行政院國家科學委員會補助專題研究計畫期中進度報告 有關鼻咽癌與 EB 病毒感染的長期追蹤研究之初步探討 計畫類別 : 個別型計畫 整合型計畫計畫編號 :NSC B 執行期間 :95 年 8 月 1 日至 96 年 7 月 31 日

Transcription

1 行 咽 精 類 行 年 年 行 立 參 理 年

2 成果報告 行政院國家科學委員會補助專題研究計畫期中進度報告 有關鼻咽癌與 EB 病毒感染的長期追蹤研究之初步探討 計畫類別 : 個別型計畫 整合型計畫計畫編號 :NSC B 執行期間 :95 年 8 月 1 日至 96 年 7 月 31 日 計畫主持人 : 王成平共同主持人 : 婁培人 陳建仁計畫參與人員 : 成果報告類型 ( 依經費核定清單規定繳交 ): 精簡報告 完整報告 本成果報告包括以下應繳交之附件 : 赴國外出差或研習心得報告一份 赴大陸地區出差或研習心得報告一份 出席國際學術會議心得報告及發表之論文各一份 國際合作研究計畫國外研究報告書一份 處理方式 : 除產學合作研究計畫 提升產業技術及人才培育研究計畫 列管計畫及下列情形者外, 得立即公開查詢 涉及專利或其他智慧財產權, 一年 二年後可公開查詢 執行單位 : 國立臺灣大學醫學院耳鼻喉科 國立臺灣大學公共衛生學院流行病學研究所 中華民國 96 年 10 月 12 日

3 計畫中文摘要就全世界而言, 鼻咽癌是一種少見的癌症 但在台灣 南中國以及東南亞一帶, 卻是常見 的癌症之ㄧ 過去的研究發現 : 某些粵式食物 職業接觸以及家族基因的遺傳傾向都是發 生鼻咽癌的高危險因子 而 EB 病毒的感染更是與鼻咽癌有密切的關係 我們過去所做的 研究證明 : 血液中的 anti-ebv VCA IgA 和 anti-ebv DNase 呈陽性反應時, 罹患鼻咽癌的 危險性會明顯升高 不過, 過去有關 EB 病毒的血清抗體檢查與鼻咽癌的關係, 都僅採用 Case-series 和 casecontrol 的研究方法 換句話說這些研究並無法回答 EB 病毒的血清抗體是否在臨床可見 的鼻咽癌發現之前就已經有升高的現象 事實上, 目前學術界透過長期而大規模的追蹤, 來了解 EB 病毒的血清抗體變化和發現鼻咽癌的時序關係的研究非常少 而從我們過去長 期追蹤的研究發現在鼻咽癌發生之前,anti-VCA IgA 以及 EBV DNase 抗體就會出現 這 兩種抗體出現其中一種發生鼻咽癌的相對危險性是陰性者的四倍 若兩種抗體同時出現 時, 則相對危險性是兩者均為陰性者的三十三倍 而且追蹤時間越長, 有 EB 病毒抗體者 與無 EB 病毒抗體者有關鼻咽癌的累積發生率的差異會越大 不過由於出現鼻咽癌的患者 還是太少, 再者, 我們從有另一個鼻咽癌多發家族的研究中發現在一等血親且尚未罹患鼻 咽癌家屬的血液中,anti-EBV VCA IgA anti-ebv DNase 和 anti-ebna1 也有較高的陽性 率 這是否意味著, 這一群具有鼻咽癌多發性家族的人在 EB 病毒抗體的預測能力上會有 不同的結果 因此希望藉由現有的兩個不同研究族群的來進行下述的研究以回答以上的問 題 研究重點以及目的如下 : 1. 持續追蹤來先前收集來自於六個的城鎮的 9699 名, 以及 1005 名有多發性鼻咽癌家族史 的男性受試者, 以期發現新的鼻咽癌患者 2. 分析 anti-vca IgA 與 anti-dnase 抗體的高低與鼻咽癌危險性的關係 3. 藉由 anti-vca IgA 與 anti-dnase 抗體, 來比較鼻咽癌多發性家族的家庭成員與一般社 區居民罹患鼻咽癌之危險性 4. 分析個別或合併 EB 病毒抗體結果對預測鼻咽癌發生的探討 關鍵詞 : 鼻咽癌,EB 病毒, 鼻咽癌多發家族

