類別:

阻斷WNT訊息傳導以遏制KRAS引起肺癌轉移

Wed, 01 Jan 2020 00:00:00 GMT

標題: 阻斷WNT訊息傳導以遏制KRAS引起肺癌轉移; The Inhibition of Wnt Restrain KRASG12V-Driven Metastasis in Non-Small-Cell Lung Cancer 作者: Pei-Shan Hung; 洪珮珊摘要: KRAS基因變異而導致癌症的發生約佔30% ，特別是發生在胰臟癌、大腸癌、肺癌等這些高度惡性癌症。針對KRAS基因突變的治療標的是有相當的困難度。KRAS的基因變異主要為錯義突變(missense mutation)，其中，在胺基酸12、13和61的密碼子是突變的熱點。KRAS的錯義突變，會提高抵抗GTPase activating protein (GAP) 所引起的 GTP 水解能力，與降低內生性GTP 水解能力，進而導致RAS-GTP鎖定在活化狀態之下，且持續活化下游訊息傳遞。近來許多文獻指出，KRAS基因的突變亞型，並不能被視為同一種基因突變。然而，卻缺乏對於各別KRAS基因突變亞型的特性，與相關致癌機制的研究與探討。在本論文裡，我們驗證在非小細胞肺癌中，KRAS基因突變亞型KRASG12V 與 KRASG12D所引起的癌症轉移機轉差異，並且使用Wnt/β-catenin pathway 抑制劑可以降低KRASG12V所引起的非小細胞肺癌細胞轉移。我們使用H838等基因系細胞株 (isogenic cell line)，以排除癌細胞內基因背景的干擾。首先，我們發現KRASG12V 與KRASG12D，在細胞實驗 (in vitro) and與動物實驗 (in vivo) 中，觀察到不同的轉移 (metastasis) 能力。透過基因集(GSEA)的分析後，在H838KRASG12V細胞所表現的基因集與RhoA-related signaling基因集相比，比對結果呈現負相關。我們也進一步發現，在H838KRASG12D細胞中，KRASG12D反而引起較高的RhoA活性，且具有較低的Wnt/β-catenin 活化。當抑制H838KRASG12D細胞內的RhoA活性後，則可以偵測到提高的Wnt/β-catenin 的活化。此外，當給予Wnt/β-catenin抑制劑後，可降低H838KRASG12V細胞的爬行 (migration) 能力。在本論文，我們闡明了在KRAS 所引起的非小細胞肺癌裡，KRAS/RhoA/Wnt/β-catenin所調控的轉移機轉。KRASG12V所引起的轉移對於Wnt/β-catenin機制有較高的依賴。我們的研究結果證實，KRASG12V 與KRASG12D基因變異亞型所引起的非小細胞肺癌轉移，在設計治療方法上，應被視為獨立的疾病; The KRAS mutations has been an obstacle to identify therapeutic targets in cancer treatment. In this work, we clarified the distinct metastasis pattern of Non-Small-Cell Lung Carcinoma (NSCLC) induced by KRASG12V/KRASG12D mutations and inhibited the KRASG12V mediated metastasis by Wnt inhibitor. First, we found that KRASG12V induced more aggressive phenotype in Vitro and in Vivo experiments. The Gene Set Enrichment Analysis (GSEA) results of H838 KRASG12V cells showed a significant negative correlation with RhoA-related signaling. Following this clue, we observed KRASG12D induced higher activation of RhoA and suppressed activation of Wnt/β-catenin in H838 KRASG12D cells. The restored activation of Wnt/β-catenin in H838KRASG12D cells could be detected when expression with a dominant-negative mutant of RhoA or treatment with RhoA inhibitor. Furthermore, the Wnt inhibitor abolished the KRASG12V -induced migration. We elucidated the importance of the axis of RhoA/Wnt in regulatory NSCLC metastasis driven by KRAS mutations. Our data indicate that KRASG12V driven NSCLC metastasis is Wnt-dependent and the mechanisms of NSCLC metastasis induced by KRASG12V/KRASG12D is distinct.

