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Departments of Anatomical and Cellular Pathology [A. S. C. C., K. F. T., K. W. L., K. F. M., G. M. K. T., J. C. K. L., D. P. H.] and Surgery [W. P.], Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong SAR, China; Department of Laboratory Medicine-Pathology, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada [B. C.]; Department of Pathology, Anhui Provincial Hospital, Hefei, Anhui, China [M. D.]; and Department of Pathology, Chinese PLA General Hospital, Beijing, China [X. L.]
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ABSTRACT |
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Introduction |
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Materials and Methods |
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The gender and age of the above patients are listed in Table 1
. The age ranges of the NPC patients from Hong Kong,
Anhui/Beijing, and Toronto were 31–73 (mean, 50.9), 25–67 (mean,
49.3), and 35–74 (mean, 54.6), respectively. For the normal NP from
the three different geographic regions, the ages of noncancerous
healthy individuals were 11–94 (mean, 45.9), 18–50 (mean, 35.3), and
27–80 (mean, 57.4), respectively. The male:female ratios of NPC and
normal NP samples from Hong Kong, Anhui/Beijing, and Toronto are shown
in Table 1
.
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Microsatellite Analysis.
A total of five microsatellite polymorphic markers were analyzed at
loci on chromosomes 3. These include D3S659, D3S1228, D3S1067,
D3S1076, and D3S1038 mapping to the regions 3p13,
3p14.1–14.3, 3p14.3–21.1, 3p21.1, and 3p25, respectively. The
sequences of the primers and chromosomal localization were obtained
from the Genome Database (The John Hopkins University, Baltimore, MD).
Prior to PCR, 0.25 pmol of the forward primers were end-labeled with
[
-32P]ATP (Amersham Corp., Buckinghamshire,
United Kingdom) and T4 polynucleotide kinase (Amersham Corp.). One µl
of the DNA was amplified by PCR in a final reaction volume of 5 µl
containing 62.5 µM each deoxynucleotide
triphosphate, 3 µM MgCl2,
1x PCR buffer (Applied Biosystems, Foster City, CA), and 0.25
unit AmpliTag Gold DNA polymerase (Applied Biosystems, Foster City,
CA). An initial enzyme activation step of 95°C for 12 min was
followed by 40 cycles of denaturation at 95°C for 1 min, annealing at
50–60°C for 1 min, extension at 72°C for 1 min, and then a final
extension at 72°C for 7 min. The annealing temperature for each pair
of primers was optimized to give distinct patterns of alleles with
minimal nonspecific signal. The products were resolved by
electrophoresis through a 6% denaturing polyacrylamide gel containing
7 M urea and exposed to Kodak X-OMATAR film for
12–36 h. LOH was initially scored by visual inspection when there was
a 50% decrease in band intensity when comparing with the normal
alleles. In some cases, the assessment of LOH was confirmed by
densitometric analysis. The data were examined for all categories by
the use of two-tailed Fisher’s exact test with a significant level of
0.05.
EBER in Situ Hybridization.
Four of the serial formalin-fixed, paraffin sections of each
nasopharyngeal tissue was used for H&E stain and ISH. Detection of
EBERs was carried out with an EBV probe ISH kit (Novocastra, Newcastle,
United Kingdom) according to the manufacturer’s instructions. The
tissue sections were deparaffinized, rehydrated, and hybridized to a
fluorescein-conjugated oligonucleotide probe, which consists of a
mixture of the two EBERs, EBER-1 and EBER-2. Hybridization was detected
by incubation with rabbit antifluorescein antibody tagged with alkaline
phosphatase, followed by reaction with the substrate
5-bromo-4-chloro-3-indoyl phosphatase and the colorimetric indicator
nitroblue tetrazolium. The sections were counterstained with 1%
methyl-green.
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Results |
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NPC and Dysplastic Lesions from a High-Risk Region (Hong Kong).
All of the 21 (100%) undifferentiated NPCs of southern Chinese from
Hong Kong showed allelic loss on one or more loci on the short arm of
chromosome 3 (Fig. 1
; Table 2
). In the dysplastic lesions, 6 of 8 (75.0%) showed chromosome 3p LOH
on one or more loci examined. In the 8 dysplasias, 3 of 4 (75%)
low-grade and 3 of 4 (75%) high-grade dysplastic lesions showed 3p
LOH. EBV infection was detected in 21 of 21 (100%) NPCs and 4 of 8
(50%) dysplastic lesions (Fig. 1
; Table 2
). All of the EBER-positive
cases were found in high-grade (cases 5–8) but not in low-grade
dysplasias (cases 1–4), and 3 of 4 (75%) EBV-positive cases contained
3p LOH (cases 6–8; Fig. 1
). Examples of EBER-ISH in high-grade lesions
and NPCs are shown in Fig. 2, B and C
.