4 計畫英文摘要 Nasopharyngeal carcinoma (NPC) is rare in most populations around the world but common in southern China and Southeast Asia. Both genetic and environmental factors are involved in the development of NPC. Previous studies have found infection with Epstein-Barr virus (EBV), certain dietary factors, especially Cantonese salted fish consumption, occupational exposures, familial tendency, and host genetic susceptibilities are significantly associated with an increased risk of NPC.1-63 Our previous case-control studies have shown an elevated NPC risk associated with the seropositivity of antibodies against EBV viral caspid antigen IgA (anti-ebv VCA IgA) and EBV DNase (anti-ebv DNase).14, 21, 22, 39 Most previous studies on EBV and NPC were either case-series or case-control studies, which were limited in correctness of causal temporality. In other words, they failed to address whether NPC or elevated serotiter of antibodies against EBV comes first. There were only few long-term follow-up studies on associations between EBV infection biomarkers and risk of NPC. In a longterm follow-up study on NPC and EBV infection, we found strong evidence that anti-ebv VCA IgA and anti-ebv DNase may appear long before the development of NPC. The relative risk of NPC was 4 for individuals seropositive for one of the two markers, and 33 for those seropositive on both markers in comparison with those seronegative on both markers. This study also indicated that the longer the duration of follow-up, the greater the difference in the cumulative incidence of NPC between seropositive and seronegative subjects.32 However, the number of NPC cases were small and we have also observed an increased seroprevalence of anti-ebv VCA IgA, anti-ebv DNase, and antibodies against EBV nuclear antigen 1 (anti-ebna1) among firstdegree relatives of NPC multiplex families in comparison with community controls.62 That means the NPC predictive validity of the EBV infection markers are different or not. Therefore, we initiate this pilot study to evaluate these questions. Keyword: Nasopharyngeal carcinoma,epstein-barr virus,npc multiplex families

5 研究計畫內容 : 1. BACKGROUND AND LITERATURE REVIEW: (1). Ethnic variation in NPC. The NPC incidence has marked variation among geographical areas. It is less than 1 per 100,000 persons per year for either sex in most countries in the world. However, the age-standardized incidence rate is extremely high in southern China (especially in the provinces of Guangdong, Guangxi, Hunan, and Fujian) and Southeast Asia. Approximately, the annual incidence rate is 20 per 100,000 persons.1 NPC is also a common cancer in Taiwan, the age-standardized annual incidence rates for males and females in 1997 were 7.9 and 3 per 100,000, respectively.4 The significant difference in NPC incidence among various countries may be attributable to differences in their ethic composition and/or exposures to potential risk factors. For example, the strikingly high incidence rate in Hong Kong and other areas in Southeast Asia may be explained by the uniquely dietary exposure of Cantonese-style salted fish and/or early infection of EBV. There is striking ethnic variation in incidence of NPC. The ethnic difference was more striking in Los Angeles and Singapore. The highest-to-lowest ratio was 49 for males and 9.3 for females in Los Angeles and 37 for males and 14.6 for females in Singapore.5 A similarly pattern of high incidence rate of NPC has been observed in southern Chinese who have migrated to other countries.6-8 These migrant studies imply that genetic factors and inherited cultural lifestyles might play an important role in the etiology of NPC. (2). EBV and NPC. Previous studies have found infection with Epstein-Barr virus (EBV), certain dietary factors, especially Chinese salted fish consumption, occupational exposures, familial tendency, and host genetic susceptibilities are significantly associated with an increased risk of NPC.1-3 It has been suggested that EBV infection may be a major risk factor for the development of NPC. The EBV was first discovered by Epstein, Achong, and Barr from Buekit s lymphoma in the 1960s. EBV is a member of the herpesvirus family. This is one of the most successful viruses, affecting over 90 percent of the population worldwide and usually with lifelong persistence. Most EBV infections are asymptomatic, but the virus can be associated with rare malignant transformations in lymphoid cells or epithelial tissue. Malignancies linked to EBV infection include NPC and a variety of methods have been used to detect antibodies against EBV antigens in patients with this disease In 1966, Old et al. found elevated levels of antibodies against EBV in patients with NPC,11and subsequently, titers of IgG antibodies against EBV capsid antigen, early antigen, and soluble complement-fixing antigen were shown to increase with progressive clinical stages of NPC.12-13In a case-control study, titers of IgM antibodies against EBV capsid antigen and early antigen were found to be significantly higher in NPC patients than in the three comparison groups.14 IgA antibodies against EBV capsid antigen and early antigen were later found to be characteristic

