or more of diagnosed nasopharyngeal carcinomas. In the United States,
UK, and France, the corresponding proportions range from 33% to 73% (Shanmugaratnam, 1982).
3. Ethnic & Geographic Distribution
The highest rates of NPC are found among the southern
Chinese, where incidence of the order 20 - 30 per 100,000 person-years among males, and 8 - 12 per 100,000 among females are typical. These rates, age-adjusted to the world 70 population, are fifty to a hundred times higher than those recorded for most other parts of the world (table 1). In
Guangdong Province, NPC ranks third for cancer mortality among men (fourth among women), accounting for 15% of male cancer deaths and 11% of female cancer deaths (Nat'l Cancer
Control Office, 1979). Within the southern China region
itself, there is considerable variation in rates (Li et al.,
1985). The Cantonese dialect group have the highest
incidence, approximately two to three times the rates among
Fukienese, Teochew, and Hakka dialect groups. In the
Guangxi Autonomous Region, an ethnically diverse province, the contrast is especially striking -- Han Chinese
(predominantly cantonese-speaking) make up only 40% of the province's population, but account for the overwhelming majority of NPC cases diagnosed. Shanghai, which is on the coast of central China, has about a fifth the incidence of
Guangzhou (Canton).
Eskimo and other native communities in the Arctic are also at high risk (Lanier et al., 1980), and there is moderate incidence among Maghrebian Arabs in North Africa
(Muir, 1972), and among indigenous peoples in S.E. Asia
(Shanmugaratnam, 1982).
Unlike the age-specific incidence for most other cancers of the respiratory, urinary, and gastro-intestinal 71 tract which rise abruptly and steeply from the sixth decade on, NPC age-incidence in the endemic regions begins a more gentle rise from about age 20, reaches a plateau at about
45-55, and thereafter declines. There is little to suggest that this is due to a cohort effect. such an age-incidence curve is consistent with early, age-related etiologic exposures, decreasing susceptibility of older subjects, or exhaustion of susceptibles from the population. Among North
Africans, the age-incidence has a bi-modal distribution with an early peak in the second decade of life and a more pronounced one in the fifth decade (Cammoun et al., 1971).
Whether these two peaks correlate with differences in histologic features, exposure patterns, or familial tendency is not known. In low incidence regions, the age-incidence curve approximates the shape more typically seen for other cancers.
The sex differential in incidence -- two to three fold higher among males -- is seen worldwide and across the age spectrum. If this reflects a gender differential in exogenous exposures rather than a sex-related host response, it would have to be quite stable across different cultures and socioeconomic settings.
4. Migrant studies
Incidence rates among people of southern Chinese 72 descent in Hong Kong (Waterhouse 1987), Taiwan (Lin et al.,
1971), Singapore {Shanmugaratnam et al., 1987) and Malaysia
(Armstrong et al., 1979) are comparable to those recorded for southern China, when adjusted for dialect-group composition. This does not necessarily argue for a heritable disposition per se since diet and many other aspects of lifestyle have been retained by these migrant communities. It is noteworthy however that in Singapore, where immigration from southern China had virtually ceased by the 1950s and the highest-risk age group are now typically of third generation, NPC incidence has remained stable for at least the last fifteen years {Lee et al.,
1988). Indeed, Singapore-born Chinese appear to have slightly higher risk than foreign-born Chinese in Singapore.
Singaporean Indians, on the other hand, have retained the very low risk of Caucasoid populations despite a comparable length of settlement on the island. In California and
Hawaii however, NPC mortality rates among second generation
US-born Chinese is about half that of foreign-born Chinese
Americans, but still remains substantially above Caucasian rates (Zippin et al., 1962; King & Haenszel, 1973; Buell,
1974; Fraumeni & Mason, 1974; Yu et al., 1981). This on the other hand is not clearcut evidence of modifiable, nonheritable risk factors since the Chinese-American population is of more diverse province of origin and the 73 decline in NPC mortality is entirely within the range of geographic variation of NPC rates in China.
s. Inherited susceptibility
The high risk of NPC among southern Chinese is consistent with an inherited susceptibility as well as with other exposures peculiar to high-risk communities. Migrant studies have neither ruled out a strong inherited disposition nor a substantial contribution by non-inherited exposures. Likewise, family clustering of NPC patients
(Liang, 1964; Jung, 1965; Pang, 1965; Ho, 1972a,b; Williams
& de The, 1974; Lanier et a1., 1979) is consistent with heritable susceptibility as well as with other exposures common to household family members. A striking family pedigree was reported by Ho (1976) in which NPC diagnoses extending over three generations included a father and six of his seven children from one spouse.
HLA associations with NPC involving A and B locus antigens were first reported by Simons et al (1974).
Subsequent studies showed that the A2-Cw11-Bw46 haplotype was a significant risk factor among Singaporean Chinese aged
30 and above (relative risk (RR) = 3.4, p < 0.001), while the Bw58 antigen was a significant risk factor for subjects aged 30 and below (RR = 3.8, p < 0.001) (Chan, S.H. et al.,
1983) (table 2). The same A and B locus antigens were 74 consistently also markers for risk among Chinese in
Malaysia, Hong Kong, California and China (Simons &
Shanmugaratnam, 1982). The Bw46 allele in particular is common among southern Chinese (12% gene frequency among
Singaporean Chinese), less common in other parts of China, and absent among Caucasians, Indians and Africans (Chan,
S.H., 1990). Furthermore, the A2-Cwll-Bw46 haplotype is invariably associated with the DR9 antigen among controls, this being one of the extended haplotypes common among southern Chinese. Among NPC patients with the A2-Cw11-Bw46 haplotype however, more than 75% were DR9 negative (Chan,
S.H. et al, 1986). This implies that the genomic region between the B and DR loci, on chromosome 6, might be of particular importance for risk among southern Chinese.
The findings for other ethnic groups are more preliminary : among Thais, the Bw46 antigen is also associated with NPC (RR = 2.5). Among Malays, the associations were with Bw58 and B18, and among Northern
Chinese, Bw35 (RR = 3.7) (Chan et al., 1985, 1986). A
Tunisian study reported no associations with A-locus antigens, but a deficit of detectable antigens at the B locus (Betuel et al., 1975).
