胰腺导管腺癌（pancreatic ductal adenocarcinoma，PDAC）是一种侵袭性极强的胰腺恶性肿瘤，是胰腺肿瘤中最常见的组织学类型，占胰腺肿瘤病例的90%以上。PDAC患者预后极差，五年生存率约在11%。而且近些年来，PDAC患者的死亡率有呈现逐年上升趋势。目前针对PDAC的治疗手段主要为手术和辅助化疗，目前这些治疗手段对预后的改善并不明显。因此，探寻新型有效的治疗策略是很迫切的。基于抑制程序性细胞死亡受体1（programmed cell death 1, PD-1）/程序性细胞死亡配体1（programmed cell death ligand 1，PD-L1）轴的免疫检查点抑制剂疗法是癌症免疫治疗研究中一项重要的里程碑；然而，多项有关PDAC抗PD-1治疗的临床试验结果显示免疫检查点抑制剂的治疗响应率很低。基于目前这些存在的现实问题，鉴定出新的免疫检查点通路以及充分了解PDAC的免疫结构模式有助于发现新的潜在免疫治疗靶点以及改善免疫治疗反应性。由于多个新型免疫检查点的表达以及免疫结构模式在PDAC中潜在的临床应用价值还不明确；因此，本研究拟在PDAC中通过检测多个新型免疫检查的表达情况以及描绘免疫结构模式特征，并探讨和分析其与PD-L1等传统免疫分子指标、临床病理特征，预后以及术后辅助化疗反应性之间的关联。本论文主要的研究内容分为以下两个部分：

第一部分 新型免疫检查点在胰腺导管腺癌中的表达和意义

研究内容Ⅰ 新型免疫检查点TIGIT及其配体CD155在胰腺导管腺癌免疫微环境中的空间表达和意义

研究背景：针对新出现的具有免疫球蛋白和免疫受体酪氨酸抑制基序结构域的T细胞免疫受体（T cell immunoreceptor with immunoglobulin and ITIM domain，TIGIT）/CD155免疫调控轴在一些恶性肿瘤中具有恢复抗肿瘤免疫和重塑免疫微环境的作用，但对其在PDAC患者中的有关免疫表型的预后意义及其对术后辅助治疗（adjuvant chemotherapy，ACT）反应性预测价值的了解十分有限。本研究将探索免疫检查点TIGIT/CD155在PDAC免疫微环境中的表达及其临床意义。

研究方法：对包括适应免疫细胞亚群、PD-1/PD-L1轴以及TIGIT/CD155轴等多个免疫指标进行合理设计形成三个七色多光谱panel，通过多重免疫组化在272个PDAC标本中研究了TIGIT/CD155的蛋白表达模式及其与免疫微环境特征、预后以及术后辅助化疗的反应性的关联。

研究结果：我们揭示了PDAC免疫微环境的低免疫原性和高异质性，其特征是总的CD3+ T细胞和CD68+巨噬细胞数量丰富，活化的具有细胞毒性的多个T淋巴细胞功能亚群浸润的密度相对较低。通过western blotting、免疫组化和多重免疫荧光，我们发现PDAC组织中TIGIT和CD155的表达水平相对于癌旁组织中显著更高。TIGIT在肿瘤细胞（tumor cells，TCs）、肿瘤浸润淋巴细胞（tumor infiltrating lymphocytes，TILs）和CD8+ T细胞上阳性率分别为82.95%、88.83%和74.26%。CD155在TCs和CD68+巨噬细胞上阳性率分别为79.09%和61.83%。TILs上TIGIT阳性表达的与更高密度的CD45RO+ T细胞的浸润相关；TIGIT+CD8+ T细胞与更高比例的CD3+CD45RO+FOXP3+T细胞浸润相关。CD155肿瘤细胞阳性表达（CD155+TC或CD155+CK+）与更高比例CD3+T细胞和CD3+CD8+CD45RO+ T细胞相关。TIGIT+TCs、TIGIT+CD8+ T细胞以及CD155+CD68+巨噬细胞与较差的无进展生存（progression-free survival，PFS）和疾病特异性生存（disease-specific survival，DSS）相关，其中TIGIT+TCs和CD155+CD68+巨噬细胞是PFS的独立预后因素，TIGIT+CD8+ T和CD155+CD68+巨噬细胞是DSS的独立预测指标。并且它们的临床意义与PD-L1表达状态相关。基于TIGIT和CD155的共表达对患者进行精准风险分层，可以识别出四个完全不同临床结局的亚组，其中TIGIT+CK+或TIGIT+CD8+与CD155+CD68+的空间共表达提示PDAC患者的最差的临床结局。TIGIT+CK+、TIGIT+CD8+ 、CD155+CD68+和CD155-CD68+ PDAC患者接受ACT比未接受ACT生存明显获益，而TIGIT-CK+和TIGIT-CD8+ PDAC患者无论是否接受ACT无明显生存差异。在PDAC患者接受ACT的前提下，（TIGIT+CK+或TIGIT+CD8+且CD155+CD68+）联合表型的病例表现出了最差的预后状况，TIGIT和CD155的空间共表达有可能增加了PDAC患者对ACT的治疗抵抗。

