- 學位論文
EZH2的表現、H3K27三個甲基化與DNA甲基化在瀰漫性大型B細胞淋巴癌預後的影響
Prognostic impact of EZH2 expression, H3K27 trimethylation and DNA methylation in diffuse large B cell lymphoma
摘要
傳統上，淋巴癌病人對化學治療反應效果的預測只能依靠International Prognostic Index (IPI score，國際預後索引)，目前並沒有更加實用且準確的分子生物層次方面的預測因子可以使用。我們希望藉由表觀基因學 (epigenetic) 在淋巴癌上的研究來找到新的預後因子。 EZH2蛋白的過度表現與Histone 3-lysine 27三個甲基化 (H3K27me3)的減少，在乳癌、攝護腺癌等很多癌症上是一個預後變差的因子。我們的研究主要是探討EZH2蛋白，它具有甲基轉化酶的功能而且會去增加H3K27的甲基化，並藉由這個位置上三個甲基化的程度來調控基因的功能。在癌細胞上，抑癌基因的過度甲基化會強化其DNA與組蛋白的纏繞結合，進而減低抑癌基因之表達，最後導致癌症惡性度變高。 在Cytosine的5-position加上一個甲基就是 5-methylcytosine (5-mC)，它是表觀基因學上一個重要的標記。我們可以偵測 5-mC的量來代表整個DNA甲基化的程度。DNA甲基化的改變會導致人類自體免疫的疾病、神經學的病變與癌症，而癌細胞一般會呈現DNA整體去甲基化與局部基因的過度甲基化。而5-mC的hydroxylated型態則是5-hydroxymethylcytosine (5-hmC)，他在動物的中樞神經系統含量較高，此標記可能代表DNA去甲基化的程度。在Tahiliani (2010)的研究中，5-mC可以被Ten-Eleven-Translocation (TET) protein轉換成5-hmC，會去改變甲基化的程度；在TET1減少時，會增加基因的甲基化。在急性骨髓性血癌中發現TET2的突變而減少5-hmC的量，此結果在臨床上則會使急性骨髓性血癌的預後變差，而在瀰漫性大型B細胞淋巴癌（DLBCL）病人的檢體中則無相關的研究。除此之外，在老鼠的胚胎幹細胞中發現，5-hmC會特別豐富的存在於基因起始子含有H3K27me3與H3K4me3的地方，5-hmC也被推測可能有暫停轉錄的作用。 在西方國家的研究中發現，21.7% 的germinal center B cell (GCB) 類型的DLBCL中有EZH2 Tyr 641的體細胞突變，在體外試驗中發現此突變蛋白會選擇性的使甲基化的功能增加。然而，在DLBCL族群中，EZH2蛋白的過度表現、增加H3K27三個甲基化與整個DNA甲基化的程度並沒有被完整的研究。所以我的研究是採用回溯性的研究方法，先針對淋巴癌病患的病理檢體來進行研究分析，再與病人之前的治療效果與臨床表現作相關性的探討。本研究主要是希望找出分子生物層次方面的預後因子，進而改善未來淋巴癌病人治療的效果。再來，瞭解5-mC/ 5-hmC/ EZH2/ H3k27me3在淋巴癌檢體中的相關性。最後，分析EZH2 Tyr 641在亞洲的淋巴癌病患中的突變情形。 本實驗是將之前診斷為DLBCL的107位亞東紀念醫院與83位奇美醫院病患之病理檢體做兩個部份的處理：第一部份將病理檢體做特殊染色Immunohistochemistry (IHC) stain，包含anti-H3K27me3、anti-EZH2、anti-5-mC 與anti-5-hmC來定量病人檢體Histone甲基化，EZH2的表現與整體DNA甲基化的狀況; 第二部份是將此病理檢體之DNA萃取出來，來檢驗EZH2上Tyr641的突變狀態。之後做病歷資料的回顧，分析病理EZH2/ Histone methylation/ DNA methylation/ DNA hydroxymethylation/ Tyr641 mutation的表現與病人癌症的臨床狀況，進而瞭解這些基因與蛋白是否可用為預測淋巴癌病人預後的因子，最後用來改善未來淋巴癌病人治療的效果。 目前從西元2002年至2010年底的病理報告中初步篩選出190位確定診斷DLBCL的病患，只用接受標準化療（CHOP或R-CHOP）的138位病人進行生存分析。其中90位是男性，48位是女性，年紀最輕是13歲，年紀最大是86歲，平均年齡為57.2歲。癌症分期中，第一與第二期的病患最多。IPI score則是以0~2分的最多。治療的處方以R-CHOP這種標準處方的病人最多。經第一線化學治療後，達到完全緩解(complete remission, CR)的病人佔80%。平均追蹤時間是42個月，而病人的存活時間，最長3475天，最短39天(從診斷到死亡)。 初步收集的48位病人的檢體並無EZH2 基因之hot spot mutation，與文獻中的狀況不相同。在病患的臨床資料與四種IHC染色的分析中觀察到，H3K27me3的強弱表現與性別的分布有顯著的差異(P=0.044)，而5-mC的強弱也與治療的反應有相關性(P=0.006)，其他因子皆與四種IHC染色無相關性。而四種IHC染色中，H3K27me3與5-mC以及5-mC與5-hmC彼此間有正相關性。 在單變項分析下，stage、IPI score與化學治療的反應對overall survival (OS)有統計學上的意義，而四種抗體並無對OS有統計學上的意義，只有5-hmC有trend（P=0.061）。在多變項分析下，四種抗體個別的強弱仍沒有對OS有統計學上的意義。接著用H3K27me3搭配5-hmC來做分組，其中high H3K27me3/ low 5-hmC的病人存活率較高而high H3K27me3/ high 5-hmC的病人存活率較低（P=0.0497）。在多變項分析後，H3K27me3/5-hmC的分組就沒有統計學上的意義。 在subgroup 分析中進一步發現，在經標準化學治療後CR的110位病人中，H3K27me3/5-hmC的分組就對OS有統計學上的意義（P=0.013）。經多變項分析後，這種分類仍然有統計學上的意義（P=0.017），其中high H3K27me3/ low 5-hmC的病人存活率較高。 結論：我們的研究中指出在DLBCL中H3K27me3/5-hmC的分類可能與預後有關，特別是在CR的族群中，這個high H3K27me3/ low 5-hmC表現的病人存活率較高。四種IHC染色中，H3K27me3與5-mC以及5-mC與5-hmC彼此間有正相關性。而EZH2 Tyr641突變的比例在我們台灣的這個族群的確偏低。
並列摘要
Purpose International Prognostic Index (IPI) score is used to predict the prognosis in diffuse large B cell lymphoma (DLBCL) for around 20 years. However, few molecular prognostic factors were proposed. Overexpression of Enhancer of zeste homolog 2 (EZH2) and decreased histone 3-lysine 27 tri-methylation (H3K27me3) are associated with poor prognosis in many cancers. Promoter CpG island hypermethylation of tumor-suppressor genes is a common hallmark of all human cancers. 