6 of the disease. Additionally, IgA antibodies against EBV capsid antigen were associated with the prognosis of NPC High titers of anti-ebv antibodies were detected among Eskimo Children in Greenland, a high-risk area of NPC; this also implied the role of EBV in the etiology of NPC.20 Numerous studies have shown both diagnostic and prognostic utility of other anti-ebv antibodies for NPC. For example, Chen et al. also found that elevated titers of antibody against EBV-specific DNase were more frequently seen in NPC cases than in normal controls or patients with cancers of other sites. The seroprevalence of anti-ebv DNase was higher in high incidence areas of NPC than in low incidence areas Population screening studies have also conclusively shown that healthy individuals with serological patterns similar to those observed in patients with proven NPC are at elevated risk of subsequent malignancy (i.e. the EBV serology pattern is predictive of future disease occurrence). The IgA antibodies against EBV capsid antigen have been used as a screening test for NPC in massive population studies in China. The investigators found that IgA antibodies against EBV capsid antigen could be detected prior to the clinical diagnosis of NPC.23-25Besides the utility of diagnosis, these EBV markers were reported to be a good index for confirmation of clinical remission after radiotherapy or for prediction of relapse on cases in the clinical management of NPC.26-27Some expression of EBV latent genes, such as the virus-coded nuclear antigen EBNA1, latent membrane protein 1 and 2 were observed the association with NPC development in recent years Although previous studies show the association between anti-ebv antibodies and NPC, there are uncertainties regarding the direct carcinogenic effect of EBV, and it remains unclear at which stage in the pathogenesis of NPC the virus has a role. Most previous studies were based on crosssectional observations, which did not resolve the critical issue of whether serologic markers of EBV become detectable before or after the development of nasopharyngeal carcinoma. A longterm follow-up study in Taiwan found strong evidence that IgA antibodies against EBV capsid antigen and anti-ebv DNase antibodies can appear long before the development of NPC. Individuals that tested positive for one of the two markers examined had a relative risk of 4.0 compared to seronegative individuals, and those positive for both markers had a relative risk of 33. This study also indicated that the longer the duration of follow-up, the greater the difference in the cumulative incidence of NPC between seropositive and seronegative subjects. Measurement of these two specific serologic markers may be useful for the early detection of NPC in high-risk populations. This is the only cohort study that demonstrated causal temporality between EBV infection and development of NPC.32 More recently, it has been suggested that EBV viral load measured in plasma might be a very specific marker of occult NPC. In one study, cell-free EBV DNA was detectable in the plasma of 96% of NPC patients compared with 7% of healthy controls. Additionally, it showed good temporal correlation between cell-free EBV DNA and tumor recurrence in NPC patients. The results suggest that this EBV marker may be a useful clinical and research tool in the screening and monitoring of NPC

7 2. SIGNICANCE OF PROPOSED STUDY: NPC is a unique cancer among Chinese, and a variety of factors are involved in the development of this disease.63 Although the role of EBV has been suggested as the principal factor in most studies and strong evidence was showed in our population-based cohort study. 32 The sample size was relative small. In this proposed study, we are going to use two cohorts to elucidate the associations between NPC and various EBV infection markers. It will have the advantage to differentiate the predictive validity of various EBV infection markers, and to compare EBV infection markers in members of NPC multiplex families and community residents. This study may further clarify the causal association between NPC and EBV and assess the feasibility of application of EBV infection seromarkers to predict NPC risk. It will be one of the largest cohort studies to examine the NPC risk associated with EBV infection. 3. OBJECTIVES: 1) To continue the follow-up original cohorts of 9699 male residents from six townships and 1005 male family members of NPC multiplex families for their occurrence of newly-diagnosed NPC. 2) To elucidate the dose-response relationships between NPC risk and serotiters of anti-ebv VCA IgA and anti-ebv DNase. 3.) To compare the NPC risk between multiplex family members and community residents after adjustment for anti-ebv VCA IgA and anti-ebv DNase. 4.) To evaluate NPC predictive validity (sensitivity and specificity) for individual or combined EBV seromarkers. 4. RESEARCH DESIGN AND METHODS: (1). Cohort Recruitment: (1). Multiple risk factors and multiple diseases cohort ( MRND) recruited a total of 9699 male aged more than 30 years in seven townships in Taiwan from 1984 to The six townships included Yuanshan, Hsinpu, Kuanhsi, Hengshan, Chutien and Checheng. All of them were invited to participate in the study. Informed consent was obtained from study subjects for questionnaire interview, biospecimen collection, various serological and biochemical assays and medical chart review. All were examined by otorhinolaryngologists, and subjects with suspected NPC were further confirmed by pathological examination. (2). Chinese and American Genetic Epidemiology of NPC Family Study (CAGEN) recruited a total of 1005 male and 1,028 female NPC multiplex family members from 1996 to The recruitment of study subjects has been described in detail in our previous report. All serum samples were separated immediately and stored at -30 prior to the test for anti-ebv antibodies. All patients at the recruitment were examined by otorhinolaryngologists, and subjects with suspected NPC were further confirmed by pathological examination

