影響尿路上皮癌預後之分子預測因子研究

The Study of Molecular Prognostic Predictors in Urothelial Carcinoma

柯宏龍(Hung-Lung Ke)

指導教授 ： 吳文正

高雄醫學大學/醫學院/醫學研究所博士班/博士(2016年)

摘要

研究背景
上泌尿道泌尿上皮癌在泌尿系統是相對少見的疾病，可是在台灣卻有不同於西方國家的高發生率與性別分佈，其中的原因可能與飲用富含砷的地下水以及長期服用中西藥有關，但是這仍然無法解釋上泌尿道泌尿上皮癌在台灣以女性居多的現象，也無法徹底了解泌尿上皮癌化的機轉。
對於上泌尿道泌尿上皮癌的患者而言，腎臟輸尿管及膀胱袖口切除手術是最好的治療方式，癌症轉移與膀胱復發是最常見影響病患存活的因素，病理分期與惡性度是最重要的預後相關因子，但是同樣期別、接受同樣手術治療的病患，仍然有不同的復發或存活機率。因此需要研究分子生物因子，希望能提供病患治療方向的選擇與預後的預測。

研究材料與方法
第一部分研究缺氧誘導因子(HIF-1α)、骨橋蛋白(OPN)、第二型環氧化酶(COX-2)、內臟脂肪激素(visfatin)在上泌尿道泌尿上皮癌腫瘤組織裡的表現。我們收集病患臨床及病理資料和手術後的病理檢體，利用免疫組織化學染色的方式，檢驗腫瘤組織裡面上述四種蛋白質的表現，藉由病理科醫師觀察評分，再利用生物統計方式評估蛋白質表現程度與病患臨床和病理參數的相關性。
第二部分研究微小核糖核酸在上泌尿道泌尿上皮癌病患的表現，先收集病患臨床與病理資料和血液，抽取去氧核糖核酸，利用Illumina Infinium II iSelect custom SNP array platform (Illumina, San Diego, CA)平台偵測微小核糖核酸生成相關基因(DDX20, DGCR8, DICER1, EIF2C1, GEMIN4, RAN, RNASEN, XPO5)的單核苷變異，所得結果透過BeadStudio Software (Illumina)分析輸出，研究這些變異與病患腫瘤復發及惡化的相關性。其次利用ABI TaqMan microRNA Array檢驗泌尿上皮癌細胞株BFTC-909與同一病患的泌尿上皮癌組織與正常泌尿上皮組織之間微小核糖核酸表現量的差異，再利用病患手術後腫瘤組織與正常組織驗證不同表現的微小核糖核酸，挑選差異最大的微小核糖核酸進行細胞株試驗，在泌尿上皮癌細胞株加入微小核糖核酸抑制劑或擬似物，觀察細胞增生、移行及侵犯能力的改變，以及下游蛋白質的表現差異。

研究結果
66.3%的腫瘤組織有缺氧誘導因子的陽性表現，缺氧誘導因子的表現量與腫瘤分期(p<0.001)與惡性度(p<0.001)呈現正相關，缺氧誘導因子的表現量越高，病患因癌症死亡的風險越高(HR=2.23, p=0.004)，腫瘤復發機率越高(HR=1.58, p=0.036)。49.1%的腫瘤組織有骨橋蛋白的高度表現，骨橋蛋白的表現量與癌症相關的存活有關(p=0.014)。53.1%的腫瘤細胞與57%的基質細胞有第二型環氧化酶的陽性表現，如果基質細胞的第二型環氧化酶呈現陽性表現，病患因癌症死亡的風險越高(HR=6.38, p=0.0223)。內臟脂肪激素的表現量與腫瘤分期(p=0.001)及惡性度(p=0.007)有正相關性，高度表現內臟脂肪激素的病患，癌症復發機率較高(HR=3.22, p=0.009)，病患因癌症死亡的風險越高(HR=5.74, p=0.023)。
微小核糖核酸生成相關基因DDX20裡面rs197412基因變異對表淺型膀胱癌病患接受經尿道刮除手術後的腫瘤復發有最大的影響，變異型對偶基因會降低病患的復發率(HR=0.58, 95% CI=0.40-0.82, p=0.002)。DGCR8有兩個連鎖的單核苷酸基因變異(rs2073778和rs720012)會影響癌症惡化(HR=4.00, 95% CI=1.53-10.46, p=0.005)。微小核糖核酸miR-145在上泌尿道泌尿上皮癌組織裡的表現量比正常泌尿上皮組織低，相對的，乙型轉化生長因子第二型受體在腫瘤組織裡的表現量就比正常組織高，利用miR-145表現量可以準確區分腫瘤與正常組織(AUC=0.882, 95% CI=0.822-0.943, p<0.001)，miR-145擬似物可以抑制細胞增生、移行及侵犯能力。miR-210在上泌尿道泌尿上皮癌組織裡的表現量比正常泌尿上皮組織高，而且與腫瘤分期(p=0.017)及惡性度(p=0.049)呈正相關，缺氧誘導因子的表現量也與miR-210的表現量呈正相關(r=0.442, p=0.001)

