- 學位論文
第三型誘餌受體DcR3在血液惡性疾病的研究
Study of Decoy Receptor 3 in Hematologic Malignancy
摘要
第三型誘餌受體DcR3是屬於腫瘤壞死因子受體群的一種分泌性誘導死亡接受器(secretory decoy receptor)。研究證實DcR3可結合Fas ligand、LIGHT和TL1A。DcR3可抑制Fas ligand和LIGHT所引起的腫瘤細胞凋零及T細胞活化。DcR3也可以結合TL1A以促進血管新生作用。DcR3在肺癌、胃腸道腫瘤、惡性神經膠母細胞瘤和與病毒相關的惡性淋巴瘤有過度表現。研究推論腫瘤細胞可利用釋放DcR3以躲避免疫系統的監視(immune surveillance)。在血癌疾病方面未有DcR3的研究報告。研究DcR3在各種血液惡性腫瘤疾病的表現及其與臨床預後的相關性將有助於更進一步瞭解DcR3在血液腫瘤細胞生成所扮演的角色。 本實驗的研究對象為診斷為急性骨髓性白血病(Acute myeloid leukemia, AML)，急性淋巴芽細胞白血病(Acute lymphoblastic leukemia, ALL)，慢性骨髓性白血病(Chronic myeloid leukemia)，多發性骨髓瘤(Multiple myeloma, MM)，和骨髓化生異常症候群(Myelodysplastic Syndrome, MDS)的病人。以酵素免疫吸附檢測(enzyme-linked immunosorbent assay, ELISA)方法定量骨髓血漿中DcR3的濃度。部份病人再以免疫組織化學染色法(Immunohistochemical Stain, IHC)證實骨髓病理切片檢體的腫瘤細胞有DcR3蛋白質的過度表現；分析骨髓細胞DcR3 mRNA的表現是否與骨髓血漿DcR3蛋白質表現相符合。DcR3與臨床表現相關方面則分析病人臨床表現及預後是否與骨髓血漿DcR3濃度有相關。追蹤病人疾病治療前後，疾病是否得到緩解的不同時間點病人骨髓血液DcR3的濃度。 實驗總共收集95位急性骨髓性白血病的病人，21位急性淋巴芽細胞白血病的病人，16位慢性骨髓性白血病的病人，19位多發性骨髓瘤病人，18位骨髓化生異常病人和23位健康骨髓捐贈者的骨髓檢體。利用酵素免疫吸附檢測(ELISA)方法定量骨髓血漿DcR3濃度。骨髓血漿DcR3濃度的平均值及標準差(mean ± standard deviation)和最大及最小值(maximum-minimum)分別是5.97±18.12 (145.63-0) ng/ml in AML patients，21.95±82.54 (380.85-0) ng/ml in ALL patients，0.83±1.48 (5.99-0) in CML patients，0.19±0.31 (1.12-0) ng/ml in MM patients，1.40±2.06 (7.86-0) ng/ml in MDS patients，0.42±0.35 (1.11-0) ng/ml in healty bone marrow donor。如果以正常骨髓血漿DcR3濃度的平均值加三倍的標準差(1.5ng/ml)為界限，則超過此數值的人數百分比，在AML為37% (35/95)，ALL為33% (7/21)，CML為13% (2/16)，MM為0% (0/19)，MDS為33% (6/18)。將骨髓病理切片檢體以免疫組織化學染色方法染色DcR3蛋白質，證實腫瘤細胞分泌DcR3。將骨髓血液中分離出的腫瘤細胞，利用反轉錄聚合酶鏈鎖反應分析腫瘤細胞mRNA的表現，證實DcR3 protein level與DcR3 mRNA表現有明顯相關。 分析AML病人臨床表現與骨髓血漿DcR3濃度的相關性，我們分析95位AML病人的臨床表現，包括年齡、性別、乳酸脫氫酶濃度(LDH)、白血球計數、血色素、血小板計數、是否有發燒或敗血症徵兆、FAB分類和染色體變異分析。統計分析發現年齡大小與DcR3濃度有統計上的差別，年齡大於60歲以上的病人DcR3濃度大於1.5ng/ml的比例較低(p=0.004)。DcR3濃度與性別、乳酸脫氫酶濃度、白血球計數、血色素、血小板計數、是否有發燒或敗血症徵兆和FAB分類並無統計學上顯著差異。共有92位病人可分析染色體的變異。在染色體的變異中，complex chromosome changes，t(8;21)，inv16的病人骨髓血漿中DcR3>1.5ng/ml的比例在50%以上；分別是6/9(67%)，3/5(60%)，2/4(50%)。 分析AML病人治療及預後。總共有71位AML病人接受標準的引導性化療。DcR3濃度的高低與治療後是否達到完全緩解(complete remission, CR)並無統計上顯著相關(DcR3>1.5 ng/ml 及DcR3<1.5 ng/ml 的兩群病人達到CR的比例分別為69%及69%)。71位AML病人接受標準引導性化療的總存活時間(overall survival)在DcR3>1.5ng/ml與DcR3<1.5ng/ml兩群病人中並無明顯差別(median, 22 vs 24.2 months, p=0.803)。共有49位病人達到完全緩解。無病存活時間(disease free survival) 在DcR3>1.5ng/ml與DcR3<1.5ng/ml的病人中並無明顯差別(median disease free survival, 37 vs 16 months, p=0.789)。 共有7位DcR3>1.5ng/ml的AML病人於化學治療前及化學治療後有收集到骨髓血液檢體。此7位病人於引導性化學治療後達到血液學上的完全緩解。分析骨髓血漿DcR3 level在化學治療前與化學治療後，DcR3 level有明顯下降。 這是第一篇關於DcR3在血癌疾病的研究。骨髓DcR3在不同血液惡性病的表現不同。AML病人有較高的比例過度表現DcR3，但MM病人骨髓中DcR3濃度與正常人無異。AML的腫瘤細胞是否有過度表現DcR3與臨床表現及預後並無顯著相關。追蹤治療後DcR3濃度或許可做為疾病監視的指標。
並列摘要