8 (2).Follow-up of newly-diagnosed NPC incident cases. All the study subjects who were free from NPC at the recruitment were included in this followup study. The occurrence of NPC will be ascertained by computerized data linkage with the profiles of National Cancer Registry in Taiwan from January 1, 1984 through December 31, In order to check the completeness of the NPC ascertainment, we will also review the National Death Certification profiles to identify NPC cases unregistered in the National Cancer Registry System. Medical charts of NPC cases identified through death certificates were reviewed. Only pathologically confirmed incident NPC cases will be included in the data analysis. (3).Laboratory Examination of five EBV infection markers based on a case-control study nested in these MRND and CAGEN cohorts. Serum samples collected at cohort entry will be retrieved from deep freezers for all from MRND and CAGEN cohorts. Their frozen serum/plasma samples collected at the time of enrollment will be retrieved from specimen bank and tested for various EBV infection markers. Two seromarkers of antibodies against EBV will be tested. They will include IgA antibodies against EBV VCA by IFA. A titer of less than 1:10 will be considered negative. Anti-DNase detected by ELISA. Results will be obtained by reading the OD values at 405nm. 5. RESULTS AND DISCUSSION: 1. Increased cumulative risk rates in the both Cohorts, especially in the CAGEN cohort. (Figure 1 and Table 1) In the MRND cohort, we found 7 NPC prevalent cases at the recruitment and 4 subjectives getting NPC within one year after the recruitment. After that, there were 29 new-diagnosed NPCs in the MRND cohort during a follow-up of person-years (16.8 years per person). The incidence of new-diagnosed NPC was 17.9 per person-years, which is still more than that of the general male population in Taiwan, with an incidence of 8.3 per personyears. In the CAGEN cohort, who mainly recruited multiplex NPC family members, we found one NPC male patient diagnosed within one year after recruitment. After that time, there were 4 male subjectives getting new-diagnosed NPCs during a follow-up period of 4590 person-years (4.6 year per person). The incidence of new-diagnosed NPC was 87.1 per person-years, which is much more than the incidence of MRND cohort, with an incidence of 17.9 per person-years. From these analysis, the cumulative incidence of NPC in subjectives with multiplex NPC family is very high, ten folds the incidence of NPC in general population. Therefore, NPC family is one the most risk factor for NPC occurrence.