研究结论：我们的研究结果表明TIGIT和CD155在PDAC的TCs和ICs上表达均较为广泛，且我们的结果支持TIGIT和其配体为负性免疫调控分子，或许其可以成为PDAC的一个潜在治疗靶点。TIGIT和CD155阳性表达是独立预后预测指标，且根据TIGIT和CD155共表达对患者进行更精确的危险度分层，从而更精准地为PDAC患者制定随访策略；其次，发现了TIGIT/CD155的表达与术后辅助化疗是否获益之间的关联，TIGIT/CD155空间共表达可以作为辅助化疗决策的潜在预测指标，从而辅助临床医生筛选出对术后化疗明显获益的患者；还揭示了TIGIT和CD155在PDAC中的表达模式以及其临床意义均和PD-L1相关，表明单独抗TIGIT/CD155治疗和或联合PD-1免疫检查点抑制治疗在PDAC中具有潜在的应用价值，为PDAC患者的临床前和临床试验提供了潜在的治疗策略。

关键词：胰腺导管腺癌、TIGIT、CD155、PD-L1

研究内容Ⅱ 联合可替代的免疫检查点分子探究新型免疫检查点OX40及其配体OX40L在胰腺导管腺癌中的表达和意义

研究背景：阻断或激活免疫检查点调控轴在许多恶性肿瘤中是一种重要的治疗手段。包括针对OX40/OX40L共刺激信号的新型免疫检查点的靶向治疗正在开展多个相关临床试验。然而，关于OX40和OX40L在PDAC中的作用仍知之甚少。因此，我们在PDAC中检测了OX40和OX40L的表达模式，以及它们与替代免疫检查点、免疫浸润、临床病理特征和临床结局的关联。

研究方法：我们在255名PDAC患者的组织样本中对OX40、OX40L、CD8和CD68等多个免疫指标进行多重免疫荧光染色。我们分析了PD-L1、B7-H3、B7-H4、CD3和Foxp3的免疫组化数据。同时，还评估了癌症基因组图谱（The Cancer Genome Atlas， TCGA）和国际癌症基因组联盟（International Cancer Genome Consortium，ICGC）数据库中OX40/OX40L的RNA测序数据。

研究结果：肿瘤细胞（tumor cell, TCs）和免疫细胞（immune cell, ICs）上OX40的阳性率分别为8.6%和10.2%，而TCs、ICs和巨噬细胞上OX40L的阳性率分别为20%、40.4%和12.9%。OX40的阳性表达与良好的临床病理特征表型相关联。OX40+ICs、OX40L+TCs或巨噬细胞，而不是OX40和OX40L的mRNA和总蛋白水平，与更好的预后相关。且ICs上的OX40和巨噬细胞上的OX40L的阳性表达是独立的预后预测因素。此外，通过结合ICs上的OX40和TCs上的OX40L共表达，可以更准确地对PDAC患者进行危险度分层，具有（OX40+ ICs且OX40L+ TCs）表型患者表现出最长的生存时间。在PD-L1阴性的PDAC中，OX40+ICs、OX40L+TCs或OX40L+巨噬细胞与良好预后相关；然而OX40+ICs、OX40L+TCs或OX40L+巨噬细胞在PD-L1阴性的肿瘤中均无预后提示意义。此外，OX40+ICs与B7-H4在肿瘤细胞上的阴性表达、更高比例的CD3+ T细胞和Foxp3+调节性T细胞相关；OX40+TCs和OX40L+TCs分别与更低密度的Foxp3+ T细胞浸润相关。