A HLA-linkage study among southern Chinese has produced strong evidence for the existence of disease 75 susceptibility gene(s) (DSG) closely linked to the HLA region (Lu et al., 1990). A recessive genetic model which provided the best fit estimated a relative risk of 20.9 (95% confidence limits : 5.1, infinity) for the homozygous recessive. Together with the possible range of the allelic frequency, the putative DSG could account for a large portion of the high risk among southern Chinese. The mechanisms through which a putative DSG could operate are at this point speculative. If the inherited susceptibility is expressed through host response to EBV, it could operate through cellular or humoral immune responses, or viral interaction with the target cell. It is interesting to note that tumor necrosis factor (TNF) a and ~ genes map to the HLA region between the B and DR loci, although one might expect TNF dysfunction to result in broader susceptibility to a range of tumors. There is one report from Alaska that siblings of NPC patients are at higher risk for a variety of other cancers (Ireland et al., 1988). Other possibilities involving regulation of epithelial cell differentiation and control of viral gene expression, as well as tissue susceptibility to chemical carcinogens were also considered by Lu et al. studies of oncogene expression in NPC tissue, and restriction fragment length polymorphism (RFLP) analyses of the genomic regions of interest, are underway in a number of laboratories. 76
6. Epstein-Barr Virus
The EBV is a ubiquitous herpesvirus which has been shown serologically to be present in every population thus far tested (Evans, 1989). In many developing countries,
EBV-antibody prevalence exceeds 80% by age five, and becomes practically universal shortly thereafter. In the industrialised countries, or more precisely, in higher socioeconomic settings, age at infection tends to be later.
In studies of entering college students in the 1960s in the u.s., England and New Zealand for instance, EBV-antibody prevalence ranged from 26% to 60% (Evans, 1974). EBV infection in young children is mostly mild and without acute symptoms. Primary infection in young adulthood however has been causally linked to infectious mononucleosis (IM)
(Evans, 1969, 1974).
The major route of transmission of EBV is probably through intimate oral contact and exchange of saliva
(Hoagland, 1955; Evans, 1960). Among young children in poorer and more crowded settings, virus spread is probably through infected saliva on fingers, toys, and other inanimate objects, or from adult pre-chewing of infant food.
The virus life cycle is believed to involve at least two phases -- a latent phase in less differentiated lymphoid and epithelial cells, and a lytic, replicative phase in 77 terminally differentiated or senescent cells (Lemon et al.,
1977; Sixbey et al., 1983, 1984; Crawford & Ando, 1986).
Virus shedding occurs primarily from the oropharyngeal
epithelium (Lemon et al., 1977; Sixbey et al., 1984), and possibly from the cervical epithelium as well (Sixbey et
al., 1986). There is evidence from in vitro studies that the
latent virus can exist in an episomal form as well as
integrated into the host genome (Dambaugh et al., 1986).
Viral gene expression in the latent state is currently known to be restricted to six nuclear-binding proteins (EBNA's 1-
6), three membrane antigens (LMP1, TP1, TP2), and other viral transcripts yet to be fully characterised. The EBV early antigen complex (EA), and the viral capsid antigen
(VCA) are expressed during the lytic, replicative cycle.
6.1 Epstein-Barr Virus and Nasopharyhgea1 carcinoma
The association between EBV and NPC was first suggested by the reactivity of sera from NPC patients to antigens extracted from cultured Burkitt's tumor cells (Old et al., 1966). Subsequent serologic studies confirmed that
NPC patients from around the world have at diagnosis a distinctive EBV serological profile characterised by high
IgG and IgA titers against the VCA and the diffuse component of the EBV early antigen complex (EA-D) (de Schryver et al., 1969, 1974; Henle, w. et al., 1970, 1973; Lin et al., 1971b; 78
Henderson et al., 1974; Wara et al., 1975; Henle, G. et al.,
1977) (table 3). Elevated titers against the EBV-encoded
DNase and thymidine kinase -- two components of the EA complex-- have also been reported (Cheng et al., 1980; de
Turenne-Tessier et al., 1989; Littler et al., 1990). In contrast, healthy controls and patients with carcinomas of the oropharynx or hypopharynx or tumors of the nasopharynx other than carcinomas usually have much lower titers. The level of elevated titers in NPC moreover correlate with disease progression from early to late stages -- successful treatment is accompanied by loss of anti-EA(D) and a gradual decline of anti-VCA (Henle, w. et al., 1977). Nucleic acid hybridisation studies have consistently demonstrated the presence of EBV-DNA in NPC biopsies (zur
Hausen et al., 1970, 1974; Nonoyama et al., 1973), and specifically in the anaplastic or poorly differentiated carcinoma cells (Wolf et al., 1973, 1975; Desgranges et al.,
1975a, 1975b). Biopsies from other tumors of the nasopharynx (other than carcinomas) have been negative for
EBV-DNA (Andersson-Anvret et al., 1977), but viral DNA has been detected in tumors at other sites of the head and neck
--carcinomas of tonsillar (Brichacek et al., 1984), supraglottic laryngeal (Brichacek et al., 1983), parotid
(Saemundsen et al., 1982), and thymic (Leyvraz et al., 1985) epithelia. The consistency of these associations however 79 have yet to be established. The EBNA complex is also detected in anaplastic NPC cells, but not in the lymphoid elements of the tumor (Wolf et al., 1973, 1975; Huang et al., 1974; Klein et al., 1974; Desgranges et al., 1975).
6.2 EBV-Epithelial Cell Interaction
Although EBV exhibits marked B-lymphotropism, its replication in epithelial cells of the oropharynx (Lemon et al., 1977; Sixbey et al., 1984), salivary glands (Wolf et al., 1984), uterine cervix (Sixbey et al., 1986) and tongue
(Greenspan et al., 1985), and its presence in nasopharyngeal and other carcinoma cells indicates that the virus has some means of gaining entry into epithelial cells. Putative viral receptors similar to the CR2 molecule on B-cell surfaces have been found on pharyngeal and cervical epithelial cell surfaces (Sixbey et al., 1987; Young et al.,
1986, 1989). The earlier postulated scenario involving cell fusion between EBV-infected B-lymphocytes and epithelial cells (Bayliss & Wolf, 1980; Yiwan et al., 1984) is now considered less plausible.