5-methylcytosine (5-mC) levels in cancer cells can reflect the DNA hypermethylation status and measurement of 5-hydroxymethylcytosine (5-hmC) levels may estimate the DNA demethylation status in cancer cells. A unique EZH2 Tyr 641 somatic mutation was detected in DLBCL and follicular lymphoma. However, the clinical implications of DNA methylation status, DNA hydroxymethylation, EZH2 expression, the extent of H3K27me3 and EZH2 Tyr 641 somatic mutation in DLBCL patients have not been studied in a comprehensive or integrated way. We aim to see significant impact of DNA methylation, DNA hydroxymethylation, EZH2 expression levels and mutation status, and H3K27 trimethylation on the clinical and biological presentation of DLBCL. Patients and Methods We enrolled 107 consecutive patients with DLBCL in Far Eastern Memorial Hospital and 83 consecutive patients in Chi Mei Medical Center (diagnosed between 2002 and 2009). The demographic data, treatment regimens, and response of disease were reviewed retrospectively. Immunohistochemistry (IHC) was used to examine 5-mC, 5-hmC, EZH2 expression and the extent of H3K27me3 in formalin-fixed, paraffin-embedded biopsy specimens of DLBCL. DNA extraction from the tissues was examined for EZH2 Tyr641 mutation. Statistical analysis was performed with the Stata statistical software (Small Stata, version 11.0, Stata Corp, College Station, TX). Results Statistical analysis was performed in 138 patients with Rituximab-CHOP and CHOP regimen. Totally we recruited 90 male (65%) and 48 female (35%) with a median age 57.2 years. Fifty-nine percent of the patients had stage I/II disease. According to the IPI, 70% of the patients were classified as low/low-intermediate risk (IPI=0-2) and 30% as intermediate-high/high risk (IPI=3-5). Thirty-seven patients received CHOP-like regimen and 101 patients received Rituximab-CHOP-like regimen as first-line chemotherapy. The median observation time for overall survival (OS) in the 138 patients was 42 mo. Male patients tended to have lower H3K27me3 expression (p=0.044). Certain clinical parameters such as IPI score, stage and response to treatment were correlated with OS. None of 48 patients harbored EZH2 mutation at Tyr641. Low expression 5-mC patients tended to have more complete response after standard chemotherapy (P=0.006). Low expression 5-mC tended to have lower H3K27me3 expression (P=0.014). And low expression 5-mC tended to have lower 5-hmC expression (P=0.001). There was no obvious relationship between the expression of EZH2 and degree of H3K27me3 in DLBCL tumor cells. Although there was no significant prognostic impact in univariate survival analysis, low 5-hmC expression seemed to have longer OS (P=0.061). When other strong prognostic factors were included in multivariate analysis, there was no significant prognostic impact in single epigenetic marker. Then we subdivide the patients by combination H3K27me3 with 5-hmC. The high H3K27me3/ low 5-hmC patients was associated with longer OS (P=0.0497) but there was no significant prognostic impact in multivariate survival analysis. In the subgroup analysis, we enrolled the 110 patients with complete remission (CR) after standard chemotherapy and subdivide the patients by combination H3K27me3 with 5-hmC. The high H3K27me3/ low 5-hmC patients was associated with longer OS in univariate (P=0.0131) and multivariate analysis (P=0.017). Conclusion High H3K27me3/ low 5-hmC expression was a possible favorable prognostic factor in DLBCL and it showed even more significant prognostic impact in CR patients. Theincidence of EZH2 mutation at Tyr641 in our cohort is much lower compared with western countries. Low expression 5-mC tended to have lower H3K27me3 expression and low expression 5-mC tended to have lower 5-hmC in DLBCL tumor cells. Our study suggests epigenetic markers may be an informative biomarker for prognosis prediction in DLBCL.
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