- 學位論文
胃癌中之免疫反應及免疫調節因子表現:與腫瘤亞型及病患預後相關
Immune Response and expression of immunoregulatory molecules in gastric cancer: correlation with tumor subtype and patient prognosis
摘要
背景 眾所周知，胃癌是一種複雜，且異質性的實體，具有不同的組織學類型、分子特徵和生物學行為。根據基因組數據，它可以細分為四種分子類型：EBV病毒（EBV）陽性，微衛星不穩定性（MSI），基因組穩定（GS）和染色體不穩定性（CI）。儘管新靶標治療策略基於胃癌相關的分子機制改善了治療效果，但預後並未明顯改善。這四種類型的免疫微環境是否不同以及免疫狀態是否可用於預測患者的預後仍有待闡明。因此，目前迫切需要開發新的預後生物標誌物。 方法 這項研究旨在評估TCGA分類對免疫狀況的功能影響，並評估免疫調節蛋白在胃癌患者預後中的表達。在本研究中，將台灣大學醫院胃癌患者的腫瘤組織構建成組織陣列進行分析。進行錯配修復蛋白的免疫染色和EBV原位雜交，分別檢測微衛星不穩定性和EBV感染。使用福爾馬林固定石蠟包埋的樣品進行DNA流式細胞儀鑑定異常DNA含量。免疫組織化學測定法用於IDO1、Granzyme B、FoxP3、PD-1、PD-L1、Tim-3和發炎性細胞標記物。通過使用Vectra Polaris自動定量病理成像系統將載玻片掃描為多光譜圖像。細胞計數由模式識別軟件inForm Tissue Finder確定。生存分析的臨界點由X-tile軟體決定。 結果 我們發現EBV陽性和MSI患者的復發生存和總體生存比CI和GS患者更長。此外，彌散型CI患者的復發和死亡頻率比GS和腸型CI患者更高。接下來，我們發現在胃癌的四種生物學亞型中，EBV陽性和MSI腫瘤的免疫浸潤最豐富。生存分析表明，免疫調節蛋白（包括IDO1、Granzyme B、FoxP3、PD-1、PD-L1、Tim-3）的高表達在復發生存和總體生存方面均具有良好的預後。然而，只有高CD3 + T細胞、CD4 + T細胞、CD8 + T細胞和CD20 + B譜系細胞預後良好，而CD56 + NK細胞、CD68 +巨噬細胞和CD206 +巨噬細胞的計數在總體生存和復發生存中均無意義。此外，發現IDO1是復發生存或總體生存的獨立預後因素。在III和IV期患者中，僅IDO1的表達水平與預後顯著相關（P <0.05）。 結論 總之，我們發現免疫調節因子在胃癌中的預後意義。這些結果表明，免疫調節因子可能是潛在的預測指標，並可能指導胃癌免疫檢查點治療劑的選擇。
並列摘要
Abstract Background It is well known that gastric cancer is an intricate, heterogeneous entity with diverse histological types, molecular profiles, and biological behaviors. It can be sub-classified into four molecular types based on the genome data: Epstein-Barr virus (EBV)-positive, microsatellite instability (MSI), genomically stable (GS), and chromosomal instability (CI). Although new target therapeutic strategies based on molecular mechanisms related to gastric cancer have improved treatment outcomes, prognoses have not improved significantly. Whether these four types have different immune microenvironment and whether the immune status can be used to predict patient prognosis are still need to be illuminated. Hence, new prognostic biomarkers development is urgently needed. Methods This study was aimed to evaluate the functional impact of TCGA classification on immune contexture and assess the expression of immune regulatory proteins in the prognostic role in gastric cancer patients. In our study, tumor tissues of gastric carcinomas patients from National Taiwan University Hospital were constructed into tissue array for analysis. Immunostaining for mismatch-repair proteins and EBV in situ hybridization was conducted to detect microsatellite instability and EBV infection, respectively. DNA flow cytometry was conducted to identify abnormal DNA content using formalin-fixed paraffin-embedded samples. Immunohistochemistry assay was employed for IDO1, Granzyme B, FoxP3, PD-1, PD-L1, Tim-3 and inflammatory cell markers. The slides with be scanned to multispectral image by using Vectra Polaris Automated Quantitative Pathology Imaging System. Cell counts were determined by the pattern recognition software, inForm Tissue Finder. Cut off points of survival analysis were decided by X-tile software. Results We found that EBV-positive and MSI patients had a longer 5-year recurrence-free survival and 5-year overall survival than CI and GS patients. Besides, diffuse type CI patients had more frequent recurrence and death than those with GS and intestinal type CI patients did. Next, we discovered that among the 4 biological subtypes of gastric cancer, EBV-positive and MSI tumors had the most abundant immune infiltrate. Survival analysis indicated that high expression of immune regulatory proteins, including IDO1, Granzyme B, FoxP3, PD-1, PD-L1, Tim-3, yielded strongly favorable prognosis in both recurrence-free survival and overall survival. However, only high CD3+T cell, CD4+T cell, CD8+T cell, and CD20+ B lineage cell generated favorable prognosis, whereas the count of CD56+ NK cells, CD68+ macrophages and CD206+ macrophages showed no significance in both overall survival and recurrence-free survival. Furthermore, IDO1 was found to be an independent favorable prognostic factor for both recurrence-free survival or overall survival. In stage III and IV patients, only the expression level of IDO1 was significantly correlated with prognosis (P < 0.05). Conclusions In summary, we found that the prognostic significance of immune regulatory factors in gastric cancer. These results indicate that immune regulatory factors might be a potential predictive marker, and may guide the selection of immune checkpoint therapeutics in gastric carcinoma.