Attempts at elucidating the virus' activity within the host epithelial cell have been hampered by difficulties in culturing normal epithelial tissue and in establishing cell lines from NPC biopsies. In vitro immortalisation of epithelial cells by the virus has not met with much success 80 either. The available evidence suggests that EBV interacts with host epithelial cells in a differentiation-dependent manner. In fresh frozen sections of ectocervical, tonsillar and nasopharyngeal epithelia (Sixbey et al., 1987; Young et al., 1986), viral receptors are expressed primarily by the less differentiated cells, in particular cells in the proliferating, basal layer. EBNA expression too seems to be limited to mitotically active epithelial cells (Sixbey et al., 1983). The expression of replicative cycle antigens (VCA and EA) and virion production however seem to be confined to the uppermost layers of cells in stratified epithelia, and in recently desquamated cells (Sixbey et al.,
19831 1984) o It appears therefore that the virus exists in a latent state in cells which are actively dividing, the viral episome replicating in synchrony with the host cell. Upon host cell differentiation or senescence however, the virus enters the lytic replicative cycle. This is evidently an evolutionarily successful strategy, judging by the ubiquity of the virus - it furthermore has implications as to how a (mutant ?) virus might interact with a host cell to de regulate cellular growth and differentiation (Dawson et al., 1990). Perhaps too, it might explain the fact that EBV is rarely associated with the more differentiated form of NPC,
81
i.e. it might be due to self-limiting lysis of EBV-carrying
NPC cells which embark on a differentiation program.
There is little information to date on whether the virus integrates into the host genome in epithelial cells in a manner relevant to oncogenesis.
6.3 EBV Gene Expression in Host Cells
EBV gene expression in NPC biopsies and NPC-derived cell lines has been studied at the protein and RNA levels.
In the three main EBV-carrying cell prototypes -- in vitro immortalized lymphoblastoid cell lines (LCL's), EBV- carrying Burkitt's lymphoma cells, and NPC cells viral protein expression is restricted to less than 10 of the approximately 100 genes of the EBV genome. These are the latent cycle antigens of which 6 are nuclear-binding proteins (EBNA's 1-6) and three are membrane-bound proteins
(LMPl, TP1/LMP2, TP2). 82
EBV Gene Expression in Selected Cell Types
EBNA-1 EBNA-2 EBNA-3-6 LMP1 LMP2/TP1*
Lymphoblastoid + + + + + cell lines
Burkitt's l~phoma + (+/-) type 1 cellst
Nasopharyngeal + +/- (+) carcinoma cells
* anti-LMP2/IgG detected in 16/42 NPC sera but not in Burkitt's nor IM sera t early passage Burkitt's lymphoma cell lines
adapted from G. Klein (1989)
There has been some progress in delineating the functions of EBNA-1, EBNA-2 and LMP1 (table 3), but the roles of the latent cycle antigens for the most part are not well understood. Neither virions, nor antigens expressed during the lytic cycle (VCA and EA-D, EA-R) have been detected in NPC cells, with the exception of one report involving a nude-mouse passaged cell line (Trumper et al.,
1977) •
The pattern of latent expression in NPC biopsy tissues and Burkitt's lymphoma cells is much more restricted than that seen in EBV-transformed LCL's (fig. above). In 83 biopsied NPC tissue, EBNA-1 is consistently detected
(Fahraeus et al., 1988; Young et al., 1988). It binds to the site of origin of latent replication of the virus and mediates the replication and maintenance of the viral genome in its episomal state (Sugden & Warren, 1989; Yates et al.,
1985). EBNA-2 however is not detected in NPC or Burkitt's cells. This is noteworthy since the EBNA-2 deletion mutant
P3HR1 is a non-immortalising EBV strain (Dambaugh et al.,
1984), and EBNA-2 in cooperation with LMP1 induces the expression of CD23 (Wang, F. et al., 1987; 1990), a B-cell activation antigen which is cleaved and shed from the cell surface and acts as an autocrine growth factor for LCL's
(Swendeman & Thorley-Lawson, 1987). Other EBNA subtypes (3-
6) are also not detected in NPC cells.
LMP1 is expressed, but only in about half of NPC biopsies examined (Fahraeus et al., 1988; Young et al.,
1988). Since there is evidence that EBNA-2 activates the expression of LMP1 in B-lymphoma cell lines (Abbot et al.,
1990; Wang, F. et al., 1990), the NPC biopsies expressing
LMP1 seem to have an alternate route which circumvents the need for EBNA-2 expression. Transfection experiments have shown that LMP1 can transform rodent fibroblast (Wang, D. et al., 1985) and human keratinocyte cell lines (Fahraeus et al., 1990). Furthermore, LMP1 transfection and expression in a human epithelial cell line was accompanied by changes 84
in cell surface phenotype similar to those observed in NPC cells, and by severe impairment of cellular response to differentiation signals (Dawson et al., 1990).
Other transfection experiments however indicated that
sub-genomic EBV-DNA fragments which did not include coding regions for any of the EBNA subtypes nor for LMP1 could
immortalize primate epithelial cells, although a "fully transformed" phenotype was not established (Griffin & Karran, 1984; Karran et al., 1990). Hence, LMP1 may not be essential for immortalization of epithelial cells, but may play a role in establishing a fully transformed phenotype. It has been proposed from structural and biochemical analyses that the LMP1 molecule could function as an ion channel, or as a growth factor receptor (Knutson & Sugden,
1989) .
A second latent membrane protein, LMP2 (also referred to as TP1) which is encoded across the fused terminal repeats of the viral genome, is also expressed in EBV latently infected lymphoblastoid cell lines, and in
Burkitt's lymphoma cell lines (Frech et al., 1990;
Longnecker & Kieff, 1990; Rowe et al., 1990). Antibodies against LMP2 were detected in sera from 16 of 42 NPC patients, but not in sera from EBV-positive Burkitt's patients, infectious mononucleosis patients, nor from healthy EBV-infected subjects. The antibodies detected were 85 of the IgG type. Anti-LMP2/IgA was also assayed but not detected in any of the NPC sera (Frech et al., 1990).