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; Table 2
). No difference was found between these two groups
(P = 1.0). Collectively, 17 of 21
(81%) undifferentiated NPCs from the low-risk regions showed 3p LOH.
EBV was detected in all NPCs from central/northern Chinese (100%) and
7 of 9 (77.8%) NPCs from Caucasians (Table 2
; Fig. 1
). In the samples
from Caucasians, EBERs were positive in 2 of 2 cases (C-NPC 8 and 9,
Fig. 1
) without 3p LOH. The other 2 Caucasians’ samples (C-NPC 4 and
5, Fig. 1
) without EBV latent infection showed LOH on 3p.
NP from a High-Risk Region.
Among the histologically normal NP from noncancer individuals in Hong
Kong, 17 of 23 (73.9%) showed 3p LOH on one or more loci examined
(Fig. 1
; Table 2
). To obtain a better insight into the correlation of
genetic lesions and chronic exposure to our environment, we had
therefore examined the status of 3p LOH in the NP from 10 aborted
fetuses. Our results demonstrated that there was no LOH on 3p detected
in any of the nasopharyngeal epithelia from the aborted fetuses (0 of
10; 0%). All of the above NP samples were diagnosed by a pathologist
(K. F. T.) and were classified into either respiratory (Fig. 2
A, Cases 1 and 11) or squamous types (Fig. 2
A,
case 4). The presence of the EBV latent transcripts, EBERs, was also
examined in all NPs from southern Chinese using ISH. No EBER
transcripts were detected in the epithelial cells of these specimens
(Table 2)
.
NP from Low-Risk Regions.
In the low-risk regions, histologically normal epithelia were collected
for the investigation of genetic changes on 3p. Only 3 of 16 (18.8%)
NPs from central/northern Chinese (Anhui/Beijing) and 3 of 14 (21.4%)
NPs from Caucasians (Toronto) showed LOH on one or more loci on 3p
(Fig. 1
; Table 2
). No significant difference was found between these
two groups (P = 1.0). In these
low-risk regions, the combined frequency of 3p LOH was determined to be
6 of 30 (20%). Of note, microsatellite instability was observed in the
7 of 14 (50%) NP samples of Caucasians and only 1 of 16 (6.3%) of
those from the central/northern Chinese (Fig. 1)
but none of the NPs
from the southern Chinese (Fig. 1)
. All histologically normal epithelia
were found to be EBER negative (Table 2)
.
Statistical Analysis.
The frequencies of LOH on chromosome 3p in NPC and histologically
normal NPs from the high- and low-risk regions were analyzed using the
two-tailed Fisher’s exact test. For NPC specimens, no significant
difference was found between the NPC from southern Chinese and
those from central/northern Chinese (P = 0.13) or from Caucasians (P = 0.08). When
comparing the NPCs from the high-risk region (Hong Kong) and the
low-risk regions (Anhui/Beijing and Toronto), no significant difference
of frequencies of LOH on 3p was found (P = 0.1). For the histologically normal NPs, the frequency of LOH on 3p in
the samples from Hong Kong was significantly higher than that from
Anhui/Beijing and (P = 0.002) and that from
Toronto (P = 0.005). There was no significant
difference between the NP from the central/northern Chinese
(Anhui/Beijing) and those from the Caucasians (Toronto;
P = 1.0). When comparing the NPs from
the high-risk region (Hong Kong) and low-risk regions (Anhui/Beijing
and Toronto), the difference in the frequency of 3p LOH was found to be
highly significant (P = 0.0003).
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Discussion |
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The identification of chromosome 3p LOH in histologically normal NP from noncancerous, healthy individuals in the endemic region for NPC indicated the presence of genetically abnormal clones in the nasopharynx in our population, revealed for the first time. Notably, lower frequencies of LOH on 3p were observed in the normal NP from central/northern Chinese and Caucasians in the low-risk regions when compared with those from southern Chinese. Moreover, statistical significant frequencies of LOH on 3p in normal nasopharyngeal epithelia were detected between the high-risk and low-risk groups (73.9% versus 20%; P = 0.0003). The high frequency of genetic damage found in the normal NP from southern Chinese might reflect differences in chronic exposure to different environmental factors and genetic susceptibility. The significant difference in the incidence of LOH on 3p between different ethnic and geographic regions may be correlated to the distinct risk for cancer development. Of note, microsatellite instability was observed in the normal NP tissues from the Caucasians but not in those analyzed for southern Chinese and rarely for central/northern Chinese. The reason for such observation is unclear and remains to be investigated in future studies.