研究结论：我们首次确定了OX40和OX40L可以作为PDAC患者的预后生物标志物，OX40和OX40L的共表达为PDAC患者进行了更精准的预后分层，从而改善PDAC患者术后随访策略。我们的结果支持OX40及其配体OX40L在PDAC中为正向免疫调控分子，且与包括PD-L1在内的多个B7家族的负性免疫检查点分子的表达存在相关性，说明了新型免疫检查点OX40与PD-L1等B7家族分子在PDAC中可能发挥拮抗作用，同时提示了单独靶向OX40的免疫疗法和或联合抗PD-1治疗的策略在PDAC中的应用潜能。

关键词：胰腺导管腺癌、OX40、OX40L、PD-L1

研究内容 Ⅲ 新型免疫检查点Siglec-15在胰腺导管腺癌中表达和意义

研究背景：Siglec-15被认为是具有极大应用前景的二代癌症免疫治疗的新免疫检查点分子。然而，在此之前，Siglec-15在PDAC中的表达和意义完全未知。本研究首次旨在探索Siglec-15在PDAC中的表达模式及其临床意义和潜在价值。

研究方法：本研究共纳入291例PDAC患者并对这些患者的经福尔马林固定石蜡包埋的肿瘤组织样本进行Siglec-15、PD-L1等多个免疫指标及多个DNA损伤修复（DNA damage repair，DDR）分子指标进行免疫组织化学染色和多重免疫荧光染色。综合考虑各个指标的表达特征，从而选取最适的定量和定性方法进行评估，分析Siglec-15在不同类型细胞上的表达与临床病理参数、预后、PD-L1的表达、免疫细胞浸润和DDR分子之间的关联。

研究结果：在本研究中我们观察到18.6%和30.3%的PDAC患者的肿瘤细胞阳性表达Siglec-15和PD-L1，还在3.4%的患者中检测到了巨噬细胞中的Siglec-15阳性表达。在6.1%的患者中检测到Siglec-15与PD-L1的共表达。其中共有33个PD-L1阴性样本（18.0%）表达了Siglec-15；64个Siglec-15阴性样本（29.9%）有PD-L1阳性表达。Siglec-15在肿瘤细胞上的阳性表达更常见于中-高分化肿瘤。Siglec-15与更低密度FOXP3+Tregs和CD45RO 记忆性T细胞的浸润、BRCA1高表达以及更好的预后相关。另外，Siglec-15和PD-L1在肿瘤细胞上的阳性表达都是患者预后的独立因素。我们还揭示了Siglec-15与淋巴结状态和BRCA1/2表达在预后上存在交互作用。在淋巴结阴性、BRCA1高表达或BRCA2低表达的肿瘤中，Siglec-15均是良好预后的独立预测因素；而在淋巴结阳性、BRCA1低表达或BRCA2高表达的肿瘤中，没有出现类似结果。Siglec-15对于在淋巴结阴性、BRCA1表达高或BRCA2表达低的肿瘤中的预后意义更具鉴别性和区分度。Siglec-15的预后意义受到淋巴结状态和BRCA1/2表达的效应修饰。

研究结论：肿瘤细胞Siglec-15阳性表达在PDAC患者中是独立的预后提示因子。Siglec-15的表达与PD-L1在肿瘤细胞上的阳性表达有一定的互补性，说明两者在PDAC中或许有着协同作用，靶向Siglec-15对抗PD-1治疗无效的患者来说可能是一种新型治疗选择。肿瘤细胞Siglec-15阳性表达更常见于BRCA1高表达的PDAC中，Siglec-15的预后意义与BRCA1/2相关，表明联合免疫治疗与PARPi治疗可能为同时具有BRCA缺陷和Siglec-15阳性表达的PDAC患者的临床前和临床试验提供一个潜在的治疗方案。