Studies of viral gene expression at the RNA level, in a nude-mouse passaged NPC, detected transcripts corresponding to coding regions for EBNA-1, LMPl, DNase, possibly the EBV terminal protein (LMP2), antisense transcripts to EBV replicative genes, and other undefined regions of the viral genome (Hitt et al., 1989).
In a different study using cytoplasmic RNA from nasopharyngeal biopsies of healthy individuals and NPC patients, all control biopsies expressed viral RNA in a pattern similar to that for NPC biopsies, except for the absence of small species of EBV-RNA's transcribed off the
EcoRI-J fragment (Tugwood et al., 1987). Furthermore, transcripts associated with productive infection were also detected in a proportion of controls and NPC patients. An earlier study of viral gene expression in a keratinizing NPC
(Raab-Traub et al., 1983) also detected a transcription pattern suggestive of an activated but abortive state of infection. The differentiated state of the tumor may be relevant to the expression of viral replicative genes.
In all these studies, one should bear in mind the possibility that viral gene expression may evolve as the 86 tumor develops. Hence, and quite apart from cell-type specificity, viral gene expression studied in a cross section of time may not render a complete picture of the virus-cell interactions relevant to carcinogenesis.
6.4 EBV Antigens Expressed During Viral Replication :
Relevance to NPC Etiology
The above findings on EBV replicative transcripts in
NPC biopsies recall the paradoxical situation of elevated titers against lytic cycle antigens seen in NPC patients
(VCA and EA-D) at the same time that only latent viral antigens are detectable in NPC biopsies (table 3).
The etiological significance of elevated replicative titers remains unclear. It may be the result of an increasing mass of EBV-carrying tumor cells, or adjacent
EBV-carrying normal tissue, becoming permissive for EBV replication as the tumor progresses. Alternatively, if elevated replicative titers precede tumor progression, EBV lytic replication could be a promoting factor in causing epithelial tissue trauma resulting in enhanced tissue regeneration and increased likelihood of a transforming event. A further possibility is that enhanced lytic replication in the pre-diagnosis period increases the probability of infectious viral particles going on to establish latent existence in cells which have been 87 initiated for further neoplastic development. It has also been suggested that elevated anti-VCA/IgA may favor NPC progression through its blocking effect on the antibody dependent cellular cytoxicity (ADCC) which is mediated by
IgG and directed against EBV-infected tumor cells (Mathew et al., 1981).
6.5 Viral Isolates from EBV-Associated Diseases
Virus isolates from different EBV-associated diseases have not revealed any consistent disease-specific features of the viral genomes (Rymo et al., 1979; Bornkamm et al.,
1980; Lung et al., 1988). These studies employed restriction fragment length polymorphism methods to detect polymorphisms at enzyme cleavage sites. Gross alterations such as large deletions and insertions in principle should also be detectable by such methods.
Two EBV strains (EBV-1 and EBV-2) differing in the genomic region encoding EBNA-2 have been known for some years (Adldinger et al., 1985). The two strains also differ significantly in the EBNA-3A, -3B and -3C coding regions
(Rowe et al., 1989; Sample et al., 1990). The EBV-1 subtype induces higher levels of expression of the B-cell activation antigens CD21 and CD23 and more readily produces a transformed cell phenotype than the EBV-2 subtype (Wang, F. et al., 1990; Rickinson et al., 1987). Although EBV-2 is 88 more common in equatorial Africa and New Guinea than in the
West, EBV-1 is the predominant strain isolated worldwide
from Burkitt's lymphomas, NPC, infectious mononucleosis, and healthy seropositive subjects (Zimber et al., 1986; Young et al, 1987). This however could be an artifact of a virus
isolation protocol which relies on lymphoid immortalisation and therefore selects in favor of the more efficiently transforming strain. Virus strain typing using polymerase chain reaction (PCR) techniques would circumvent this bias and such studies are underway in a number of laboratories.
An EBV strain isolated from a hybrid NPC-adenoid cell line appears to have both transforming and early antigen inducing abilities (Sato et al., 1986, 1988, 1989). Unlike the prototypic P3HR1 virus which induces early antigen on superinfecting Raji cells, it does not possess defective heterogenous DNA. No deletions of unique sequences were found when compared to the B95-8 strain (another viral prototype) although multiple distinctive restriction enzyme polymorphisms were found.
7. Dietary Factors
On the assumption that lower NPC rates among local born Chinese in the United States and Australia implied a modifiable etiologic factor, Ho suggested in 1971 that
Cantonese salted fish, a popular food among southern 89
Chinese, might be a risk factor for NPC. Its pattern of consumption matched the geographic and social class
correlates of NPC --infrequent consumption among northern
Chinese, but a frequent and cheap accompaniment to rice based diets especially among the poorer southern Chinese.
Mixed in with rice gruel, it was a common staple for infants
in the weaning or post-weaning period (Topley, 1973), a
feature pertinent to the relatively early rise in NPC age specific incidence. It was also the kind of dietary practice more persistent among migrant southern Chinese in S.E. Asia than among Chinese in the u.s. or in Australia.
Six case-control studies have been carried out (five in Hong Kong and China, one in Malaysia), all reporting significant associations between childhood consumption of salted fish and risk for NPC (Geser et al., 1978; Armstrong et al., 1983; Yu et al., 1986, 1988, 1989, 1990) (table 4).