Study of Decoy Receptor 3 Expression in Hematologic Malignancies Decoy receptor 3 (DcR3), a soluble decoy receptor, belongs to the tumor necrosis factor receptor superfamily. Decoy receptor 3 binds Fas ligand, LIGHT, and TL1A, and inhibits Fas ligand- and LIGHT-mediated cell apoptosis and TL1A-medaited angiostatic reaction. DcR3 is expressed in several types of malignant tumors, and is postulated to endow tumor cells to escape immune surveillance. We investigated the bone marrow DcR3 expression in patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), multiple myeloma (MM), and myeloid dysplastic syndrome (MDS), and also evaluated the clinical relevance of DcR3 expression in AML patients. We examined plasma DcR3 levels of bone marrow blood with enzyme-linked immunosorbent assay (ELISA) in 23 healthy bone marrow donors, 95 AML patients, 21 ALL patients, 16 CML patients, 19 MM patients, and 18 MDS patients. Bone marrows from 23 healthy marrow transplantation donors were used as normal controls. The DcR3 protein expression in malignant cells was demonstrated by immunohistochemical staining (IHC). We also examined the DcR3 mRNA expression of leukemic cells by RT-PCR and compared with β-actin expression. The DcR3 levels (mean±S.D.) were 0.42±0.35ng/ml in healthy bone marrow donors, 5.97±18.12 ng/ml in AML patients, 21.95±82.54ng/ml in ALL patients, 0.83±1.48 in CML patients, 0.19±0.31ng/ml in MM patients, 1.40±2.06ng/ml in MDS patients. The IHC showed positive staining of DcR3 in the leukemic cells from the AML patients with elevated serum level of DcR3. The DcR3 mRNA expression of leukemic cells was also correlated with the plasma DcR3 levels. We used the DcR3 level of 1.5ng/ml as cut-off level. There is no significant correlation between DcR3 levels and clinical parameters, except the age, in AML patients. 71 AML patients received induction chemotherapy. The DcR3 levels have no significant association with overall survival in these patients. 49 AML patients achieved hematologic complete remission after induction chemptherpay. The DcR3 levels have no significant association with disease free survival in these patients. We examined the DcR3 levels before and after induction chemotherapy in 7 AML patients who had hematologic complete remission. The DcR3 levels significantly decreased after induction chemotherapy. This is the first study to investigate the DcR3 expression in AML, ALL, CML, MM, and MDS. A significant portion of AML patients showed higher levels of marrow DcR3 than normal controls. In contrast, the marrow levels of DcR3 in MM patients were not increased compared with that in normal controls. The plasma DcR3 level of bone marrow blood may be a potential marker for monitoring of treatment response of AML.