Several studies have also demonstrated the presence of genetic changes in the normal-looking epithelia, such as breast terminal ductal-lobular units and bronchial epithelium. Genetically abnormal clones were found in normal breast tissues and in normal bronchial epithelia (16, 17, 18, 19) . Moreover, in the study by Wistuba et al. (19) , a significant difference in frequencies of chromosomes 3p/9p deletion was found between the normal bronchial epithelia of smokers and nonsmokers. Mao et al. (18) suggested that such genetic damages might be a consequence of chronic exposure to carcinogenic agents present in cigarette smoke. The high frequency of genetic changes found in bronchial epithelia from former smokers indicates that such damages may persist for years after smoking cessation. Similarly, the nasopharynx may also be exposed to various carcinogenic agents that are suspected to be present in the environment, such as dietary factors and smoking. Smoking may also contribute as one of the environmental factors to NPC development (8) . Unfortunately, the smoking histories of the noncancerous individuals in the present study were not available. Chronic exposure to various carcinogens may lead to genetic instability/damage of the epithelial cells in the nasopharynx and may give rise to the development of multiple abnormal cell clones in the epithelium. Differences of frequencies in 3p deletion in the normal nasopharyngeal epithelia among different ethnic and geographic groups may indicate deviated exposure of environmental carcinogens pertaining to different lifestyles. Several studies have reported that the incidence of NPC declined between the first and the third generations after migration. These observations readily reflect that the environmental factors, living habits, and intermarriage may be the important determinants of cancer risk. Traditional diet, such as early age of exposure to salted fish, has been suggested as one of the environmental factors and major risk factor for NPC in the southern Chinese population for years (1 , 5 , 6) . However, genetic susceptibility, for example, genetic polymorphism of the CYP2E1 gene, may also be implicated (4) . Our preliminary results suggest that molecular epidemiological studies on the association of the genetic changes in normal NP, environmental factors, and genetic factors in the endemic and nonendemic regions will provide a clearer picture of the carcinogenesis of NPC.
High frequencies of 3p deletion were detected in the normal-looking NP epithelia and precancerous lesions from the southern Chinese. No significant difference of LOH on 3p was found between these two types of samples (P = 1.0). It is suspected that this genetic abnormality may occur at a very early stage before any phenotypic change commences. The definition of "normal NP" may be worth extending to the molecular level. Our pilot study on the aborted fetus NP epithelia showed us that the above genetic abnormality was not inherited but rather acquired after birth. Chronic exposure to specific environmental carcinogens may give rise to the development of genetically abnormal clones in the NP.
We have also investigated the presence of EBV latent infection in the normal epithelia, precancerous lesions, and invasive carcinoma of nasopharynx. By EBER ISH, EBV latent infection was detected in all NPC and high-grade precancerous lesions but not in the low-grade lesions and normal epithelia from southern Chinese. Similar findings were reported previously by Pathmanathan et al. (20) Their study also demonstrated the clonal EBV genome in both NPC and precancerous lesions. Of note, we illustrated in the present study that EBV latent infection of the nasopharyngeal epithelial cells may occur during progression of low-grade to high-grade precancerous lesions. These findings suggested that the clonal expansion of the EBV latent infected NP cells is an early event in NPC development.
In summary, the present study demonstrated that 3p deletion is common among NPCs from high- and low-risk geographic regions. The presence of such genetic alteration in the histologically normal NP and the dysplastic lesions suggests that it may be an early event. We also showed that the EBV latent infection occurred in the NPC and high-grade precancerous lesions but not in the low-grade lesion and normal NP. Our results suggested that EBV latent infection, together with abnormal genetic changes, might play crucial roles in NPC tumorigenesis. The higher frequency of 3p LOH found in the normal NP from southern Chinese compared with other low-risk groups may be related to the distinct incidence rate of this cancer among these populations. On the basis of the above findings, it may be worthwhile to carry out a large-scale molecular epidemiological survey by the inclusion of other geographic regions/ethnic groups and correlation to their respective environmental factors and genetic susceptibility to NPC.
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FOOTNOTES |
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1 Supported in part by the Research Grants Council
(RGC), Hong Kong SAR (RGC Earmarked Grant CUHK261/96 M, RGC Central
Allocation, 1999–2000), and The Kadoorie Charitable Foundations. 
2 To whom requests for reprints should be
addressed, at Anatomical and Cellular Pathology, Prince of Wales
Hospital, Chinese University of Hong Kong, Statin, Hong Kong SAR,
China.
3 The abbreviations used are: NPC, nasopharyngeal
carcinoma; NP, nasopharyngeal epithelia; LOH, loss of heterozygosity;
EBER, EBV-encoded RNA; ISH, in situ hybridization.
Received 1/17/00. Accepted 8/15/00.
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