The weaning period appeared to be the time of greatest susceptibility -- relative risks (RR) were mostly in the range 2-3 for ever-consumed versus never-consumed. Very high RR's -- 17.4 and 37.7 -- for daily consumption of salted fish in childhood were reported by one group of researchers (Armstrong et al., 1983; Yu et al., 1986). The possibility of interviewer bias and recall bias cannot be excluded, since the salted fish hypothesis was widely known 90 among health interviewing personnel, if not among the general populace. This would have been particularly likely for the later studies. Nonetheless, the plausibility of salted fish as etiological agent gains support from the confirmation that malignant nasal cavity tumors can be induced in Wistar rats fed on salted fish at concentrations comparable to that in cantonese diets (Yu et al., 1989b, Huang et al., 1978). The carcinogenic moieties in salted fish remain unknown. Low levels of pre-formed volatile nitrosamines have been detected in salted fish samples from Hong Kong (Huang et al., 1981). Animal studies indicated that these volatile nitrosamines can induce tumors of the respiratory tract including the nasal and paranasal cavities (Althoff et al., 1974; Pour et al., 1973; Haas et al., 1973; Cardesa et al., 1976). Biochemically, nitrosamines are well known mutagens which achieve their effect through alkylation of nucleotides, particularly at the 0 6-position of guanine, giving rise to 0 6 -alkylguanines which in turn introduce base-pairing errors during DNA replication (Loveless, 1969). The levels of pre-formed volatile nitrosamines in salted fish however are comparable to the levels in cured meats and fish products in Europe and Japan (Tannenbaum et al., 1985; Kawabata et al., 1979). Because N-nitroso compounds can also be produced endogenously from secondary and tertiary 91
amines in fish, and nitrite from bacterial fermentation of nitrate (Ohshima & Bartsch, 1981), volatile nitrosamine
levels after nitrosation have also been assayed in foods
collected from NPC high incidence regions (Poirier et al.,
1987, 1989). Large increases were seen for hard salted and dried grouper, soft salted and dried Japanese mackerel, harissa spice mixture from Tunisia, turnips fermented in brine, and dried fish varieties from Greenland.
The variety of foods assayed was prompted by the
suggestion that dietary risk for NPC might extend to a broader range of salted or preserved foods (Yu et al., 1988,
1989a). In southern China, such foods included salted vegetables, pickled vegetables, salted duck eggs, fermented black bean and soybean pastes, fermented bean curd,
fermented fish sauce, and preserved plums. Such a variety of salted, preserved foods being associated with NPC risk raises the possibility that they may be indicators for low socio-economic status and associated lifestyles, i.e. they may be proxies for other exposures associated with poor livelihood. This is more plausible for low-grade salted fish, and salted and pickled vegetables. Frequent consumption of other preserved foods and condiments however are not necessarily indicators of poor socio-economic standing.
Two of the case-control studies reported reduced risk 92 associated with increased consumption of fruits and vegetables (Yu et al., 1988; Ning et al., 1990). A prospective study of dietary risk factors for NPC is being planned for Singapore. Dietary sources of presumed EBV-activating substances were also investigated following the observation that diterpene esters with in vitro EBV-activating activity were derived from plants whose geographical distribution were co extensive with NPC high-incidence regions in China (Hirayama & Ito, 1981). Many of these plants are members of the
Euphorbiaceae family (C. tiglium, E. lathylis, A. fordii, J. curcas,
E. antiquorum, E. milii, E. pekinensis, E. kansui, D. odora), some of which are used in formulating herbal medicines. None of the preserved vegetable items assayed were significant EBV activators (Shao et al., 1988). More pronounced effects were seen with a few varieties of preserved seafoods from southern China (dried squid and dried salted Japanese mackerel), and harissa spice mixture and qaddid mutton preserve from Tunisia.
Tung oil, extracted from A. fordii, is widely used in southern China and in S.E. Asia for polishing, preserving and waterproofing of wood products, and for waterproofing cloth, paper, umbrellas, bamboo hats, nets, sails and many other consumer items. The suggestion that ecologic exposures of EBV-activators are important -- via airborne 93
particulates and waterborne contaminants -- is hard to
reconcile with the high NPC rates in S.E. Asia, unless the
exposure is more indirect through food or medicaments
originating from southern China.
a. Occupational and Social Class Correlates: Inhalants Case-control studies undertaken in the late 1970s
began to define a social profile of risk -- low
socioeconomic status, eating few fresh foods, little variety
in meals, living in old, poor-quality housing (Armstrong et
al., 1978), belonging to the four lowest occupational
classes, practising a more traditional lifestyle
characterised by Buddhist or ancestor worship, having
religious altars in the house, not eating canned food and bread, and non-use of spices (Geser et al., 1978).
The early attention given to domestic atmospheric pollutants (Clifford, 1967) -- smoke and benzpyrene containing soot in the dwellings of high-incidence Kenyan communities -- or at the workplace, cannot by themselves account for the gross features of NPC epidemiology.
Nonetheless, Henderson and colleagues (1976) found workplace exposure to fumes (RR 2.0, p = 0.006), smoke (RR 3.0, p = 0.008), and chemicals (RR 2.4, p = 0.006) to be risk factors in the Californian population they studied (table 5). Other findings reported from Malaysia (Armstrong et al., 1983) 94
included smoke (RR 6.0, p = 0.006), dusts (RR 4.0, p < 0.001), and from Guangzhou (Yu et al., 1990), products of
combustion (RR 2.4, p = 0.001). studies among low-incidence
European and N. American populations suggested that
occupational exposures to chlorophenols (Hardell et al.,
1982), formaldehyde (Roush et al., 1987; Blair et al., 1987) might also be risk factors.
A serological survey in Guangxi province, China
reported that three NPC cases were diagnosed from a chemical
factory workforce of 216 (Zeng et al., 1983). The factory
workforce also had a high prevalence of anti-VCA/IgA seropositivity (22/216) and all 3 patients came from among
the seropositives. No follow-up to these findings has been
reported however.
The findings on cigarette smoking are variable. No
association was reported by some investigators
(Shanmugaratnam & Higginson, 1967; Henderson et al., 1976;
Shanmugaratnam et al., 1978; Armstrong et al., 1983; Yu et
al., 1986). A subsequent study by Yu et al. (1990) found
significant risk associated with lifetime consumption of 30+ pack-year equivalents-- RR 3.7 (1.2, 11.8), while Lin et
al. (1973) found a greater than twofold significant risk
among subjects smoking 20+ cigarettes a day, compared to nonsmokers. They also found NPC risk associated with 95
working under poorly ventilated conditions, having nasal
symptoms, use of nasal balms and oils, and use of herbal
drugs. Among low-incidence subjects, Mabuchi et al. (1985)
found a significant trend for maximum number of cigarettes
smoked per day previously. Yu et al. (1988) in a subsequent
study found that having a smoker in the household around the time of birth was a risk factor (RR 2.0, p = 0.004).