关键词：胰腺导管腺癌、Siglec-15、PD-L1、预后

第二部分 免疫结构模式在胰腺导管腺癌中的意义研究

研究内容Ⅳ B细胞亚群及其相关的三级淋巴结构的空间分布计算在胰腺导管腺癌中预后价值及术后辅助治疗反应性预测的研究

研究背景：B细胞在某些特定类型的肿瘤中显示出抗肿瘤免疫和促进免疫疗法响应的作用，但它们在PDAC中的精确免疫表型及其意义仍然很少被描述，尤其是当B细胞联合其他免疫细胞形成三级淋巴结结构（tertiary lymphoid structures，TLS）时。因此，本研究将探索不同B细胞分化亚群及其相关三级淋巴结构在PDAC免疫微环境中的空间分布和意义。

研究方法：合理使用三个定量多重免疫组化panel，以单细胞分辨率原位检测282名PDAC患者组织样本中五种不同发育阶段的B亚群的密度、空间分布和与肿瘤细胞之间的空间组织结构及其相关TLS的位置及细胞组成。通过空间邻近性分析测量B细胞亚群与PDAC细胞之间的距离。使用多因素Cox回归确定它们与患者预后和辅助化疗（adjuvant chemotherapy，ACT）益处的关系。且上述所有分析均根据PD-L1表达进行分层。

研究结果：五种不同发育阶段B细胞亚群在TLS内外的分布高度异质，并且与PD-L1表达状态相关。我们还表征了TLS的丰度、空间位置和细胞成分。高比例的IgM+记忆B细胞（IgM+ memory B cells，IgM+ Bm）以及TLS阳性而不是数量，是独立预后预测因素。B细胞亚群和TLSs在PD-L1阳性和PD-L1阴性组预后意义也是有差异的。在PD-L1 CPS<1的PDAC中，肿瘤周围的TLSs（peritumoral-TLSs，P-TLSs）或肿瘤内-TLSs（intratumoral-TLSs，In-TLSs）阳性以及高密度的CD20+ B、CD27+异型转换记忆B细胞（CD27+ isotype-switched memory B cells，CD27+ SwBm）、 CD27−异型转换记忆B细胞（CD27− isotype-switched memory B cells，CD27− SwBm）、初始B细胞（naive B cells，Bn）和IgM+ Bm与预后相关；而在PD-L1 CPS≥1的PDAC中，只有In-TLSs的存在与预后相关。在PD-L1 CPS≥1组中，癌细胞周围20μm半径内的浆细胞（plasma cells，PCs）数量高于PD-L1 CPS<1组的。总体上，PD-L1 CPS≥1的患者中各个肿瘤浸润B细胞亚群与PDAC细胞间的空间距离相对于PD-L1 CPS<1的患者是要更小。除了细胞密度外，IgM+ Bm、Bn和CD20+ B与PDAC细胞的空间距离更近与患者更好的预后相关。我们还发现了P-TLSs或In-TLSs阳性的PDAC患者更有可能从ACT中获益。

研究结论：我们确定了B细胞及其相关TLS的空间分布的临床意义，且IgM+ Bm和TLSs是独立预后预测因子，我们的结果可能支持B细胞和TLSs成为改善临床管理潜在的新指标；不同发育阶段的B细胞与肿瘤细胞间的空间距离与预后有关联，且与PD-L1相关，这可能为PDAC肿瘤免疫生物学提供新的见解和认识。还发现了TLSs阳性与术后辅助化疗益处的关联，TLSs可能作为辅助化疗决策的潜在预测指标，从而为PDAC患者的选取制定更有效更精准的个体化治疗方案。

关键词：胰腺导管腺癌、B细胞、三级淋巴结构、PD-L1

研究内容Ⅴ 中性粒细胞及其胞外捕获网在胰腺导管胰腺癌中的意义研究

研究背景：细胞外捕获网（extracellular traps，ETs）和肿瘤浸润免疫细胞在肿瘤进展中起着至关重要的作用。然而，在胰腺导管腺癌（pancreatic ductal adenocarcinoma，PDAC）中，关于肿瘤浸润性中性粒细胞和巨噬细胞以及相关ETs的分布及临床意义知之甚少。本研究调查了中性粒细胞或巨噬细胞浸润或ET形成与PDAC的临床病理特征、免疫检查点分子、临床结局和对辅助化疗（adjuvant chemotherapy，ACT）的抵抗之间的关联。