阻斷Wnt/β-catenin訊息傳遞路徑以治療肝細胞癌

Sun, 01 Jan 2017 00:00:00 GMT

標題: 阻斷Wnt/β-catenin訊息傳遞路徑以治療肝細胞癌; Targeting the Wnt/β-catenin Signaling Pathway in the Treatment of Hepatocellular Carcinoma 作者: Hsiao-Hui Lin; 林曉慧摘要: 肝細胞癌(Hepatocellular Carcinoma，簡稱HCC)為十分致命且預後非常差的癌症之一。過去幾十年來，在肝細胞癌的篩檢、早期診斷、以及局部治療上，雖然已有長足的進步；但大多數的肝細胞癌病人仍終將發展至晚期(advanced stage)，而必須考慮接受全身性藥物治療。目前晚期肝細胞癌的標準治療，也是唯一經衛生主管機關核可的藥物是sorafenib (商品名為Nexavar【蕾莎瓦】)。Sorafenib是一種多重激酶抑制劑，可抑制Raf激酶及對抗血管內皮生長因子受體。整體而言，sorafenib在肝癌的治療上，腫瘤縮小的機率不高，腫瘤控制時間短暫，其治療效果仍有相當進步的空間。本論文研究鎖定「Wnt/β-catenin訊息傳遞路徑」做為肝細胞癌嶄新的治療標的。Wnt/β-catenin訊息傳遞路徑在胚胎發育、組織結構的恆定、以及肝細胞癌癌化的過程上，都扮演十分重要的角色。本論文研究嘗試驗證以下兩個假說：一、抑制Wnt/β-catenin訊息傳遞路徑的活性可促進sorafenib對於肝細胞癌的治療療效；二、肝細胞癌β-catenin 基因上特殊的活化性突變，可能導致該癌細胞對特定的Wnt/β-catenin訊息傳遞路徑抑制劑特別敏感。在本論文的第一部分中，我們證實了無論於細胞培養上或活體內，抑制Wnt/ β-catenin訊息傳遞路徑的活性可促進sorafenib對於肝細胞癌的治療效果。ICG-001是Wnt/β-catenin訊息傳遞路徑的小分子抑制劑，它經由阻斷的β-catenin與轉譯共同激活因子CBP之間的交互作用而達到抑制效果。在多株肝細胞癌的體外培養實驗中，我們發現伴隨著ICG-001使用的濃度提高，肝細胞癌細胞株生長受到sorafenib治療後生長抑制的情況及走向細胞凋亡的細胞比例隨之增加。進一步以RNA干擾的技術抑制β-catenin後，我們亦發現肝細胞癌細胞株Huh7對sorafenib作用的敏感度明顯提升；反之將β-catenin過度表現，則可降低Huh7對sorafenib作用的敏感度。此外，我們發現經短髮夾RNA(shRNA)處理導致β-catenin表現低下之Huh7對sorafenib作用的敏感度提升，再將該細胞的β-catenin過度表現之後，其敏感度則再度降低。就機制上而言，合併使用sorafenib與ICG-001可促使更多肝細胞癌細胞株走向細胞凋亡，並導致Mcl-1表現的更顯著下降。在Huh7肝細胞癌細胞小鼠皮下腫瘤的動物模式，合併使用sorafenib與ICG-001的組別相較於單獨使用sorafenib亦或ICG-001的組別，更能顯著地抑制腫瘤的生長。在本論文的第二部分中，我們則闡明了CTNNB1基因之第三外顯子具有錯義突變(missense mutation)之肝細胞癌細胞株，對特定的Wnt/β-catenin訊息傳遞路徑抑制劑的抑制作用有較高的敏感度。我們使用SNU398與Huh6(分別於CTNNB1基因的第三外顯子的S37C及G34V有錯義突變)、Huh7及HepG2(在CTNNB1基因第三外顯子處有缺失的片段[interstitial deletion])、及PLC5、Hep3B、HLE及SK-Hep1 (在第三外顯子的位置無基因異常)等肝細胞癌細胞株；並分別處理包括ICG-001、XAV939 (為tankyrase的抑制劑，可藉此達到AXIN-1的穩定，以抑制β-catenin的訊息傳遞活性)、以及LGK974 (為porcupine的抑制劑；porcupine可促使Wnt ligands的成熟與分泌)等Wnt/β-catenin訊息傳遞路徑抑制劑。在XAV939及LGK974的處理下，不同肝細胞癌細胞株生長受到抑制的程度差異並不顯著；然而在ICG-001的處理下，我們發現SNU398的生長抑制作用最為顯著，緊追其後為Huh6。除此之外， ICG-001對於SNU398的細胞聚落形成的抑制力、經ICG-001處理後誘發sub-G1波峰提升的程度，皆高於其他肝細胞癌細胞株。最後，不同的化學治療藥物，包含doxorubicin、cisplatin、及5-fluorouracil對不同肝細胞癌細胞株的抑癌效果，並無發現顯著的差異。我們的實驗研究結果顯示抑制Wnt/β-catenin訊息傳遞路徑的活性可促進sorafenib對於肝細胞癌的治療療效，而具有β-catenin第三外顯子有錯義突變的肝細胞癌細胞株對ICG-001的抑癌效果最為敏感。本論文研究的發現支持持續鎖定Wnt/β-catenin訊息傳遞路徑研發抑制劑(無論是與sorafenib合併使用，或針對特定分子標記族群做單一治療)以治療肝細胞癌。; Hepatocellular carcinoma (HCC) is one of the most lethal cancers in the world. Although a lot of advances in screening, early diagnosis, and loco-regional therapies for HCC have been made over the past decades, most HCC patients would eventually develop advanced diseases that need systemic therapy. Currently, sorafenib, a multikinase inhibitor targeting Raf kinase and vascular endothelial growth factor receptor, is the only approved drug for HCC. However, its efficacy is limited, with low tumor response rate and short tumor control duration. The current thesis work focuses on developing new therapeutic strategies for HCC by targeting the Wnt/β-catenin signaling pathway, which plays an important role in embryonic development and tissue homeostasis, as well as in hepatocarcinogenesis. The thesis work has tested two hypotheses: (1) inhibition of the Wnt/β-catenin signaling pathway could improve the anti-tumor effects of sorafenib in HCC; (2) activating mutations of β-catenin