9. Early Detection of NPC by EBV seroloqical screeninq
Elevated antibody titers against EBV antigens in the
sera of NPC patients have prompted much interest in the
potential of EBV serologic markers -- in particular IgA
antibodies against VCA -- as screening aids in early
detection of NPC (Zeng, 1985a). The data on EBV serology
prior to diagnosis however is sparse, and the temporal
relationship between altered EBV serological profile and
emergence of disease remains obscure.
Ho et al. (1978b) reported on the pre-diagnosis anti
VCA/IgA status of three patients from Hong Kong. One patient had an elevated titer of 1:80 at thirty-five months before diagnosis. The other two patients had pre-diagnosis titers of 1:5 and 1:20 at fifty-five months and thirty months respectively.
Lanier et al. (1980) conducted a study in which sera
from seven Alaskan NPC patients had been collected two to 96 ten years before diagnosis of disease. Three separate sets of controls were chosen for each case the first set matched on age, race, sex, village of residence; the second set comprised Alaskan Natives aged 40 years and above living in an area geographically similar to the case; and the third set were Alaskan Natives with other head and neck tumors, mixed tumors of the parotid, or persons without tumors from whom two separate sera or more were available for testing. With the exception of anti-EBNA, the pre-diagnosis anti-EBV spectra and titers for six of the seven cases did not differ from those of controls. Anti-EBNA titer was as high as, or higher, for every case than its closest matched control. However, the validity of the comparison is unclear since the control sera were collected 6 to 13 years after the index case sera. For the seventh case, sera obtained at 17 and 21 months before diagnosis showed antibody patterns characteristic of NPC patients. since this patient was admitted in stage IV and died soon thereafter, the authors considered it likely that the tumor was already advanced when the sera were collected. In 1980, Chinese researchers conducted a mass screening in Wuzhou City of the Guangxi Autonomous Region. Sera were obtained from 20,726 individuals aged 40 years and above. 1,136 anti-VCA/IgA positive subjects were followed up with clinical and histological examinations once a year. 97
After four years, 17 incident cases of NPC were diagnosed
5 in stage I, 11 in stage II and 1 in stage III (Zeng et
al., 1985b). The incidence rate for this cohort was 7.5
times the age-adjusted rate for the general Wuzhou
population. This result however overstates the RR
associated with pre-diagnosis anti-VCA/IgA positivity, since
follow-up subjects were subjected to close otolaryngological
examinations while incident cases who emerge from the
general population, for any particular year, would include
fewer of the early stage clinically less evident cases.
This bias could be attenuated in subsequent years of follow
up. The active follow-up also results in a higher
proportion of low-stage cancers diagnosed among the
seropositives. In another follow-up of 1,138 anti-VCA/IgA
positive subjects in Zangwu County, 15 of 81 individuals whose anti-VCA/IgA titers increased fourfold or more during
the 3-year follow-up, developed NPC. The 81 individuals
represented 7.1% of the IgA/VCA positive screens, but the 15
NPC cases accounted for 71.5% of NPCs diagnosed in this
cohort (de The & Zeng, 1986).
A fourth study, conducted in Taiwan, reported pre
diagnosis anti-VCA/IgA titers for four NPC patients (Chen et
al., 1985). Two of the subjects had undetectable titers at
9 months and 29 months prior to diagnosis. A third subject had a titer of 1:10 at twelve months pre-diagnosis, and the 98
remaining subject had a titer of 1:40 (twenty-eight months
pre-diagnosis) which increased to 1:160 over the next
fourteen months and maintained at that level till diagnosis.
In a serologic case-control study based on four serum
banks with specimens from over 240,000 persons, seven cases
of undifferentiated or poorly differentiated NPC were
diagnosed in a period after serum collection which ranged
from 26 months to 154 months (Chan, C.K. et al., submitted).
Two controls per case were randomly selected from a risk set matched on serum bank, age, sex, race, and date of serum
collection. For anti-VCA/IgG, geometric mean titer for
cases (88.3) was significantly higher than that for controls
(75.5) (p < 0.05). No significant differences were found
for the other four antibodies (anti-VCA/IgA, anti-EA(D),
anti-EA(R), anti-EBNA). When case titers and their matched
controls were plotted out by duration prior to diagnosis,
six of seven case titers for anti-VCA/IgA fell within the range of matched control titers, or lower. For anti-VCA/IgG titers, it was also six of seven, and for anti-EA(D), five of seven. No time effects were evident.
The studies reviewed above do not clearly establish that elevated pre-diagnosis anti-EBV titers are risk factors
for subsequent development of NPC. A more definitive study of the pre-diagnosis EBV serological profile is needed 99
before the potential for EBV serologic screening can be
properly assessed.
Generally speaking, the usefulness of screening for
early detection and treatment of disease presupposes that
the screening will identify with high likelihood a pre
clinical phase which is detectable but usually not diagnosed
by the existing patterns of health-seeking behavior
(Morrison, 1985). It furthermore assumes that early
treatment, or foreknowledge of risk of subsequent overt
disease, will have beneficial consequences. In particular,
serologic screening for NPC seeks to identify individuals
with high likelihood of pre-clinical neoplastic lesions, or
patients already in stage I of the disease when the tumor is
small and localized to the nasopharynx, but not otherwise
symptomatic enough to cause the person to seek treatment.
The Wuzhou screening exercise indicates a detectable
prevalence of pre-symptomatic disease of 18 cases out of
1,136 anti-VCA/IgA seropositives. The predictive value of a
positive test result is thus low -- 1.6%. The predictive value for the prevalence of detectable disease plus disease that became detectable within four years was 3.1%. The predictive value of a negative test result was not determined.
The earlier stage-at-diagnosis of NPC patients detected from among the seropositives, compared to cases 100
emerging from the general population, is not surprising
given the active nature of case detection among the
seropositives (Zeng 1983, 1985b). In regard to NPC survival
rates, five-year relapse-free survival for disease diagnosed
and treated at stage I is 63%; for stage II 50%; stage III
27%; and for stage IV, 14% (Ho, 1978a). It should be
remembered though that the longer median survival of
patients diagnosed at early stages is due in part to lead
time contributed by early diagnosis. The benefits of early
detection of NPC on improved survival needs to be documented
further.