研究方法：我们对205名PDAC患者的组织样本进行多重免疫荧光染色，检测中性粒细胞、巨噬细胞及其相关ET形成，并联合使用了本队列的PD-L1、PD-L2、B7-H3和B7-H4的免疫组化数据。

研究结果：巨噬细胞和中性粒细胞中的ET阳性率分别为23.9%和45.4%。高密度的中性粒细胞和中性粒细胞ET阳性表达的患者表现出较短的无进展生存（progression-free survival，PFS）和疾病特异性生存（disease-specific survival，DSS），而巨噬细胞及其ETs与PFS和DSS均无关。通过多因素Cox回归分析，确定了中性粒细胞浸润和其ET形成是独立预后预测因子。中性粒细胞浸润比例低或中性粒细胞ET阴性染色的PDAC患者接受ACT生存时间可以显著延长。根据中性粒细胞的浸润和中性粒细胞ETs的共存情况，识别出四个不同临床结局的PDAC亚组，更精准地对PDAC患者进行风险分层，其中中性粒细胞浸润比例较低且中性粒细胞ET阴性染色的患者表现出最佳的生存状况。在PD-L1 TPS <1%或IC<1%亚组中，中性粒细胞ET阳性表达提示不良预后。然而，在PD-L1 TPS≥1%或IC≥1%亚组中，中性粒细胞ET的阳性表达与预后无关。

研究结论：中性粒细胞浸润和及其相关ETs是PDAC疾病进展和生存的独立预测标志物，且基于中性粒细胞的浸润和其相关ETs的共存，能更精准地对PDAC患者进行风险分层，为患者随访提供帮助。中性粒细胞及其ETs可以用于预测PDAC术后ACT的效果，可以帮助指导临床医生制定更有效的个体化PDAC治疗方案。中性粒细胞ETs阳性表达及其意义与PD-L1的表达存在一定关联，说明它们在PDAC中可能发挥互斥或协同作用，联合PD-1阻断和DNase I抑制中性粒细胞ET形成可能为PDAC的临床前和临床试验治疗提供一种潜在的治疗策略。

关键词：胰腺导管腺癌、中性粒细胞、中性粒细胞胞外捕获网、PD-L1

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive type of pancreatic malignancy and the most common histological type among pancreatic tumors, accounting for over 90% of pancreatic cancer cases. The prognosis for PDAC patients is extremely poor, with a five-year survival rate of about 11%, and the majority of patients are diagnosed at an advanced local stage or with metastasis. Moreover, in recent years, there has been an increasing trend in the mortality rate of PDAC patients. The current treatments for PDAC primarily include surgery and adjuvant chemotherapy, but these treatments have limited effectiveness and do not significantly improve prognosis. Therefore, exploring new and effective treatment plans and strategies is urgent. The development of immune checkpoint inhibitor therapy targeting the programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) axis is a significant milestone in cancer immunotherapy research. However, clinical trials of anti-PD-1 treatment in PDAC have shown that the response rate to immune checkpoint inhibitors is very low. Given these realities, identifying new immune checkpoint pathways and fully understanding the immune architecture of PDAC might reveal new potential immunotherapeutic targets and improve the response rate and efficacy of immunotherapy. The potential clinical application value of the expression of multiple novel immune checkpoints and the immune architecture pattern in PDAC remains unclear; thus, this study aims to investigate the expression of multiple new immune checkpoints in PDAC, characterize and delineate the distribution features of the immune architecture pattern, and analyze its association with traditional immune molecular indicators such as PD-L1, clinicopathological characteristics, prognosis, and the response to postoperative adjuvant chemotherapy. The main content of this thesis is divided into the following two parts:

Part Ⅰ The Expression and Clinical Significance of Novel Immune Checkpoint in Pancreatic Ductal Adenocarcinoma

Project Ⅰ The spatial coexistence of TIGIT/CD155 defines poorer survival and resistance to adjuvant chemotherapy in pancreatic ductal adenocarcinoma

Background: Targeting emerging T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT)/CD155 axis shows promise for restoring anti-tumor immunity, but its immune phenotypes and prognostic significance in a large cohort of pancreatic ductal adenocarcinoma (PDAC) are limited.

