計畫性凋亡配體-1在肺癌的表現與臨床病理特徵、腫瘤微環境、肺癌常見基因突變、及預後之關聯性

Programmed Death-Ligand 1 Expression in Lung Cancer and its Association with Clinicopathological Characteristics, Tumor Microenvironments, Driver Mutations, and Clinical Outcomes

楊景堯(Ching-Yao Yang)

指導教授 ： 張逸良

國立臺灣大學/醫學院/病理學研究所/博士(2017年)

https://doi.org/10.6342/NTU201800043

摘要

肺癌長久以來一直居癌症死亡率的第一名，不管是台灣還是全球都有這個現象。目前對於晚期肺癌治療的發展，已經從傳統的化學治療、小分子藥物標靶治療，進展到免疫治療蓬勃發展的年代。在免疫治療之中，發展迅速且具有顯著療效的，當屬於免疫稽核點(immune checkpoints)的抑制劑。所謂的免疫稽核點，原本是正常生理中用來控制發炎反應的機轉，許多癌細胞可以藉由表現免疫稽核點來抑制宿主的免疫反應，將T細胞去活化以逃避免疫系統的攻擊。在眾多免疫稽核點裡，計畫性凋亡因子-1(programmed death-1, PD-1)及其配體，計畫性凋亡配體-1(programmed death-ligand 1, PD-L1)，為目前研究最多且臨床實證最豐富的免疫稽核點標的。在肺癌及許多其他的癌症中，都有發現計畫性凋亡配體-1有過度表現的現象，並與病人預後有一定相關性。可見計畫性凋亡配體-1對腫瘤的發生與存活上，均有相當的重要性。因此，研究癌細胞表計畫性凋亡配體-1的模式，不僅可了解癌細胞與宿主之間的免疫平衡關係，也可評估其是否可應用於免疫治療的療效預測，可說是相當重要的課題。
我們在兩組第一期非小細胞肺癌(non-small cell lung cancer, NSCLC)病人的腫瘤樣本上做了一系列的研究。以免疫組織化學染色的方式，評估計畫性凋亡配體-1的表現模式。在第一組肺腺癌的病人裡 (共有163位病人)，如果以大於百分之五為切點(免疫組織化學染色在百分之五以上的癌細胞，且主要染色位置為細胞膜)，我們發現共有65位病人(65/163, 39.9%)其腫瘤表現計畫性凋亡配體-1為陽性，且計畫性凋亡配體-1的表現，與癌細胞較惡性的分化、及是否有癌細胞血管內侵犯(vascular invasion)成正相關。另外，計畫性凋亡配體-1的表現與肺腺癌常見的基因突變如表皮生長因子受體(epidermal growth factor receptor)或間變性淋巴瘤麟酸酶(anaplastic lymphoma kinase, ALK)突變等無明顯關聯。藉由多變數分析，我們發現計畫性凋亡配體-1的表現，是無疾病存活期(Disease free survival, DFS)改善的獨立預後因子；而較惡性的分化及較高的腫瘤指標(癌症胚胎抗原(Carcinoembrionic antigen, CEA)則為無病存活期惡化的獨立預後因子。而關於整體存活率(Overall Survival, OS)，唯一的獨立預測因子為疾病分期 (1a與1b相比存活期較佳)。
在第二部分，我們在另一組第一期肺部麟狀細胞癌病人(共105人)的腫瘤樣本裡，同樣用組織化學染色的方法去分析除了計畫性凋亡配體-1的表現。除此之外，我們更分析腫瘤微環境的相應變化，特別針對各種腫瘤浸潤淋巴球(tumor infiltrating lymphocyte)的組成。用同樣的切點(組織化學染色在百分之五以上的癌細胞，且主要染色位置為細胞膜)，我們發現在59位病人檢體上(59/105, 56.2%)計畫性凋亡配體-1表現為陽性。整體表現比率，鱗狀細胞癌顯著的比肺腺癌高(adenocarcinoma vs squamous cell carcinoma, 39.9% vs 56.2%, p<0.001)。分析腫瘤微環境的結果顯示，肺癌細胞的計畫性凋亡配體-1的表現與腫瘤內部的CD8+ T淋巴球、及腫瘤周遭CD4+ T淋巴球的浸潤呈現正相關。另外，我們發現在腫瘤周遭，有許多計畫性凋亡配體-1呈現陽性的免疫細胞(PD-L1+ immune cells)，而這些細胞的浸潤程度則與腫瘤細胞本身呈現計畫性凋亡配體-1的量呈現負相關。另外，各種腫瘤浸潤淋巴球各自與臨床預後有關：腫瘤內的CD8+ T淋巴球及腫瘤周遭CD4+ T淋巴球均與無病存活期及整體存活期的延長相關，而腫瘤周遭的調節性T淋巴球(Regulatory T cell)浸潤，則會呈現較差的預後。藉由多變數的存活分析，我們發現不管是無病存活期還是整體存活期，計畫性凋亡配體-1的表現均呈現較好的預後(整體存活期達到顯著水準，p=0.021；無病存活期僅呈現趨勢，p=0.088)。
我們的研究結果顯示，在早期的非小細胞肺癌裡，計畫性凋亡配體-1的表現很一致的預測了較好的臨床預後。這暗示了在這些病人身上，腫瘤細胞表現計畫性凋亡配體-1，很可能是一種適應性抵抗(adaptive resistance)的現象，代表宿主的免疫系統仍有能力對腫瘤細胞並造成的壓力，促使腫瘤細胞產生抵抗的機轉。而表現計畫性凋亡配體-1，便是這些早期肺癌細胞足以抵抗的重要機制。藉由在腫瘤微環境的分析結果，我們發現在鱗狀細胞癌中，腫瘤細胞表現計畫性凋亡配體-1與特定的免疫細胞組合高度相關(高的腫瘤內部CD8+ T淋巴球及腫瘤周遭CD4+ T淋巴球)，而此種淋巴球的組成亦呈現較好的臨床預後。這樣的結果更佐證了早期肺癌表現計畫性凋亡配體-1的機轉，很可能便是透過適應性抵抗的模式。雖然腫瘤細胞已經表現計畫性凋亡配體-1嘗試抵抗T細胞的攻擊，但反映的可能是一個強有力的宿主免疫反應，因此造就了病人有比較好的臨床預後。我們希望藉由此研究成果，可以對肺癌的免疫治療、腫瘤的免疫微環境有更進一步的理解，也希望經由持續的努力，可以對正在受苦的肺癌病人們帶來一些幫助。

