貓乳癌中HIF-1α和VEGF蛋白質表現與臨床因子、患貓預後之間的關係

Abstract

在惡性腫瘤組織發生過程中，內部發生缺氧是常見的現象，而在這個過程中，新血管生成扮演了重要的角色。其中，缺氧誘導因數-1α（HIF-1α）和血管內皮生長因數（VEGF）在缺氧時會改變其活性和表現量以促進血管新生，使癌症細胞適應惡劣環境。本實驗據此設計以研究HIF-1α和VEGF在貓乳癌組織的免疫組織化學染色特性，探究兩者的表現與臨床、病理因素和預後的關係。共收集72例貓乳癌病例的石蠟組織，除調查臨床資訊如年齡、性別、腫塊大小、臨床是否發現淋巴轉移等，也回溯性地視檢其組織病理學因子，記錄其組織學分型、管腔形成的範圍、核多形性及有絲分裂數量，同時亦記錄了區域壞死、鱗狀上皮化生、間質反應（stromal response）程度，淋巴管血管侵犯和淋巴結轉移的存在與否。結果顯示絕大多數的病例為絕育母貓，腫塊直徑多大於3 cm；根據四項組織學分類標準，結果顯示導管乳突狀上皮癌（tubulopapillary carcinoma）類別最多（52/72, 72.2%）；視檢各腫瘤侵犯情況，在35個病例中發現淋巴管血管入侵；對於送檢的26個淋巴結，16個出現轉移病灶；另外，大多數的病例皆出現明顯的壞死、可見鱗狀細胞化生、腫瘤內間質反應（stromal response）。根據EE分級系統，多數的病例被分為等級II （43/72, 59.7%）。以這72例病例製作成組織晶片，進行HIF-1α和VEGF的免疫組化染色。結果顯示HIF-1α的表現主要出現在腫瘤細胞的細胞核，在貓乳癌組織的陽性表現率為69.4% （50/72）。VEGF的陽性訊號可在腫瘤細胞和結締組織細胞中發現，過度表現率為77.8%（56/72）。藉由卡方檢驗（Chi-square test）或費希爾精確檢驗（Fisher exact test），HIF-1α的表現未顯示與臨床及病理因數相關。VEGF的過度表現與組織學類型（P=0.020）、間質反應程度大小（P=0.040），及鱗狀化生程度（P=0.000），淋巴管血管入侵（P=0.007）有關。利用斯皮爾曼等級相關（Spearman Rank Correlation）分析HIF-1α和VEGF的關係，兩者並不具線性相關（P=0.057，r=0.26）。在這72例貓乳癌病例中，38例可被調查到1年後存活情況，其中12例存活，26例死亡。對於此38例具有一年存活情況資訊的貓乳癌組織，又進行了全切片的免疫組織化學染色，得出HIF-1α的表現與預後情況具顯著相關性（P = 0.033），而VEFG的染色結果均未顯示與預後有顯著相關。這樣的結果顯示HIF-1α之表現在貓乳癌可能可以作為預後較差的生物標記，同時也為標靶治療貓乳癌疾病提供了一個可能的治療方向。

Anoxic microenvironment is a hallmark of solid tumors including feline mammary gland carcinomas (FMGCs). Intratumoral hypoxia is caused by the lack of functional blood vessels in rapidly proliferating neoplastic tissue. Malignant cells can undergo genetic and adaptive changes allowing them to escape from dying of oxygen deprivation through expression of Hypoxia-inducible factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF). The purpose of this study was to evaluate the expression of HIF-1α and VEGF in feline mammary carcinomas, and to analyze the association of these two factors with the clinical and pathological characteristics, such as clinical stage, tumor grade, regional metastasis and one-year survival rate. Paraffin-embedded tissue samples collected from 72 cats with FMGCs were retrospectively studied. Histopathologic pattern, tubule formation, nuclear pleomorphism, and mitoses were recorded for grading the cases. The results showed that tubulopapillary carcinomas and histologic grade II cases (43/72, 59.7%) prevailed in the study. Most cases also showed obvious necrosis, discernable squamous metaplasia in the neoplastic cells and intratumoral stromal response. According the results of immunochemical stainings performed in TMA, the expression rates of HIF-1α and VEGF were 69.4% (50/72) and 77.8% (56/72) in the patients, respectively. Through Chi-square test and Fisher exact test, HIF-1α expression was insignificantly associated with examed clinicopathological factors. As for VEGF overexpression, statistically significant associations were observed with the histopathologic pattern (P=0.020), stromal response (P=0.040), squamous metaplasia (P=0.000), and lymphovascular invasion (P=0.007). However, neither HIF-1α nor VEGF status was correlated with tumor grade and metastases. Of 38 cats with 1-year follow-up, the expression of HIF-1α was negatively associated with the survival time of patients (P < 0.05) (log-rank test) through the results from immunochemistry performed in whole sections. There was no significant association between VEGF expression and the feline survival time. Correlation between HIF-1α and VEGF was not significant through Spearman analysis. It is asserted that HIF-1α could be used as a biomarker indicating poor prognosis in cats with FMGCs. Developing compounds that inhibit HIF-1α might be a potential approach in FMGCs treatment.