比較HBV與HCV相關肝癌腫瘤浸潤白血球基因表現與預後關聯之差異

Abstract

癌症至今為國人十大死因之首已蟬聯35年，而又肝癌於我國十大癌症死亡率中名列第二，且國際世界衛生組織(WHO)公布2015年全球死於肝癌人數在所有癌症中排名第二(約78萬人死亡)。據國民健康署統計，死於肝癌的病患中，約有70%的人為B型肝炎帶原者，而20%為慢性C型肝炎感染者，無疑地，肝癌是亟待解決的衛生議題。 腫瘤浸潤白血球(TILs)指的是浸潤在腫瘤組織的白血球，正常來說他們有能力去辨識並且在腫瘤變為臨床可辦別以前殲滅這些轉化細胞。調節人體部分免疫系統來抵抗癌症的免疫療法成為近期抗癌新趨勢。然而，免疫療法主要的阻礙就是在腫瘤的微環境中發現局部免疫抑制的現象。已有許多研究顯示，腫瘤浸潤白血球顯著影響肝癌病人的預後，例如，調節性T淋巴球 (Tregs)主要會產生免疫抑制的反應，創造出腫瘤微環境的耐受性，所以若我們可以瞭解浸潤白血球在腫瘤免疫中扮演的角色，這可能可以成為抗癌治療的突破。而過去不少研究已針對肝癌中不同的腫瘤浸潤白血球做研究，但目前少有全面性將HBV、HCV相關之肝癌做免疫細胞之間差異的研究。本研究目的為探究HBV、HCV肝癌和肝癌中腫瘤浸潤白血球與預後之關聯性，並比較不同病毒狀態肝癌間腫瘤浸潤白血球影響之差異。 故本研究共收錄6組資料集，共有828患者，並將之分為B型肝炎肝癌(B-HCC; n=313)、C型肝炎肝癌(C-HCC; n=135)和肝癌(HCC; n=828)共三組。利用近期內新發展之ESTIMATE和CIBERSOT這兩種方法，藉由基因表達微陣列資料，經演算法計算出組織中免疫細胞的量和22種免疫細胞在組織的相對比例，如此一來，我們可以全面性的了解腫瘤浸潤白血球在組織的分布概況，和了解腫瘤浸潤白血球在不同病毒感染的肝癌之間影響預後的差異。 本研究觀察到在三組中，非腫瘤組織的免疫細胞都較腫瘤組織多，但浸潤的情況不太一樣，在B-HCC、C-HCC和HCC組中分別有12、4和12種白血球在腫瘤和非腫瘤組織中的比例有顯著差異，這可能和病毒種類的不同或有無有關，而目前發現M0 巨噬細胞和CD8 T細胞在這三種類型的肝癌中的浸潤情形是一致的。在另一方面，影響存活之免疫細胞於三組之間卻不盡相同，而在B-HCC和HCC組擁有相同浸潤情形且影響存活的免疫細胞有:活化的樹突細胞和M0巨噬細胞，並顯示此兩免疫細胞對存活有負向影響。 我們在三組之中各別顯示在腫瘤微環境的影響下參與癌變的過程並且會直接影響病患的存活的免疫細胞，代表這些免疫細胞為日後免疫治療具有潛力之標的，且未來肝癌免疫治療可以針對不同病毒狀態做不同的考量和調整，以提升治療的療效。

Cancer have been a leading cause of death lasting over 35 years in Taiwan, and hepatocellular carcinoma (HCC) ranks second among top ten cancer mortality rate. Furthermore, World Health Organization announced that HCC causing 788,000 deaths worldwide in 2015 which is in the second place in all types of cancer. According to the investigation of Health Promotion Administration, Ministry of Health and Welfare, HBV-related and HCV-related accounts respectively for 70% and 20% of HCC. Undoubtedly, HCC is an urgent problem in public health. Tumor infiltrating leukocytes (TILs) are immune cells which surrounding around tumor and which are capable of distinguishing and targeting transformed cells, and eliminate tumor before becoming clinically apparent. Immunotherapy is the new trend for anti-tumor strategy which modulates human body immune response. However, tumor-drive immunosuppression was found in microenvironment which is the main impediment of immunotherapy. There were many research have shown that TILs have effect on prognosis of HCC patients. For example, regulatory T cells (Tregs) generate immunosuppressive response and create the microenvironment tolerance. It may a breakthrough in anti-cancer therapy by realizing the role of TILs in tumor immunity. In the past, the majority of published research focused on different TILs in HCC, relatively, very few studies had comprehensively addressed HCC separated into HBV-related or HCV-related. This research will explore the association between TILs and prognosis in B-HCC, C-HCC and HCC and compare the difference of TILs influence on HCC with different virus status. There are 828 patients among six datasets in this study, and divided into three groups, B-HCC (HBV-related HCC; n=313), C-HCC (HCV-related HCC; n=135) and HCC (n=828). This study proposes developed methods, ESTIMATE and CIBERSORT, to assess the immune score which implicated the quantity of leukocytes and the relative proportion among 22 immune cell in tumor/non-tumor tissue through gene expression microarray profile. Thus, we can understand the overview of TILs in tissue and the prognostic difference in HCC with different virus status. We observed that the density of infiltrating leukocytes in non-tumor is more than in tumor, and the fraction of TILs between B-HCC, C-HCC, HCC are quite different, may related to the virus status. The percentage of TILs in tumor is significantly different from in non-tumor which was found 12, 4 and 12 kinds of immune cells in B-HCC, C-HCC and HCC, respectively. As can be seen that M0 macrophage and CD8 T cell have the same pattern in these three groups. On the other hand, there are different immune cells affecting survival within three groups, whereas, activated dendritic cell and M0 macrophage in B-HCC and HCC groups have the same infiltrating scenario and both have negative effect on survival simultaneously. We demonstrate the immune cells which participate in the process of carcinogenesis under the influence of tumor microenvironment and directly affect survival in three groups may represents potential targets for future immunotherapy, meanwhile, it suggests that we make different considerations and adjustments regarding different types of virus status to improve therapeutic efficacy.