In another ongoing serologic study in Guangdong
Province, PRC, 67,891 adults aged 30-59 were screened and
48 prevalent cases of NPC were diagnosed from the 6,102
anti-VCA/IgA positive subjects (Sham et al., 1990). This
amounted to a prevalence of 0.8 % of diagnosable disease by
routine clinical examination of the seropositive subjects.
The prevalence of pre-clinical neoplastic lesions detectable by fibreoptic endoscopy and multiple biopsy was higher --
5.4 % (7 cases detected from 130 subjects randomly selected
from the remaining seropositive screens). As in Wuzhou however, the prevalence of clinically diagnosable disease and of pre-clinical neoplastic lesions among the
seronegative subjects was not determined.
The combined results from the Hong Kong and Taiwanese 101
studies (Ho et al., 1978; Chen et al., 1985) suggest that
sensitivity is also low -- only 2 out of 7 subjects who
developed NPC had elevated anti-VCA/IgA titers in a pre
diagnosis period ranging from 9 to 55 months. Quite apart
from the issues of test sensitivity and test specificity,
the Wuzhou screening indicates that even among a high-risk
age-group (40 years and older), the relatively low
prevalence of detected pre-clinical disease (18/20,726) weakens the case for population-wide serologic screening.
If the usefulness of anti-VCA/IgA (or other EBV serologic marker) is eventually established, a more suitable strategy may be to screen high-risk individuals in the endemic
regions, e.g. persons aged 40 and above with first degree
relative(s) diagnosed with NPC. This however would limit
the benefits of screening to only 10-15 % of persons who
eventually develop NPC.
10. Further Research
The pre-diagnosis EBV serological profile is of
interest from an etiological standpoint, as well as for purposes of screening and early detection of NPC. The studies reviewed earlier indicate a need to clarify the ambiguous situation regarding pre-diagnosis EBV replicative titers-- anti-VCA/IgA, anti-VCA/IgG, and anti-EA(D). An adequate-sized serum bank from a high-incidence population, 102 established prospectively and utilising a nested case control design for pre-diagnosis serum samples, would be very useful in resolving some of these issues. Such a study would also provide the data for a more thorough evaluation of the feasibility and utility of EBV serologic screening for early detection of NPC. The same serum bank could also be used to examine antibody titers against the more recently defined EBV antigens LMP1, TP1, DNase, and thymidine kinase. Initial studies have indicated that TP1 seropositivity at diagnosis was confined to NPC among EBV-associated illnesses (Frech et al., 1990). Anti-TP1/IgA however was undetectable in sera from NPC patients. Whether this is also true of salivary anti-TP1/IgA is not known. There is also a preliminary report that antibodies against the EBV zebra gene product, an early replicative antigen, was detectable in 87% of NPC sera but was absent in control sera. Epidemiologic studies of NPC could benefit from molecular genetic studies of the putative NPC susceptibility gene(s) (Lu et al., 1990). A 10-kb deletion between the B and DR loci of the HLA region has been tentatively identifed among approximately half of NPC patients, but not among controls (Chan, S.H. personal comm.). DNA probes and other biological markers of risk could make possible molecular epidemiologic studies as well as provide a screening tool, 103
if not for population-wide studies, then at least for
counselling for early detection among first-degree relatives
of NPC patients (Taylor, 1989). The availability of
biologic markers of NPC susceptibility may also be useful in
the study of early onset NPC such as encountered in the
Maghreb region. It would also facilitate the study of
interactions between inherited susceptibility and the viral,
dietary and other components of NPC etiology.
Descriptive epidemiology relating to EBV strain
differences (e.g. EBV-1 and EBV-2 subtypes) will benefit
from recent advances in molecular techniques such as the
polymerase chain reaction. This would make possible more
definitive studies on associations between viral subtypes
and EBV-associated diseases including NPC. Quite apart from
the possible bias of earlier virus-isolation protocols (see
section 6.5), the existing reports have only attempted to
correlate viral subtypes with the different EBV-associated
illnesses. The relative prevalence of viral subtypes among diseased subjects versus healthy controls has not been
systematically examined.
The epidemiologic findings on preserved food consumption (in particular, weaning use of Cantonese salted
fish) need to be confirmed by prospective studies to exclude the possible recall and interviewer bias of the existing case-control studies. Retrospective information on weaning 104 practices could be obtained on a disease-free cohort entering the high-risk age bracket (40 years and older). This could be a component of a broader, prospective study of diet and chronic disease in an NPC-endemic region. The well-developed social infrastructure of Singapore, which includes a population-based cancer registry, would lend itself well to such a study. The possibility that salted, preserved foods are markers for other exposures associated with poorer, traditional lifestyles also needs to be addressed more rigorously. 105
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Table 1 Age-Standardised NPC Incidence Rates For Selected Countries
Incidence (per 100,000 per year) Male Female
Hong Kong 30.0 12.9
Singapore Chinese 18.1 7.9 Malay 4.0 1.5 Indian 0.3 1.3
China : Shanghai 4.4 2.0 Tianjin 1.7 0.9
U.S.A. (Bay Area) White 0.6 0.3 Black 1.1 0.5 Chinese 14.8 8.0 Filipino 3.6 1.1 Japanese 1.1
U.S.A. (Hawaii) White 1.2 0.1 Japanese 1.0 0.2 Hawaiian 0.6 1.1 Filipino 2.4 1.6 Chinese 11.2 4.9
Japan (Osaka 0.5 0.2
India (Madras) 0.7 0.3
Brazil (Sao Paulo) 1.4 0.4
Spain (Tarragona) 0.8 0.2
Italy (Varese) 1.3 0.4
Fed. Rep. Germany (Hamburg) 0.2 0.1
Poland (Warsaw) 0.3 0.1
Sweden 0.5 0.2
U.K. (England & Wales) 0.4 0.2
Source : Cancer Incidence in Five Continents (vel. 5, 1987) Lyon : !ARC. 138
Table 2 HLA Antigens Associated With Risk For NPC
HLA Antigen/Haplotype Relative Risk Reference
A2-Cw11-Bw46 3.4 (p < 0.001) Chan et al., 1983 (Singaporean Chinese aged > 30)
Aw33-Cw3-Bw58-DR3 2.2 (p = ?) Chan et al., 1983 (Singaporean Chinese aged ~ 30)
A2 1. 5 ( p < 0 . 0 1) Chan et al., 1983 (Singaporean Chinese) Bw46 2.0 Simons et al., 1975 3 1. 9 (p < 10- ) Chan et al., 1983 (Singaporean Chinese) A2, Bw46 RR unspec. Malaysian Chinese } Simons et al., 1977 Hong Kong Chinese } " Californian Chinese Jing et al., 1977
4 B17 2.1 (p < 10- ) Chan et al., 1983 (Singaporean Chinese) 139
Table 3 Epstein-Barr Virus Antigens Detected in Nasopharyngeal Carcinoma
EBV Antigen Findings Reference
EBV Replicative Antigens :
Viral capsid elevated IgG (> 1:320), IgA (> 1:80) Henle & Henle, 1982 antigen (VCA) in patients' sera at diagnosis.
