唾液腺黏液表皮樣癌血管生成、腫瘤細胞增生、細胞凋亡之半定量分析及其與臨床病理參數的相關性

Abstract

背景：黏液表皮樣癌是最常見的唾液腺惡性腫瘤。腫瘤血管生成、細胞增生、及細胞凋亡在許多人類癌症中與腫瘤大小、淋巴轉移、腫瘤復發、疾病患存活率，有顯著的相關性。然而，在黏液表皮樣癌中，相關臨床病理參數，尚未被討論。 方法: 本實驗選取35例黏液表皮樣癌與14例正常唾液腺組織檢體，使用免疫組織染色方法，分別以CD34、Ki67、及caspase 3直接偵測。腫瘤血管生成以計數腫瘤周圍及腫瘤間顯微血管密度數量做評估；細胞增生以細胞增生指數(PI = 計數Ki67染色細胞數量/計數所有細胞數量)評估；細胞於細胞凋亡前期，以caspase 3免疫染色分析，caspase整體染色分數(OSS = 染色分數 X 染色強度)； 組織內細胞凋亡小體以TUNEL 分析且其表現為凋亡指數。腫瘤周圍或腫瘤內顯微血管數目、細胞增生指數、凋亡指數、或caspase 3整體染色分數、與臨床病理參數，以Student’s-t test或ANOVA 分析二種或三種變項之間的差異性。而任二組之間變項的相關性，以Pearson’s correlation coefficient 方法分析。 結果:於黏液表皮樣癌，腫瘤周圍顯微血管密度為28.2±11.2、腫瘤間顯微血管密度為21.5±10.7、細胞增生指數為14.0±15.5、細胞凋亡指數為39.9±26.3、caspase 3整體表現分數為5.2±4.3，均顯著與正常唾液腺的顯微血管密度(12.2±1.3,P=0.001)、細胞增生指數(0.03±0.05, P=0.000)、細胞凋亡指數(1.4±2.1, P=0.000)、caspase 3整體表現分數(0.38±0.51, P=0.001)有顯著差異。腫瘤間與腫瘤周圍血管密度均與黏液表皮樣癌臨床分期有顯著相關性(P值分別為0.032與0.045)，腫瘤周圍血管密度於黏液表皮樣癌患者有遠處轉移者較無遠處轉移者數量顯著減少(P=0.001)；細胞凋亡指數在黏液表皮樣癌病人有淋巴轉移者較無淋巴轉移者低(P=0.001)，且於主唾液腺較次唾液腺高(P=0.003)。腫瘤內顯微血管數目於有淋巴轉移的黏液表皮樣癌中顯著較無淋巴轉移者多（P值為0.002），細胞凋亡指數於主唾液腺較次唾液腺高（P值為0.03），細胞凋亡指數與caspase 3整體表現分數於黏液表皮樣癌有淋巴轉移的病患，表現較無淋巴轉移病患高(P值分別為0.0023 及0.0001)。腫瘤周圍顯微血管密度與腫瘤間顯微血管密度、腫瘤周圍顯微血管密度與細胞凋亡指數；腫瘤間顯微血管密度與細胞凋亡指數、腫瘤間顯微血管密度與caspase 3整體表現分數；細胞凋亡指數與caspase 3整體表現分數，均有顯著的正相關性。 結論:腫瘤周圍與腫瘤間顯微血管數量於黏液表皮樣癌均顯著與臨床分期有關，顯微血管密度於黏液表皮樣癌顯著與細胞凋亡指數與caspase 3整體表現分數有顯著相關，和細胞增生較不具相關性。細胞凋亡在黏液表皮樣癌與血管增生有顯著關係，細胞凋亡或caspase 3整體表現分數於黏液表皮樣癌與淋巴轉移有顯著關係。

Background Mucoepidermoid carcinoma is the most common malignant salivary gland tumor. Tumor angiogenesis, cell proliferation, and cell apoptosis have been shown to have a significant correlation with tumor size, lymph node metastasis, tumor recurrence, and survival of patients in a variety of human cancers. However, their correlations to the clinicopathological parameters of mucoepidermoid carcinomas (MECs) have not been studied. Methods: In this study, tumor angiogenesis, cell proliferation, cell apoptosis in 35 specimens of MECs and 14 specimens of normal salivary glands (NSGs) were examined by immunohistochemical staining using antibodies directed against CD34, Ki-67, and caspase 3, respectively. Tumor angiogenesis was evaluated by counting peritumoral and intratumoral microvessel densities (MVDs). Cell proliferation was assessed by proliferation index (PI = Ki-67-positive cells/total cells counted). Cells in the pre-apoptotic phase were evaluated by caspase 3 immunostaining and were expressed as caspase overall staining score (OSS = labeling score × staining intensity). The apoptotic bodies in tissue section were detected by TUNEL assay and were expressed as apoptotic indices (AI). The correlations between the peritumoral or intratumoral MVD, PI, AI, or caspase 3 OSS and clinicopathological parameters were analyzed with Student’s t-test and analysis of variance (ANOVA), where appropriate. The correlations between any 2 variables of the peritumoral and intratumoral MVDs, PI, AI, or caspase 3 OSS were analyzed by Pearson’s coefficient of correlation. Results: The peritumoral MVD (28.2±11.2), intratumoral MVD (21.5±10.7), PI (14.0±15.5), AI (39.9±26.3), and caspase 3 OSS (5.2±4.3) in MEC samples were significantly higher than the MVD (12.2±1.3, P=0.001), PI (0.03±0.05, P=0.000), AI (1.4±2.1, P=0.000), and caspase 3 OSS (0.38±0.51, P=0.001) in NSG samples. Both peritumoral and intratumoral MVD were significantly associcated with clinical staging of MECs (P=0.045 and P=0.032, respectively). Peritumoral MVD was significantly lower in MECs with distant metastasis than in MECs without distant metastasis (P=0.001). Intratumoral MVD was significantly higher in MECs with regional lymph node metastasis than in MECs without regional lymph node metastasis (P=0.002). AI was significantly higher in major salivary glands than in minor salivary glands (P=0.03). Both AI and caspases 3 OSS were significantly higher in MECs with lymph node metastasis than in MECs without lymph node metastasis (P=0.002 and P=0.000, respectively). A significant correlation was found between peritumoral MEC MVD and intratumoral MEC MVD or AI, between intratumoral MEC MVD and AI or caspase 3 OSS, as well as between AI and caspase 3 OSS. Conclusions: Both peritumoral and intratumoral MVD are significantly associated with clinical staging of MECs. MVD in MECs is significantly associated with AI and caspase 3 OSS rather than PI. Tumor cell apoptosis in MECs has a significant association with tumor angiogenesis. There is a significant correlation between intratumoral MEC MVD, AI or caspase 3 OSS and lymph node metastasis in MECs.