替代性端粒延長機制以及ATRX/DAXX蛋白質之失去表現於肉瘤之關係及臨床病理特性

Alternative Lengthening of Telomeres and Loss of ATRX/DAXX Expression in Sarcomas with Clinicopathological Features

廖肇裕(Jau-Yu Liau)

指導教授 ： 鄭永銘

國立臺灣大學/醫學院/病理學研究所/博士(2018年)

https://doi.org/10.6342/NTU201801570

摘要

替代性端粒延長機制是一種無涉端粒酶而能使端粒長度維持，進而使癌細胞持續具有細胞分裂能力的一種機制。目前推測此機制是利用染色體的同源重組及修復機制，複製其他染色體上的端粒及其他DNA序列來維持另一條染色體的端粒長度。使用此機制來維持端粒的細胞特徵為端粒長度長短不一，並且某些染色體可能具有極長的端粒。這些現象可用南方墨點法或端粒的螢光原位雜合實驗觀察。軟組織肉瘤和膠狀細胞瘤為主要使用此機制的癌症，之前的胰臟神經內分泌腫瘤瘤研究發現此機制和ATRX或DAXX的失去活性有顯著相關。我們使用端粒的螢光原位雜合以及ATRX/DAXX的免疫組織化學染色來探討此機制在各種軟組織腫瘤的發生率，其和ATRX/DAXX之表現的相關性，以及可能的臨床病理特性。
我們總共分析了535例軟組織腫瘤，包含456例軟組織肉瘤(含23例胃腸道間質瘤及20例卡波西氏肉瘤)以及79例組織學上有不典型表現但不足以診斷為惡性之子宮平滑肌腫瘤。在456例軟組織肉瘤中，共有67例失去ATRX表現(15%)，其中有二例同時部分失去DAXX表現(均為血管肉瘤)。就組織型態學上來看，未分化肉瘤(12/35, 34%)、平滑肌肉瘤(30/92, 33%)、骨肉瘤(4/18, 22%)及血管肉瘤(16/88, 18%)為失去ATRX表現比例最高的數種腫瘤。少部分之胚胎性橫紋肌肉瘤(1/9, 11%)、上皮樣血管內皮瘤(1/11, 9%)、惡性周邊神經鞘瘤(1/17, 6%)、及粘液纖維肉瘤(1/27, 4%)也可見失去ATRX表現。在23例胃腸道間質瘤中有一例部分失去ATRX表現(1/23, 4%)。在非惡性之平滑肌腫瘤中，有一例惡性潛能未定型平滑肌瘤(1/12, 8%)失去ATRX表現。其餘腫瘤之ATRX/DAXX表現為正常。
共有405例腫瘤得到可判讀之端粒螢光原位雜合，共計有334例軟組織肉瘤及71例非惡性之平滑肌腫瘤。我們的結果顯示共有123例為替代性端粒延長機制陽性，包含118例軟組織肉瘤(118/334, 35%)、4例惡性潛能未定型平滑肌瘤(4/12, 33%)以及1例非典型平滑肌瘤(1/25, 4%)。就軟組織肉瘤的形態學上來看，替代性端粒延長機制在未分化肉瘤(22/34, 65%)、平滑肌肉瘤(51/86, 59%)、粘液纖維肉瘤(19/25, 76%)中最常見，在一部分的血管肉瘤(17/70, 24%), 放射治療後肉瘤(3/15, 20%)、惡性周邊神經鞘瘤(3/14, 21%)、胚胎性橫紋肌肉瘤(1/8, 13%)、上皮樣血管內皮瘤(1/7, 14%)及胃腸道基質瘤(1/16, 6%)也可見到替代性端粒延長機制。除了2例平滑肌肉瘤外，其餘ATRX失去表現之腫瘤均為替代性端粒延長機制陽性(P < 0.001)。在321例細胞遺傳學複雜的軟組織肉瘤中，共有65例(20%)失去ATRX表現，且有116例(116/252, 46%)為替代性端粒延長機制陽性，二者之比例均遠較基因融合之肉瘤為高(失去ATRX表現及替代性端粒延長機制分別為1%及2%；二者之P值均小於0.001)。在平滑肌腫瘤及血管肉瘤中我們也探討了替代性端粒延長機制及失去ATRX的臨床病理意義。我們發現替代性端粒延長機制在平滑肌肉瘤中為一不良之預後因子，在血管肉瘤中則兩種特性均特別常見於肝臟之血管肉瘤。我們也嘗試探討端例螢光原位雜合對於組織學上有不典型表現但不足以診斷為惡性之子宮平滑肌腫瘤的診斷預後判斷價值。在4例替代性端粒延長機制陽性的惡性潛能未定型平滑肌瘤中，有3例後來復發或轉移，顯示此方法有輔助診斷及預後判斷的潛力。
總結為替代性端粒延長機制對於軟組織肉瘤，特別是對於細胞遺傳學複雜的肉瘤是一個重要的維持端粒長度的機制。此機制和失去ATRX高度相關，並且在平滑肌腫瘤中是一個不良的預後因子。最近已有文獻報告指出抑制ATR酵素可能可針對性地殺死替代性端粒延長機制陽性之細胞，因此此種療法或許有潛力在將來作為對抗這些預後不良的腫瘤的希望。

