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研究生：	陳建欽 Chen, Chien-Chin
論文名稱：	Epstein-Barr病毒與種痘樣水泡症的相關性 The Association of Epstein-Barr Virus Infection and Hydroa Vacciniforme
指導教授：	李玉雲 Lee, Yu-Yun
學位類別：	碩士 Master
系所名稱：	醫學院 - 臨床醫學研究所碩士在職專班 Institute of Clinical Medicine(on the job class)
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	39
中文關鍵詞：	種痘樣水泡病、Epstein-Barr病毒、皮膚T細胞淋巴瘤
外文關鍵詞：	Hydroa vacciniforme, Epstein-Barr virus, cutaneous T cell lymphoma
相關次數：	點閱：137 下載：0
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種痘樣水泡病(Hydroa vacciniforme)是一種極為罕見的光敏性皮膚疾患，病因仍未明，臨床特色通常是兒童期開始發病，青春期後才逐漸減輕消退，皮膚損害主要表現為暴露部位(如面頰、額部、耳廓、下肢、手背部)反復發作的丘疹、水泡、潰瘍及結痂。最近十多年來的文獻上的病例探討逐漸發現，種痘樣水泡病有相當比例與Epstein-Barr病毒的感染有關連，且可能增加發生惡性淋巴增生性疾病的風險。本研究的目的就在於透過回顧臨床病例的追蹤結果並針對其皮膚切片的組織進行進一步評估，試圖想了解Epstein-Barr病毒的潛在感染與種痘樣水泡病之間是否存在病理性的關連。
透過成大醫院皮膚部的南十字星系統搜尋1989年到2009年二十年間的病例，總共找到六位經病理切片確認且臨床特徵符合的種痘樣水泡病病患。我們再透過病歷回溯性地整理病患的臨床表現、治療與檢驗、復發情況，並重新閱片及進一步評估病人的皮膚切片。臨床上來講，這六位病患一開始都是在暴露部位(如臉或四肢)有反復發作的水泡、潰瘍、及結痂而來求診。診斷時的年齡中位數是18.5歲，平均追蹤時間是每位病人103個月，且六位當中有五位是男性。以原位雜合法分析所有病患最初的皮膚切片組織，發現其中四位(66.67%)有Epstein-Barr病毒的感染。這四位有Epstein-Barr病毒感染的病人經治療後都仍復發。透過CD3和EBER原位雜交反應的雙重染色，證實了Epstein-Barr病毒的主要感染細胞為T細胞。值得一提的是其中一位有Epstein-Barr病毒感染的病患，在長達209個月的追蹤過程中，反覆不斷地復發丘疹、水泡、潰瘍結痂，並且從一開始的臉及手背等暴露部位，進一步延伸到了不常受日光照射的大腿及軀幹等處，最後發生種痘樣水泡病樣T細胞淋巴瘤併發噬血症候群而往生。
為了分析種痘樣水泡病病患，能發展出T細胞淋巴瘤的相關過程，我們以皮膚切片的石蠟包埋組織，萃取DNA，再利用BIOMED-2多重聚合酶連鎖反應來測定是否有單株T細胞。除了兩位病患的DNA因萃取後含量不足而放棄，其他四位最早的皮膚切片組織並沒有顯示單株T細胞接受器的基因重組，這當中竟也包含那位追蹤209個月後發生T細胞淋巴瘤併發噬血症候群而往生的病人，但在他死亡前的口腔與骨髓切片檢體卻都顯示有單株T細胞接受器的基因重組。可見分析切片檢體是否顯現單株T細胞接受器的基因重組，搭配病患的臨床表現與顯微證據，或許可以做為一種重要的輔助工具，找出較為可能發展為T細胞淋巴瘤的病人來積極追蹤與治療。
相對於已發表的文獻，本研究的病例群具較高的診斷年齡、同樣高比例有Epstein-Barr病毒感染、且Epstein-Barr病毒感染者有相對較嚴重的病程。CD3和EBER原位雜交反應的雙重染色，證實在種痘樣水泡病病患中Epstein-Barr病毒的主要感染細胞為T細胞。種痘樣水泡病雖然屬於罕見的光敏性皮膚疾患，但在Epstein-Barr病毒盛行的地區，必須特別注意臨床表現與追蹤復發狀況，作皮膚切片以輔助診斷，且測定是否有Epstein-Barr病毒感染，來找出可能需積極防止惡化的病人。分析切片檢體是否顯現單株T細胞接受器的基因重組，也可以做為一種重要的輔助工具，找出較為可能發展為T細胞淋巴瘤的病人來積極追蹤與治療。