gene may confer sensitivity to specific Wnt/β-catenin pathway inhibitors in HCC. In the first part of the current thesis work, we demonstrated that inhibition of the Wnt/β-catenin signaling pathway could improve the anti-tumor effects of sorafenib against HCC in vitro and in vivo. ICG-001, a small molecule that blocks the interaction of β-catenin with its transcriptional coactivator CBP, dose-dependently enhanced the growth-suppressive and apoptosis- induction effects of sorafenib in multiple HCC cell lines. Downregulation of β-catenin by RNA interference increased sorafenib sensitivity, whereas overexpression of β-catenin reduced sorafenib sensitivity in Huh7 cells. The sorafenib-sensitization effect of short hairpin RNA (shRNA)-mediated β-catenin downregulation in Huh7 cells was attenuated by β-catenin overexpression. Mechanistically, sorafenib combined with ICG-001 or shRNA- mediated β-catenin downregulation augmented the induction of apoptosis, and resulted in a significant downregulation of Mcl-1 in HCC cells. In Huh7 cell mouse xenograft model, the combination of ICG-001 and sorafenib showed a more significant growth-retarding effect than single agent treatment of sorafenib or ICG-001. In the second part of the current thesis work, we demonstrated that HCC cells with point mutation at exon 3 of the CTNNB1 gene exhibited an increased sensitivity to antitumor effects of certain Wnt/β-catenin inhibitors in vitro. SNU398 and Huh6 are HCC cells harboring missense somatic mutations in exon 3 of the CTNNB1 gene, resulting in S37C and G34V mutation, respectively; Huh7 and HepG2 are HCC cells with interstitial deletion of exon 3; while other HCC cells including PLC5, Hep3B, HLE, and SK-Hep1 are HCC cells containing no mutations in exon 3. Several classes of Wnt/β-catenin pathway inhibitors were tested including ICG-001, XAV939 (an inhibitor of tankyrases which would stabilize AXIN-1 and thus β-catenin transactivation activity), and LGK974 (an inhibitor of porcupine which could help maturation and secretion of Wnt ligands). The sensitivity to growth suppressive effects of XAV939 and LGK974 did not vary significantly among all tested HCC cells. However, the growth-suppressive effect to ICG-001 was most sensitive in SNU398 cells, followed by Huh6 cells, and less sensitive in other HCC cells. ICG-001 also induced a more significant suppression of colony-formation, and a more significant increase of sub-G1 fraction by flow cytometry in SNU398 than other HCC cells. Finally, the cytotoxic effects of multiple chemotherapy drugs including doxorubicin, cisplatin, and 5-fluorouracil did not significantly differ among tested HCC cells. Our studies have demonstrated that targeting of Wnt/β-catenin signaling pathway improves the anti-tumor effects of sorafenib against HCC and the presence of a missense mutation in exon 3 of β-catenin may confer increased sensitivity to ICG-001 in certain HCC cells. These data support further investigations on developing Wnt/β-catenin inhibitors as potential cancer therapeutics for HCC by either combination with sorafenib or single-agent approach focusing on biomarker-enriched population.