關鍵字

肉瘤 ； ATRX ； DAXX ； 替代性端粒延長機制 ； 端粒

並列摘要

Alternative lengthening of telomeres (ALT) is a telomerase-unrelated telomere maintenance mechanism that maintain the cell division ability in cancer cells. Currently it is supposed that homologous recombination and DNA repair are utilized in this mechanism, which copy the telomeres and other DNA sequences from other chromosomes to maintain the telomere length. ALT positive cells are characterized by telomere length heterogeneity and some chromosomes may have extremely long telomeres. These phenomena can be visualized by Southern blotting or telomere fluorescent in situ hybridization (FISH). Among the cancers, soft tissue sarcomas and gliomas are frequently ALT positive. In previous studies ALT has been shown to be highly correlated with loss of ATRX or DAXX in pancreatic neuroendocrine tumors. We used telomere FISH and ATRX/DAXX immunohistochemistry to determine the frequencies of this mechanism in different sarcoma types, its correlation with ATRX/DAXX expression, and potential clinicopathological features.
In total, we analyzed 535 soft tissue tumors, including 456 soft tissue sarcomas (including 23 gastrointestinal stromal tumors [GIST] and 20 Kaposi sarcoma) and 79 cases of uterine smooth muscle tumors that exhibit atypical histological features but fall short of the diagnosis of leiomyosarcoma. Loss of ATRX expression was seen in 67 tumors (15%) of the 456 soft tissue sarcomas, and partial loss of DAXX was observed in 2 ATRX-deficient tumors (both were angiosarcomas). By the histological subtype, undifferentiated sarcoma (12/35, 34%), leiomyosarcoma (30/92, 33%), osteosarcoma (4/18, 22%) and angiosarcoma (16/88, 18%) were the major tumor types most frequently exhibited loss of ATRX. Loss of ATRX expression was also seen in small proportions of embryonal rhabdomyosarcoma (1/9, 11%), epithelioid hemangioendothelioma (1/11, 9%), malignant peripheral nerve sheath tumor (MPNST, 1/17, 6%) and myxofibrosarcoma (1/27, 4%). One GIST exhibited focal loss of ATRX expression (1/23, 4%). In non-leiomyosarcomatous smooth muscle tumors, loss of ATRX expression was observed in 1 smooth muscle tumor of uncertain malignant potential (STUMP). Other tumors exhibited retained ATRX/DAXX expression.
Interpretable telomere FISH was obtained in 405 tumors, including 334 soft tissue sarcomas and 71 non-leiomyosarcomatous smooth muscle tumors. The results showed that 123 tumors were positive for ALT, including 118 soft tissue sarcomas (118/334, 35%), 4 STUMPs (4/12, 33%) and 1 atypical leiomyoma (1/25, 4%). By the histological subtype, ALT was most commonly seen in undifferentiated sarcoma (22/34, 65%), leiomyosarcoma (51/86, 59%) and myxofibrosarcoma (19/25, 76%). ALT was also observed in subsets of angiosarcoma (17/70, 24%), post-irradiation sarcoma (3/15, 20%), MPNST (3/14, 21%), embryonal rhabdomyosarcoma (1/8, 13%), epithelioid hemangioendothelioma (1/7, 14%) and GIST (1/16, 6%). Except for 2 leiomyosarcomas, all ATRX-deficient tumors were ALT positive (P < 0.001). In 321 cytogenetically complex sarcomas, 65 cases were ATRX deficient (20%), and 116 cases were ALT positive (46%). Both features were much more commonly seen than in gene fusion-associated sarcomas (both P < 0.001). We also studied the clinicopathological significance of ALT and loss of ATRX in leiomyosarcomas and angiosarcomas. We found that ALT was an unfavorable prognostic factor in leiomyosarcomas, and in angiosarcomas these two features were particularly common in primary hepatic tumors. We also attempted to investigate the diagnostic and prognostic values of telomere FISH in non-leiomyosarcomatous uterine smooth muscle tumors. Among the 4 ALT positive STUMPs, 3 exhibited tumor recurrence and/or metastasis. Our results demonstrated that this method has potential utilities in the diagnosis and prognostication.
In summary, our results showed that ALT is an important telomere maintenance mechanism in soft tissue sarcomas, particularly in cytogenetically complex sarcomas. This mechanism was highly associated with loss of ATRX and was an unfavorable prognostic factor in smooth muscle tumors. Recent studies have shown that inhibition of ATR can selectively kill the ALT positive cells. This therapy may be promising in the future to treat these tumors.

並列關鍵字

sarcoma ； ATRX ； DAXX ； alternative lengthening of telomeres ； telomere

參考文獻

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