9 2. There are no associations between subsequent NPC occurrence and age, race, education and cigarette smoking. (data no shown) 3. Increased cumulative risk for subsequent NPC in positive two EBV serological titers in both MRND and CAGEN cohorts, especially in the latter. (Table 1 and Table 2) In the MRND cohort, about anti-ebv DNase serological test, 20 new-diagnosed NPC were seronegative at the recruitment with the cumulative risk for subsequent NPC is 14.0 per person-years. (Table 1) However, 8 new-diagnosed NPC were seropositive at the recruitment, with the cumulative risk is 42.9 per person-years, which is much more than that of seronegative group, with a risk ratio of 3.1. (Table 2) About anti-ebv VCA IgA test, 24 newdiagnosed NPC were seronegative at the recruitment with the cumulative risk for subsequent NPC is 15.0 per person-years. However, 4 new-diagnosed NPC were seropositive at the recruitment, with the cumulative risk for subsequent NPC is per person-years, which is much more than that of seronegative group, with a risk ratio of If we divided the titer level into low titer group and high titer group, the risk ratios are very high in the high serological titer group with 17.8 in Anti-EBV DNase and 96.3 in Anti-EBV VCA IgA. Combined Anti-EBV DNase and Anti-EBV VCA IgA, the risk ratio is about 25.3, which is very high. That means that in general population this two serological test are helpful in prediction of subsequent NPC occurrence, even not very specific. In the CAGEN cohort, about anti-ebv DNase serological test, 3 new-diagnosed NPC were seronegative at the recruitment with the cumulative risk for subsequent NPC is 72.8 per person-years. However, 1 new-diagnosed NPC were seropositive at the recruitment, with the cumulative risk for subsequent NPC is per person-years, which is much more than that of seronegative group, with a risk ratio of 2.9. (Table 2) About anti-ebv VCA IgA test, 3 new-diagnosed NPC were seronegative at the recruitment with the cumulative risk for subsequent NPC is 67.4 per person-years. However, 1 new-diagnosed NPC were seropositive at the recruitment, with the cumulative risk for subsequent NPC is per person-years, which is much more than that of seronegative group, with a risk ratio of Combined Anti-EBV DNase and Anti-EBV VCA IgA, both seropositive risk ratios for subsequent NPC is about 38.1, which is more than that of MRND cohort. That means that in NPC family this two serological test are also helpful in prediction of subsequent NPC occurrence. 4. Still higher relative risk for subsequent NPC in CAGEN cohort compared to MRND cohort after adjustment of EBV serological data. As the table 3 shows, without EBV serology adjustment, the relative risk for subsequent NPC in CAGEN cohort is 15.3 folds than that in MRND cohort. After adjustment of either Anti-EBV DNase and anti-ebv VCA IgA,, the relative risks for subsequent NPC in CAGEN cohort are

10 15.1 and 12.0 folds than those in MRND cohort, respectively. After adjustment of both Anti- EBV DNase and anti-ebv VCA IgA, the relative risk for subsequent NPC in CAGEN cohort is still 13.1 folds than that in MRND cohort. That means that certain mechanism leads to higher incidence of subsequent NPC, besides EBV infection and several environment such as age, race, education, cigarette smoking. It may be the genetic susceptibility in CAGEN cohort because subjectives in CAGEN cohort are all family members with more than 2 NPC patients in their family, whereas subjectives in MRND cohort are not. Therefore, from this epidemiological comparison between CAGEN cohort and MRND cohort, the genetic susceptibility itself plays a very important in occurrence of NPC in Taiwan.

11 Figure 1. Cumulative risk for subsequent NPC in MRND and CAGEN cohorts. Table 1. Cumulative risk for subsequent NPC in MRND and CAGEN cohorts according to two EBV serological tests.

12 Table 2. The relative risk for subsequent NPC occurrence between two EBV serological tests in MRND cohort and CAGEN cohort. Table 3. Relative risk for subsequent NPC occurrence in comparison between MRND and CASEN cohorts after multivariate adjustment such as age, race education and EBV serological tests.

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15 Cho, E.Y. et al. Nasopharyngeal carcinoma and genetic polymorphisms of DNA repair enzymes XRCC1 and hogg1. Cancer Epidemiol. Biomarker Prev. 12: (2003). 62. Pickard, A. et al. Epstein-Barr virus seroreactivity among unaffected individuals within high-risk nasopharyngeal carcinoma families in Taiwan. Int J Cancer 111: (2004). 63. Chien, Y.C. & Chen CJ. Epidemiology and etiology of nasopharyngeal carcinoma: Gene-environment interaction. Cancer Rev. Asia-Pacific. 1: 1-19 (2003). 計劃自評 : 1. 基本上與原計畫內容相符 唯受限於經費有限, 原先計畫做更多的 EBV markers 沒有足夠經費作完本計畫收錄的 個受試者的檢體 但仍完成兩大 cohorts 的追蹤以及至少完成了兩個 EBV serological tests 的完整分析 2. 發現無論在ㄧ般族群或是鼻咽癌家族者在長期追蹤下發生鼻咽癌的確實發生率, 以及 EBV titers 的應用性 3. 可發表於期刊中, 目前準備中