鈣網蛋白突變在骨髓增生性腫瘤的角色

Sun, 01 Jan 2017 00:00:00 GMT

標題: 鈣網蛋白突變在骨髓增生性腫瘤的角色; The Roles of Calreticulin Mutations in Myeloproliferative Neoplasms 作者: Ken-Hong Lim; 林建鴻摘要: 骨髓增生性腫瘤是克隆性造血幹細胞疾病，並且可以分類為“典型”和“非典型”骨髓增生性腫瘤。典型骨髓增生性腫瘤通常會在周邊的血液中表現出終端骨髓細胞的擴增，包括真性紅血血球增多症、原發性血小板增多症、原發性骨髓纖維化和慢性骨髓性白血病。近年來，在大約30％的JAK2/MPL-未突變的骨髓增生性腫瘤中發現鈣網蛋白(Calreticulin, CALR)突變。目前，鈣網蛋白突變已經成為診斷原發性血小板增多症和原發性骨髓纖維化的重要克隆標誌物。這項研究的第一個目的是開發一種快速和敏感的篩選工具，用於檢測鈣網蛋白突變。我們使用CFX Connect即時系統成功開發了高分辨率熔解分析，以檢測原發性血小板增多症病人的鈣網蛋白第9外顯子突變。我們的高分辨率熔解分析系統在識別病人基因組DNA中的鈣網蛋白第1型和第2型突變體的最大敏感性為2.5%。我們的高分辨率熔解分析系統假陽性率為3%，並且無假陰性出現。本研究的第二個目的是評估台灣原發性血小板增多症病人鈣網蛋白突變和JAK2 /CALR共突變的臨床和預後意義。我們在92例成年原發性血小板增多症病人中利用高分辨率熔解分析篩選了鈣網蛋白第9外顯子的改變，隨後並且進行了TA克隆。我們在21例（22.8％）病人中鑑定出典型的鈣網蛋白插入/刪除型突變。鈣網蛋白突變與年齡較輕（p = 0.025），血小板數較高（p <0.001）和較低的血紅素（p = 0.016）有相關。有趣的是，我們檢測到在59例JAK2突變的原發性血小板增多症病人中有13例（22％）的鈣網蛋白第9外顯子的改變。與這些具有JAK2 /CALR共突變的原發性血小板增多症病人有相關的因子包括年齡較大（p = 0.025），診斷後發生血栓事件較多（p = 0.048），診斷後主要動脈血栓事件較多（p = 0.022），且較多屬於血栓出血併發症高風險組病人（p = 0.023）。我們的研究顯示JAK2突變的原發性血小板增多症病人可以出現頻繁的鈣網蛋白第9外顯子的改變，並且界定出一群具有血栓事件風險增加的病人亞群。　本研究的第三個目的是要探討鈣網蛋白突變之原發性血小板增多症病人的B細胞免疫特徵。我們篩選了54例台灣成年原發性血小板增多症病人的鈣網蛋白突變並評估了其B細胞免疫特徵。在這54例原發性血小板增多症病人中有19例（35.2％）具有8種不同類型的鈣網蛋白笫9外顯子突變，其中包括4例（7.4％）同時伴有JAK2V617F共突變的病人。經過年齡、性別、追踨期和血液學參數校正後，我們進行的多變量分析證實，與健康成年人相比，活化的B細胞在JAK2突變，鈣網蛋白突變和三陰性原發性血小板增多症病人中都有增加的現象。因此，活化的B細胞增加在不同突變亞組的原發性血小板增多症病人中是一種普遍存在的現象。本研究的第四個目的是使用斑馬魚動物模型研究鈣網蛋白突變的分子發病機制。我們確定了3種與人類鈣網蛋白直系同源的斑馬魚基因，稱為calr，calr3a和calr3b。CALR-del52和CALR-ins5突變體的表達使斑馬魚早期的造血幹/前驅細胞增加，並進而造成血小板球增多但不影響正常的血管生成。我們發現使用morpholino降低mpl但不是epor或csf3r可以顯著的減弱CALR突變體對血小板球增多的影響。此外，CALR突變體的表達也活化jak/stat信息傳遞路徑，而使用JAK抑製劑（ruxolitinib和fedratinib）可以抑制此活化現象。這些研究結果說明CALR突變體通過mpl依賴機制活化jak信息傳遞路徑導致斑馬魚致病性之血小板球生成。我們研究的結果也說明與突變型CALR腫瘤發生相關的訊息傳遞機制在人與斑馬魚之間是保守的。; The myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders and can be classified into 'classic' and 'atypical' MPNs. Classic MPNs usually exhibit terminal myeloid cell expansion in the peripheral blood and include polycythemia vera, essential thrombocythemia (ET), primary myelofibrosis and chronic myeloid leukemia. Calreticulin (CALR) mutations have recently been discovered in about 30% JAK2/MPL-unmutated myeloproliferative neoplasms (MPN), and have become an important clonal marker for the diagnosis of essential thrombocythemia (ET) and primary myelofibrosis. The first aim of this study is to develop a rapid and sensitive screening tool for the detection of CALR mutations. We successfully developed a high-resolution melting analysis (HRMA) with the CFX Connect real-time system to detect CALR exon 9 mutations in ET patients. The maximal sensitivity of our HRMA system in identifying both CALR type 1 and type 2 mutants from patients’ genomic DNA was 2.5%. Our HRMA has a false positive rate of 3% and no false negative. The second aim of