Methods: Three seven-color multispectral panels were rationally designed to investigate the protein expression, immune-microenvironmental feature, prognostic value, and the response to adjuvant chemotherapy of TIGIT/CD155 in 272 PDAC specimens using multiplex immunohistochemistry.

Results: We revealed low immunogenicity and high heterogeneity of the PDAC immune microenvironment featured by abundant CD3+ T cells and CD68+ macrophages and low infiltration of activated cytotoxic T lymphocytes. TIGIT and CD155 were highly expressed in PDAC tissues compared to paracancerous tissues. Tumor-infiltrating lymphocytes expressing TIGIT were correlated with high densities of CD45RO+ T cells; TIGTI+CD8+ T cells were associated with high infiltration of CD3+CD45RO+FOXP3+. CD155+CK+ were significantly related to high densities of CD3+ and CD3+CD8+CD45RO+ T cells. High positive rates for TIGIT in TCs, CD8+ T cells, and CD155 in macrophages were correlated with poor progression-free and disease-specific survival, respectively, and their clinical significance was correlated with PD-L1 status. Notably, spatial co-existence of TIGIT+CK+ or TIGIT+CD8+ and CD155+CD68+ indicated poor survival and resistance to adjuvant chemotherapy response in patients with PDAC.

Conclusion: Our findings suggest that targeting TIGIT/CD155 immunosuppressive axis may guide patient stratification and improve the clinical outcome of PDAC.

Keywords: Pancreatic ductal adenocarcinoma; TIGIT/CD155; PD-L1

Project Ⅱ Analysis of the OX40/OX40L immunoregulatory axis combined with alternative immunecheckpoint molecules in pancreatic ductal adenocarcinoma

Background: Immune checkpoint modulation has been a vital therapeutic option in many malignancies, and targeting of novel immune checkpoints, including OX40/OX40L costimulatory signaling, is being assessed in clinical trials. However, little is known about the role of OX40 and OX40L in pancreatic ductal adenocarcinoma (PDAC). Thus, we investigated the clinical significance of OX-40 and OX40L and their associations with alternative immune checkpoints, immune infiltrates, clinicopathological features, and clinical outcomes.

Methods: We performed multiplexed immunofluorescence staining for OX40, OX40L, CD8, and CD68 using tissue microarrays from 255 patients. Immunohistochemistry data for PD-L1, B7-H3, B7-H4, CD3, and Foxp3 were analyzed. And the RNA sequencing data of OX40/OX40L in The Cancer Genome Atlas and International Cancer Genome Consortium databases were also evaluated.

Results: The positive rates for OX40 on tumor cells (TCs) and immune cells (ICs) were 8.6% and 10.2%, respectively, and the positive rates for OX40L on TCs, ICs, and macrophages were 20%, 40.4%, and 12.9%, respectively. OX40 was associated with favorable clinicopathological features. OX40+ on ICs, OX40L+ on TCs, or OX40L+ on macrophages, rather than the total gene and protein levels of OX40/OX40L, were associated with improved survival. OX40+ on ICs and OX40L+ on macrophages were independent factors of clinical outcomes. Moreover, we could more accurately stratify patients through the combination of OX40 on ICs and OX40L on TCs, and patients with OX40+ ICs and OX40L+CK+ showed the best outcome. And we demonstrated that patients with OX40-ICs and low CD8+ T cells infiltration had unfavorable survival. Intriguingly, OX40+ ICs or OX40L+ macrophages demonstrated superior survival in patients with PD-L1 negativity than in those with PD-L1 positivity. Furthermore, OX40+ ICs were correlated with negative B7-H4 on TCs, high densities of CD3 T cells, and high densities of Foxp3 T cells; OX40+ TCs and OX40L+ TCs were associated with low densities of Foxp3 T cells.

Conclusions: We identified OX40 and OX40L as promising predictors for prognosis in PDAC.The expression of OX40/OX40L correlates with the expression of multiple B7 family immune checkpoint molecules, including PD-L1, suggesting that  OX40 and  PD-L1 may have antagonistic roles in PDAC. It also indicates the potential for developing and applying immunotherapy strategies that target OX40 alone or in combination with anti-PD-1 therapy in PDAC.