Early antigen elevated IgG (> 1:80), IgA (> 1:20) --do-- (diffuse) (EA-D) in patients' sera at diagnosis.
EBV-DNase high levels anti-DNase (> 6 units) in Cheng et al., 1980; NPC sera at diagnosis; anti-DNase Chen et al., 1985. detectable in sera of 4 NPC patients 9 to 29 mo. pre-diagnosis.
Thymidine kinase anti-TK detected in NPC sera, less de Turenne-Tessier (TK) activity in sera of IM, BL or healthy et al., 1989; Littler seropositives. et al., 1990.
EBV Latent Antigens :
Epstein-Barr consistently detected in NPC biopsies. Fahraeus et al., 1988; nuclear antigen 1 mediates maintenance & replication of Young et al., 1988 (EBNA-1) viral episome.
Latent membrane detected in about half of NPC biopsies. --do-- protein (LMP1) transforms human keratinocyte cell line. Dawson et al., 1990 confers resistance to cell diffn. signals.
Terminal protein 1 anti-TP1 detected in 16/42 NPC sera. Frech et al., 1990; (TP1/LMP2) not detected in sera from IM, Longnecker & Kieff, 1990; Burkitt's, or healthy seropositives. Rowe et al., 1990. 140
Table 4 Salted Fish Consumption & Risk For NPC
Age Group Relative Risk* Reference
Weaning 2o6 (p < Oo01) Geser et alo , 1978
(Hong Kong) 7 0 5 ( 3 0 9, 14 0 8) Yu et alo, 1986
Weaning 2o6 (1.2, 5o6) Yu et alo, 1988 (Guangxi, PRC)
Weaning 2o1 (1.2, 3o6) Yu et alo, 1989 (Guangzhou, PRC)
Age 1-2 2o0 (1.1, 3o6) Yu et alo, 1989 (Guangzhou, PRC) (weekly vso rarely)
Age 10 37o7 (14o1, 100o4) Yu et alo, 1986 (Hong Kong) (~ 1xjwk vso ~ 1xjmo)
Age 10 2o4 (1.0, 6o0) Yu et alo, 1989 (Guangzhou, PRC) (daily vso rarely)
Childhood 3 0 0 (p = 0 0 04) Armstrong & Chan, 1983 (Malaysian Chinese) (ever vs o never) 17o4 (2o7, 111o1) (daily vso never)
Childhood 2o2 (1.3, 3o7) Ning et alo, 1990 (Tianjin, PRC)
Adolescence 3o5 (1.2, 10o7) Armstrong & Chan, 1983 (Malaysian Chinese) (daily vso < weekly)
* p-values or 95% confidence limits in parentheses 141
Table 5 Environmental Inhalants & Risk For NPC
Exposure Relative Risk* Reference
Cigarette smoking No assoc. Shanmugaratnam & Higginson, 1967; Henderson et al., 1976; Shanmugaratnam et al., 1978; Armstrong et al., 1983; Yu et al., 1986. lifetime cig. consump. 3.7 Yu et al., 1990 30+ pack-yr. equivalents, (1.2, 11.8) vs. nonsmokers (Guangzhou, PRC) smoking 20+ cigarettes a day, > 2 Lin et al., 1973 vs. nonsmokers (Taiwan) cigarette smoking signif. trend for max. Mabuchi et al. (1985) (USA low incidence pop.) number cigarettes per day smoker in home around 2.0t Yu et al. (1988) time of birth (Guangxi)
Occupational exposures
Fumes (Calif.) 2. ot Henderson et al., 1976
Smoke (Calif. ) 3. ot II
Smoke (Malaysia) 6. ot Armstrong et al., 1983
Combustion products 2 .4t Yu et al., 1990 (Guangzhou, PRC)
Dusts (Malaysia) 4.ot Armstrong et a1., 1983
Chemicals (Calif.) 2 .4t Henderson et al., 1976
Chlorophenols (Sweden) 6. 7* Hardell et al. , 1982 (2.8, 16.2)
Formaldehyde : 2.3 Roush et al., 1987 high exposure 20+ yrs (0.9, 6.0) Blair et al., 1987 prior to death (USA)
* 95% confidence limits in parentheses t p < 0.01 t relative risk for nasal and nasopharyngeal cancers combined 142
Table 6 EBV Antibody Titers Preceding Diagnosis of NPC
study Subjects Findings
Lanier et al. 7 Alaskan NPC cases case anti-EBNAs higher; (1980) case sera collected 2-10 yrs pre-Dx; all other antibodies similar control sera collected 6-13 yrs after index case sera.
Zeng et al. 20,726 adults screened (Guangxi, PRC) 7.5 fold incidence among (1985) 1,136 VCA/IgA (+) followed 4 years sero(+) relative to age-adj. population rate.
Chan et al. 4 Asian and 3 white NPC cases matched 6 of 7 case titers for VCA/ (1990) to 2 controls each on age, sex, race, IgG, VCA/IgA within matched time of serum collection (range 26-154 control titer range, or months pre-diagnosis) lower. GMT for VCA/IgG marginally higher among cases.
Months Chen et al. 4 Taiwanese subjects IgA/VCA titer Pre-Dx (1985) patient 1 10 12 patient 2 < 10 9 patient 3 160 14 patient 4 < 10 29
Ho et al. 3 Hong Kong subjects (1978) patient 1 5 55 patient 2 20 30 patient 3 80 35