this study is to evaluate the clinical and prognostic significance of CALR mutations and JAK2/CALR co-mutations in Taiwanese ET patients. We screened for CALR exon 9 alterations with HRMA followed by TA-cloning in 92 adult ET patients. We identified classic CALR indel mutations in 21 (22.8%) patients. CALR mutations were associated with younger age (p=0.025), higher platelet count (p<0.001) and lower hemoglobin level (p=0.016). Interestingly, we detected various CALR exon 9 alterations in 13 (22%) of 59 JAK2-mutated ET patients. JAK2-mutated ET patients with concomitant CALR alterations were associated with oldest age (p=0.025), higher thrombotic events after diagnosis (p=0.048), higher major arterial thrombotic events after diagnosis (p=0.022) and more patients being high risk group for thrombo-hemorrhagic complications (p=0.023). Frequent CALR exon 9 alterations in JAK2-mutated ET patients define a specific subgroup of patients with increased risk of thrombotic events. The third aim of this study is to determine the B cell immune profiles in CALR mutated ET patients. We screened for CALR mutations and evaluated B cell immune profiles in a cohort of 54 adult Taiwanese ET patients. 19 (35.2%) of 54 ET patients harbored 8 types of CALR exon 9 mutations including 4 (7.4%) patients with concomitant JAK2V617F mutations. Multivariate analysis adjusted for age, sex, follow-up period and hematological parameters confirmed that increased activated B cells were universally present in JAK2-mutated, CALR-mutated and triple-negative ET patients when compared to healthy adults. In conclusion, increased B cell activation is present in ET patients across different mutational subgroups. The fourth aim of this study is to investigate the molecular pathogenesis of CALR mutations using zebrafish animal models. We identified 3 zebrafish genes orthologous to human CALR, referred to as calr, calr3a and calr3b. Expression of the CALR-del52 and CALR-ins5 mutants caused an increase in the hematopoietic stem/progenitor cells followed by thrombocytosis without affecting normal angiogenesis. The expression of CALR mutants also perturbed early developmental hematopoiesis in zebrafish. Importantly, morpholino knockdown of mpl but not epor or csf3r could significantly attenuate the effects of mutant CALR. Furthermore, expression of mutant CALR caused jak-stat signaling activation in zebrafish that could be blocked by JAK inhibitors (ruxolitinib and fedratinib). These findings showed that mutant CALR activates jak-stat signaling through an mpl-dependent mechanism to mediate pathogenic thrombopoiesis in zebrafish, and illustrated that the signaling machinery related to mutant CALR tumorigenesis are conserved between human and zebrafish.