結論
缺氧誘導因子、骨橋蛋白、第二型環氧化酶、內臟脂肪激素與上泌尿道泌尿上皮癌病患的復發及存活皆有相關性，可以成為預後預測因子，提供臨床醫師篩選病患給予更積極的追蹤治療。
微小核糖核酸在泌尿上皮癌化及癌症惡化皆扮演重要角色，將來有潛力成為治療上泌尿道泌尿上皮癌的重要標的。

關鍵字

尿路上皮癌 ； 微小核糖核酸 ； 環氧化酶缺氧誘導因子 ； 骨橋蛋白 ； 血管內皮生長因子

並列摘要

PURPOSE
Upper tract urothelial carcinoma (UTUC) is a relatively rare disease in western countries. There are especially high incidence and gender distribution of UTUC in Taiwan. Long-term intake of arsenic-contaminated underground water and regularly usage of Chinese herb medicine may be the causes. These reasons mentioned above can’t fully explain why female is more than male in UTUC patients. The underlying mechanisms of UTUC carcinogenesis remains unclear, either.
Radical nephroureterectomy and bladder-cuff excision is the treatment for UTUC patients. Distant metastasis and bladder recurrence are the most important factors of patients’ survival. Cancer stage and grade are important prognostic predictors. But there are still divergent recurrence and survival rates in the patients with the same stage or grade. Therefore, in order to provide more precise factors for selecting treatment modality and predicting prognosis, further researches about molecular biological markers are needed.

MATERIALS AND METHODS
First, we studied protein expressions of hypoxia-inducible factor α (HIF-1α), osteopontin (OPN), cyclooxygenase 2 (COX-2) and visfatin in UTUC tissues. UTUC patients’ clinicopathological parameters were collected from reviewing medical records and tumor tissues from surgical specimens. Immunohistochemistry staining was applied and the scoring of protein expressions was performed by pathologists. The relationships between protein expressions and patients’ clinicopathological parameters were analyzed statistically.
Second, we studied microRNA biogenesis and expression in UTUC patients. Patients’ blood samples were collected for DNA extraction. Single-nucleotide polymorphisms in microRNA biogenesis pathway genes (DDX20, DGCR8, DICER1, EIF2C1, GEMIN4, RAN, RNASEN, XPO5) were examined. The relationships between genotypes and patients’ survival were analyzed statistically. Then we compared microRNA expression profiles between UTUC cell line and normal urothelial cell line, and validated those microRNAs with significantly different expression in paired UTUC tissues and normal urothelia. We added specific microRNA mimic or inhibitor in UTUC cell line and observed cell proliferation, migration and invasion. Proteins translated by microRNA targeted mRNAs were also examined.

RESULTS
Positive HIF-1α expression was found in 66.3% of the cancer specimens. Tumor HIF-1α expression score was significantly correlated with tumor T stage (p<0.001), N stage (p<0.001) and grade (p=0.004). Higher HIF-1α score was a significant predictor for cancer-specific survival (Cox regression hazard ratio=2.23, p=0.004), and tumor recurrence (Cox regression hazard ratio=1.58, p=0.036). High OPN expression was found in 49.1% of the cancer specimens. High OPN expression was a significant predictor for cancer-specific survival (p=0.014). Positive COX-2 expression was more frequent in stromal cells (57.0%), than in tumor sites (53.1%). Up-regulation of COX-2 was strongly associated with higher cancer-specific death (HR=6.38, p=0.0223). High visfatin expression was significantly correlated with tumor stage (p=0.001) and grade (p=0.007). High visfatin expression was associated with poor recurrence-free and cancer-specific survival. Cox regression analysis also revealed that visfatin is an independent predictor of recurrence-free (HR=3.22, p=0.009) and cancer-specific survival (HR=5.74, p=0.023).
Two SNPs were significantly associated with tumor recurrence in TUR only subgroup after adjustment for multiple comparisons (Q<0.1). The most significant SNP was rs197412 in DDX20: the variant allele conferred a decreased risk of recurrence [hazard ratio (HR)=0.58, 95% confidence interval (95% CI)=0.40-0.82, p=0.002]. Two linked SNPs (rs2073778 and rs720012) in DGCR8 showed significant association with tumor progression (HR=4.00, 95% CI=1.53-10.46, p=0.005). MiR-145 expression was significantly decreased in cancer tissues compared to non-cancer urothelium, but on the contrary, transforming growth factor beta receptor 2 was up-regulated in cancer tissues. The area under curve of low expression of miR-145 to diagnose UTUC was 0.882 (95% CI=0.822-0.943, p<0.001). MiR-145 mimics could inhibit cell proliferation, migration and invasion in vitro. MiR-210 was overexpressed in UTUC compared to non-cancer urothelium (p<0.001), and also upregulated in high stage and high grade tumors (p=0.017 and 0.049, respectively). HIF-1α was overexpressed in UTUC and positively correlated with miR-210 expression (r=0.442, p=0.001).

CONCLUSIONS
HIF-1α, OPN, COX-2 and visfatin were correlated with UTUC patients’ survival. They could serve as prognostic predictors and provided a reliable method for urologists to select high risk patients for more aggressive treatment and follow-up.
microRNAs played important roles in UTUC carcinogenesis and progression. They could be potential targets of UTUC treatment.

並列關鍵字

urothelail carcinoma ； microRNA ； cyclooxygenase ； hypoxia-inducible factor ； osteopontin ； vascular endothelial growth factor

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