關鍵字

肺癌 ； 計畫性凋亡配體-1 ； 免疫稽核點 ； 免疫治療 ； 腫瘤微環境

並列摘要

Lung cancer is the leading cause of cancer-related death worldwide despite a lot of advances in cancer treatment have been achieved in recent decades. Currently, the treatment strategy for advanced lung cancer has been moved forward from conventional platinum based chemotherapy, molecular target therapy, to the era of immunotherapy. Immune checkpoint inhibitors are now the most promising immunotherapy which can unleash the immunosuppression cancer cell posed on host immune system. Among the various immune checkpoints, programmed-death 1 (PD-1) and its ligand, programmed death-ligand 1 (PD-L1) are the most predominating ones which are found in lung cancer and many other types of neoplasms. Expression of PD-L1 on cancer cells can deliver an inhibitory signal through PD-1 on T cells to down-regulate the activity of T cells and prompt cancer escape from host immune systems. Therefore, the expression pattern of PD-L1 on cancer cells becomes an important issue, either for recognition of tumor-host immune balance, or for potential prediction of anti-PD-1/PD-L1 immunotherapy.
We conducted serial studies to examine tumor PD-L1 expression in two stage I cohorts of non-small cell lung cancer (NSCLC). The first one is an adenocarcinoma cohort consisting of 163 patients. Using ≥ 5% membranous positive staining cells as cutoff value, we found PD-L1 expression was positive in 65 of 163 patients (39.9%). Higher grade of differentiation and vascular invasion were associated with PD-L1 expression. The multi-variate analysis for survival showed PD-L1 expression was an independent good prognostic factor of disease free survival (DFS), while higher grade of differentiation and abnormal carcinoembryonic antigen (CEA) were poor prognostic factors. The only favorable and independent prognostic factor for overall survival was earlier stage (IA vs IB).
In the subsequent study, we conducted a deeper and thorough evaluation of PD-L1 expression in another cohort of stage I squamous cell carcinoma (SqCC) (n=105). Because tumor microenvironments are quite crucial for immune-oncology, we analyzed the composition of tumor infiltrating lymphocytes (TIL) in addition to tumor cell PD-L1 expression and clinicopathological features. Using the same cutoff value of 5%, we found that PD-L1 expression was positive 59 of 105 patients (56.2%), which was more prevalent than the ADC cohort (SqCC vs ADC, 56.2% vs 39.9%, p<0.001). The exploration of TIL composition showed that PD-L1 expression positively correlated with tumor epithelial CD8+ T cell and tumor stromal CD4+ T cell infiltration, and negatively correlated with PD-L1+ immune cells infiltrations in tumor stroma. For the individual TIL, tumor epithelial CD8+ T cell and tumor stromal CD4+ T cell infiltrations were associated with a better DFS and OS; while tumor stromal regulatory T cells infiltrations were associated with a poor DFS and OS. By multivariate analysis, PD-L1 and tumor stromal CD4+ T cell infiltrations were independent prognostic factors of OS but only had a trend toward better DFS.
Our results may suggest that in early stage NSCLC, PD-L1 expression reflects a phenomenon of adaptive resistance that an effective host immunity may still exist and is powerful enough to force cancer cell develop an escape mechanism, especially by expressing PD-L1. However, the existence of PD-L1 expression in early stage lung cancer may not point to a status of successful immune escape. We hope our serial studies may help the delineation of cancer immunology and tumor microenvironment of NSCLC.

並列關鍵字

Lung cancer ； Programmed death-ligand 1 ； immune checkpoint ； immunotherapy ； tumor microenvironment

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