腫瘤免疫微環境在食道鱗狀細胞癌預後的意義

Wed, 01 Jan 2025 00:00:00 GMT

標題: 腫瘤免疫微環境在食道鱗狀細胞癌預後的意義; Tumor Immune Microenvironment in the Prognosis of Locally Advanced Esophageal Squamous Cell Carcinoma 作者: 黃大成; Ta-Chen Huang 摘要: 食道鱗狀細胞癌是亞洲的一個主要癌症，佔比全球食道癌超過85%。大部分的病人在診斷時已是局部晚期，而前輔助同步化放療加上隨後的根除性食道切除術是目前局部晚期食道鱗狀細胞癌的標準治療。然而，仍有大約70%的病人會遭受癌症復發。因此，我們需要一個有效的預後生物標記，才能將病患依復發風險加以分層、甚至施以不同的治療，希望能進一步改善這群病人的預後。人體對抗腫瘤的免疫力可以降低癌細胞的侵略性與生長，這也是包括化學治療與放射治療等許多抗癌治療策略，能達到治療腫瘤療效的重要關鍵之一。因此，免疫相關的生物標記是許多癌症重要的研究主題；針對食道鱗狀細胞癌，過去也有許多相關的研究。PD-L1是PD-1 (為一個免疫檢查點)的配體，可以造成T淋巴細胞的衰竭進而抑制免疫。PD-L1在腫瘤中的表現不但代表對腫瘤免疫力的拮抗、也代表著免疫力的存在。過去關於PD-L1的表現在食道鱗狀細胞癌預後意義雖有許多研究，其結果並不十分一致。另外，第三級淋巴結構是在慢性發炎的組織(包括腫瘤，也可視為一種慢性發炎組織)發生、由免疫細胞組成的一種淋巴組織。一般認為，這種淋巴組織在腫瘤中具有對抗腫瘤的免疫力。目前關於第三級淋巴結構在食道鱗狀細胞癌預後的研究仍相當有限。本論文研究針對接受前輔助化放療的局部晚期食道鱗狀細胞癌病患，聚焦其治療前的食道癌腫瘤免疫微環境，首先探討腫瘤細胞PD-L1、或免疫細胞PD-L1的表現，接著探討第三級淋巴結構的形成與成熟，對局部晚期食道鱗狀細胞癌病患的預後意義。本論文研究的第一個部分探討PD-L1 的預後意義。這一部分研究收納了100位接受以每週太平洋紫杉醇和鉑金化療藥物為處方前輔助同步化放療的局部晚期食道鱗狀細胞癌病患。以治療前上消化道內視鏡取得的食道鱗狀細胞癌組織，進行PD-L1 的免疫組織化學染色，以半定量的方式分析PD-L1表現量為0、1+、2+或3+：在免疫細胞上的表現量0或1+定義為低表現、表現量2+或3+則為高表現；在腫瘤細胞上的表現量0定義為無表現、表現量1+、2+或3+則為有表現。我們發現免疫細胞PD-L1表現的高低與病患的無惡化存活以及整體存活都有強烈的正相關，而腫瘤細胞PD-L1表現的有無則與無惡化存活以及整體存活有顯著的負相關。多變項分析也確認了上述的發現。本論文研究的第二部分，根據第一部分的研究結果提出了一個假說：腫瘤細胞表現的PD-L1與對抗癌藥物的抗藥性有相關性。我們測試五株食道鱗狀細胞癌細胞株對鉑金化療藥物的藥物敏感性與這些細胞株上PD-L1表現量的相關性。我們發現KYSE150是一株帶有PD-L1基因重複（重複數：7）的細胞株，其PD-L1表現量是五株細胞株最高的；KYSE150也五株細胞株中是對鉑金化療藥物抗藥性最高的細胞株。KYSE510是PD-L1表現量最低的，同時也是對鉑金化療藥物最敏感的細胞株。這五株細胞株PD-L1的表現量與其在一系列白金濃度下存活的比例，具有中等度的線性相關性。這個結果支持了PD-L1的表現與化療抗藥性相關的假說。然而，詳細的機轉還需要更進一步的研究。本論文研究的第三部分探索局部晚期食道鱗狀細胞癌黏膜層的第三級淋巴結構的預後意義。這一部分研究收納了一組137位接受以每週太平洋紫杉醇和鉑金化療藥物為處方前輔助同步化放療的局部晚期食道鱗狀細胞癌病患。以治療前上消化道內視鏡取得的食道鱗狀細胞癌組織，進行CD20以及CD23的免疫組織化學染色，分析第三級淋巴結構的成熟狀態。我們發現成熟的第三級淋巴結構和對前輔助化放療的完全病理反應成顯著的負相關（p=0.031）。雖然在單變項分析中，成熟的第三級淋巴結構和不良的整體存活間只存在不顯著的相關趨勢，但在多變項分析中，成熟的第三級淋巴結構成為了獨立而顯著的不良預後因子（HR: 2.91, p<0.001）。這個結果與我們進行本研究之前的假說並不吻合。本論文研究的第四個部分，我們嘗試驗證以內視鏡切片檢體判讀第三級淋巴結構狀態的可信度。我們利用nanoString平台的Human Pan-Cancer Immune Panel來分析44位局部晚期食道鱗狀細胞癌病患的腫瘤檢體，探討第三級淋巴結構中，無、不成熟與成熟三種狀態下，腫瘤免疫微環境的差異。我們發現第三級淋巴結構的形成與成熟相關於較活躍的免疫環境，包括較少的衰竭CD8 T細胞和較高的白血球、B細胞、T細胞、NK細胞、細胞激素、化學激素、腫瘤壞死因子、toll-like受器以及抗原呈現等免疫功能。我們接著利用對比成熟第三級淋巴結構與無第三級淋巴結構的腫瘤組織中免疫基因表現的差異，發展出第三級淋巴結構的基因標記（gene signature）。我們首先針對TCGA資料庫中食道鱗狀細胞癌的族群（82人），證實這個基因標記可以有效地分別出腫瘤組織中淋巴濾泡組織的有無。我們也針對一個接受免疫檢查站抑制劑治療的轉移復發食道鱗狀細胞癌族群（35人），證實這個基因標記可以有效地預測免疫檢查站抑制劑治療的療效。這些發現都支持我們使用內視鏡切片的腫瘤檢體所進行的分析方式，可以有效地區分食道癌腫瘤粘膜第三級淋巴結構的成熟狀態。總結來說，PD-L1在免疫細胞或者腫瘤細胞的表現、粘膜中成熟的第三級淋巴結構是接受前輔助同步化放療局部晚期的食道鱗狀細胞癌獨立的預後生物標記。PD-L1在腫瘤細胞的表現和化療的抗藥性有正相關，這可能是PD-L1在腫瘤細胞的表現導致不良預後的機轉之一。然而，同步放化療是否對第三級淋巴結構造成傷害而導致不良的預後，則需要進一步的研究。; Esophageal squamous cell carcinoma (ESCC) is a major cancer in Asia, and accounts for more than 85% of global burden of patients diagnosed with esophageal cancer. Most ESCC patients had locally advanced cancer at diagnosis. Neoadjuvant chemoradiotherapy (CRT) followed by radical esophagectomy is a current standard treatment for locally advanced ESCC. However, about 70% of patients suffered from recurrence. Efficient prognostic biomarkers are thus important for helping stratify