Hydroa vacciniforme (HV) is one of the rarest, usually quite severe, photodermatosis of unknown etiology, characterized by the childhood onset of necrotic vesiculopapules on exposed areas, with resolution by early adult life. Association with Epstein-Barr virus (EBV) infection and a possibly increased risk of lymphoproliferative malignancy have been raised. Our study aims to determine the association of latent EBV infections and HV by clinical surveying the complications and outcomes of patients and evaluation of biopsy specimens.
A total of six patients of HV were retrospectively recruited from the dermatopathology database at National Cheng Kung University Hospital (NCKUH), during 1989 to 2009. The clinical features, the findings of laboratory examinations, and the skin biopsy specimens were retrospectively reviewed. Clinically, all six patients initially had recurrent vesicles, necrotic ulcers, or scars on sun-exposed areas (face or four extremities). The median age at diagnosis was 18.5 years, and five of them were male. The symptom onset time before diagnosis varied from one week to 10 years. The mean follow-up time was 103 months per patient. Four of six HV patients (66.67%) were positive for EBV infection by in situ hybridization technique among initial diagnostic skin biopsy specimens. All of four EBV associated patients had relapsing course, but only one of two EBER-negative patients relapsed. Double staining of CD3 and EBV-encoded RNA (EBER) in situ hybridization proved EBV active infection in T cells. Moreover, one of EBV-associated HV patients had 209-month history of recurrent papulovesicular eruption, spreading to non sun-exposed areas, and died of HV-like T-cell lymphoma with hemophagocytosis.
To investigate the evolution of T cell lymphoma among HV patients, we performed BIOMED-2 multiplex PCR methods for detecting T-cell clonality. We gave up two patients’ specimens owing to their poor DNA quality, and the T-cell clonality assay was performed in the biopsy specimens from the rest of four patients. All initial biopsy specimens showed no T-cell monoclonality, including the man who underwent 209-month HV progression, had T-cell monoclonality in his final biopsy specimen, and turned out to be HV-like T-cell lymphoma with hemophagocytosis.
Our series reveal older disease diagnosis age, high EBV association rate, and relatively aggressive disease course in the EBV associated patients. Double staining of CD3 and EBER in situ hybridization proves EBV active infection in T cells. Although HV is a rare type of photodermatosis, clinical follow-up, surveillance of active EBV infection, and biopsy confirmation are necessary for possibly poor prognosis, especially in EBV endemic areas. To analyze T-cell clonality may be one of important ways to detect atypical HV progression and lymphoma changes.

ABSTRACT IN CHINESE  ------------------------  I
ABSTRACT IN ENGLISH  ------------------------  III
ACKNOWLEDGEMENT -----------------------------   V
CONTENTS ------------------------------------  VII
1.INTRODUCTION -------------------------------  1
1.1 Hydroa vacciniforme ----------------------  1
1.2 Biological nature of EBV infection -------  2
1.3 Hypothesis and study designs -------------  3
2.MATERIALS AND METHODS ----------------------  4
2.1. Recruitment of HV cases -----------------  4
2.2. Histopathology and immunohistochemical stains -----  4
2.3. Detection of Epstein Barr virus-encoded small nuclear RNA+ cells by in situ hybridization and dual staining with CD3  -----------------------------------------  5
2.4. Preparing samples and extracting DNA-----  5 
2.5. T-cell clonality determination by polymerase chain reaction -------------------------------------  5
3.RESULTS  -----------------------------------  6
3.1  Clinicopathologic features of patients --  6
3.2  T lymphocytes are the EBV-infected cells in HV patients by EBER ISH and CD3 double stain  ------------  9
3.3  T-cell monoclonality presents in atypical HV or HV-like lymphoma -------------------------------------  9
4.DISCUSSION ---------------------------------  9
4.1 HV patients are highly associated with EBV infection ----------------------------------------------  9
4.2 Demonstration of T-cell monoclonality combined with clinical presentation helps diagnose atypical HV or HV-like lymphoma ------------------------------ 10
5.REFERENCES ---------------------------------- 11
6.TABLES -------------------------------------- 15
Table 1. The clinicopathologic summary of patients ---- 15
7.FIGURES
Figure 1. The EBV infection and immune response ------- 16
Figure 2. The hypothetical disease model of HV & atypical HV & HV-like lymphoproliferative disorder ---------------- 17
Figure 3. The clinical pictures of patient No.1 ------- 18
Figure 4. The histology and EBER ISH of patient No.1--- 19
Figure 5. The early clinical presentation of patient No. 2 ------------------------------------------------------- 20
Figure 6. The late clinical presentation, histology, EBER ISH, and bone marrow smear of patient No. 2 ----------- 21
Figure 7. The clinical pictures of patient No. 3 ------ 22
Figure 8. The histology and EBER ISH of patient No.3--- 23
Figure 9. The clinical pictures of patient No. 4 ------ 24
Figure 10. The histology and EBER ISH of patient No.4-- 25
Figure 11. The clinical pictures of patient No. 5------ 26
Figure 12. The histology and EBER ISH of patient No.5-- 27
Figure 13. The clinical pictures of patient No. 6 ----- 28
Figure 14. The histology and EBER ISH of patient No.6-- 29
Figure 15. The serial EBER ISH results of patient No.2--30
Figure 16. The histology, CD4, CD8, and double stain of EBER ISH/CD3 of patient No. 2 ------------------------------ 31
Figure 17. The DNA quality test of initial skin biopsy specimens from all patients --------------------------- 32
Figure 18. The PCR results for TCR-γ gene rearrangement of initial skin biopsy specimens of patient No. 2, 4, 5, 6 ------------------------------------------------------- 33
Figure 19. The positive and negative controls of PCR -- 34
Figure 20. The DNA quality test of skin specimens of patient No. 2 ------------------------------------------------- 35
Figure 21. The PCR results for TCR-γ gene rearrangement of skins and bone marrow of patient No. 2 ---------------- 36
Figure 22. The positive and negative controls for the second PCR --------------------------------------------------- 37
8.CURRICULUM VITAE ------------------------------------ 38
                                    

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