patients with different risks of recurrence and for developing novel treatment strategies to improve their outcome. Anti-tumor immunity hinders the aggressiveness and growth of cancer and may contribute to the efficacy of various anti-cancer therapy, including chemotherapy and radiotherapy. Immune-related biomarkers have been investigated for their prognostic significance in patients with locally advanced ESCC before. PD-L1, a ligand of an immune checkpoint, programmed cell death protein 1, suppresses immunity by causing exhaustion of T cells. The expression of PD-L1 in tumor, an antagonist of anti-tumor immunity, may represent the existence of anti-tumor immunity. Multiple previous studies focusing on the expression of PD-L1 in tumors yielded conflicting results in patients with ESCC. Tertiary lymphoid structure (TLS)—aggregates of immune cells forming lymphoid structures in or around tumor tissues—is considered as an origin of antitumor immunity. The prognostic significance of TLS has been only reported in a limited number of studies involving patients of locally advanced ESCC. The current thesis, focusing on tumor immune microenvironment (TME) of ESCC, has explored the prognostic significance of PD-L1 expression on immune cells (ICs) or tumor cells (TCs) and the prognostic significance of mucosal TLS in locally advanced ESCC patients treated with neoadjuvant CRT with weekly paclitaxel/platinum chemotherapy. For the first part of the thesis, the prognostic significance of PD-L1 expression was investigated. A total of 100 locally advanced ESCC patients treated with neoadjuvant CRT with weekly paclitaxel/cisplatin chemotherapy were enrolled. Their pre-treatment ESCC tumor tissues, obtained by endoscopic biopsy, were analyzed for the expression of PD-L1 by immunohistochemistry (IHC) and scored semiquantitatively. PD-L1 expression on ICs (PD-L1 IC) was defined as low (0 or 1+) vs high (2+ or 3+), and PD-L1 expression on TCs (PC-L1 TC) was defined as negative (0) vs positive (1+~ 3+). We found that high PD-L1 IC expression was strongly associated with better progression free survival (PFS) (HR: 0.44, p=0.0025) and overall survival (OS) (HR: 0.44, p=0.0024), and positive PD-L1 TC was significantly associated with worse PFS (HR: 1.7, p=0.029) and OS (HR: 1.63, p=0.035). Multivariate analysis demonstrated both PD-L1 TC and PD-L1 IC as independent prognostic factors. For the second part of the thesis, we hypothesized that PD-L1 expression on TCs may contribute to chemoresistance, thus underlying the finding of positive PD-L1 TC as a poor prognostic factor in patients with locally advanced ESCC treated with neoadjuvant CRT. We tested the chemosensitivity to cisplatin and the expression level of PD-L1 in 5 human ESCC cells lines. We found that KYSE150, which has PD-L1 amplification (copy number=7), had higher expression of PD-L1 compared to other cell lines and was more resistant to cisplatin than other cells. KYSE510 had the lowest baseline expression among the 5 cell lines and was the most chemosensitive cell line. Linear relationships can be identified between PD-L1 expression level and survival percentage of the 5 cell lines under serial concentrations of cisplatin. These findings support our hypothesis about the association of PD-L1 expression on tumor cells with resistance to chemotherapy. However, the detailed mechanisms warrant additional studies. For the third part of the thesis, to investigate the prognostic significance of mucosal TLS in locally advanced ESCC, we recruited a cohort of locally advanced ESCC patients who received neoadjuvant CRT with weekly paclitaxel/platinum chemotherapy (n= 137). Pretreatment endoscope-biopsied primary esophageal tumor tissues were analyzed for the maturation status of TLS according to the IHC-defined expression of CD20 and CD23. The status of mature TLS had significant negative association with pathological complete response to neoadjuvant CRT (p=0.031). Although mature TLS only had a trend of association with worse OS (HR: 1.45, p=0.15) in univariate analysis, mature TLS turned out to be a strong independent unfavorable prognostic factor in the multivariate cox regression analysis (HR: 2.91, p<0.001). The result rejected our initial hypothesis that TLS is a favorable prognostic factor. For the fourth part of the thesis, in order to validate the reliability of TLS status identified by endoscope-biopsied tissues, we explored the TME in tumors with no, immature, or mature TLS status of patients with locally advanced ESCC. We investigated the expression profiles of immune-related genes of ESCC tumor tissues by Pan-Cancer Immune Panel of nanoString® (n=44). We found that the maturation of TLS was associated with more active TME, including less exhausted CD8 T cells and more immune functions of leukocytes, B cells, T cells, NK cells, cytokines, chemokines, tumor necrosis factors, toll-like receptors, and antigen presentation. Based on genes with highly differentiated expression between ESCC tumors with mature TLS vs no TLS, we constructed a TLS signature and demonstrated that this TLS signature can significantly differentiate tumor tissues with from without lymphoid follicles in the ESCC cohort of TCGA database (n=82); the TLS signature can also significantly differentiate ESCC patients benefited from anti-PD-1 based immunotherapy in our cohort of metastatic or recurrent ESCC patients (n=35). These findings support that the TLS status identified by IHC on pre-treatment endoscope-biopsied tumor tissues is accurate. Overall, we found that PD-L1 IC, PD-L1 TC, and mucosal mature TLS were independent prognostic biomarkers for locally advanced ESCC patients who received neoadjuvant CRT. Positive PD-L1 TC is associated with chemoresistance, which could lead to unfavorable prognosis. It needs further investigations whether the damage from CRT changes the TME of TLS and leads to unfavorable prognosis.