Keywords: OX40; OX40L; PD-L1; PDAC

Project Ⅲ Analysis of a novel immune checkpoint, Siglec-15, in pancreatic ductal adenocarcinoma

Background: Siglec-15, a novel immune checkpoint, is an emerging target for next-generation cancer immunotherapy. However, the role of Siglec-15 in pancreatic ductal adenocarcinoma (PDAC) remains poorly understood. We investigated the expression of Siglec-15 and its association with clinicopathological characteristics, programmed cell death-ligand 1 (PD-L1), immune cells, and DNA damage repair (DDR) molecules in PDAC.

Methods: 291 PDAC patients who underwent surgical treatment at our hospital were included. Immunohistochemical staining and multiplex immunofluorescence staining were performed on their formalin-fixed, paraffin-embedded tumor tissue samples to assess various immune markers such as Siglec-15 and PD-L1, as well as multiple DNA damage repair (DDR) molecular markers

Results: Positive tumoral expression of Siglec-15 and PD-L1 was observed in 18.6% and 30.3% of the samples, respectively. We also detected Siglec-15 positivity in macrophages in 3.4% of patients. Co-expression of Siglec-15 with PD-L1 was observed in 6.1% of the patients. A total of 33 PD-L1-negative samples (18.0%) were Siglec-15-positive. Siglec-15 was observed more frequently in moderate-to-well-differentiated tumours. Siglec-15 was associated with a low density of Tregs and CD45RO T cells, high BRCA1 expression, and improved survival. Both Siglec-15 and PD-L1 are independent factors of patient outcomes. The prognostic significance of Siglec-15 for survival was more discriminative in lymph node-negative, high BRCA1 expression, or low BRCA2 expression tumours than in lymph node-positive, low BRCA1 expression, or high BRCA2 expression tumours, respectively.

Conclusions: We identified Siglec-15 as a promising predictor for prognosis combined with different DDR molecular statuses and complex tumour-infiltrating cells in PDAC. Targeting Siglec-15 may be a novel therapeutic option for patients who are unresponsive to anti-PD-1 therapy. Future studies are needed to validate the prognostic significance of Siglec-15 and to investigate its regulatory mechanisms in this disease.

Key words: pancreatic ductal adenocarcinoma; Siglec-15; PD-L1; prognosis

Part Ⅱ The Role of Immunoarchitectural Patterns in Pancreatic Ductal Adenocarcinoma

Project Ⅳ Spatial computation of B cell subsets and B-cell lymphoid aggregates associates with survival and response to adjuvant chemotherapy of patients with pancreatic ductal adenocarcinoma

Background: Although B cells show promise for restoring anti-tumor immunity and promote immunotherapy response, their precise role of immune phenotypes and prognostic significance in pancreatic ductal adenocarcinoma (PDAC) remains poorly characterized with regards to their composition, location, density, and spatial organization, particularly when assembled into tertiary lymphoid structures (TLSs).

Methods: Three quantitative multiplex immunohistochemistry panels were used in situ at single-cell resolution to decipher the density and spatial distribution of five distinct B subpopulations with different developmental stage and their related TLSs in tissue samples obtained from 282 PDAC patients. The distances between B cell subpopulations and PDAC cells were measured by spatial proximity analyses. Immune microenvironmental characteristics, patient outcomes, and response to adjuvant chemotherapy (ACT) stratified by PD-L1 expression were analyzed using multivariate Cox regression.

Results: The distribution of the five B cell subsets was highly heterogeneous inside and outside the TLSs and differed by PD-L1 status. We also characterized the abundance, presence, spatial location and cellular components of TLSs. A high number of IgM+ memory B cells and the presence, not numbers of TLSs were independently correlated with improved survival. The B cell subsets and TLSs exerted distinct clinical outcomes between the PD-L1-positive and PD-L1-negative groups. The number of plasma cells surrounding cancer cells within a 20-μm radius was higher in the PD-L1 CPS≥1 than in the PD-L1 CPS<1 group. In addition to cell density, the closer proximity of IgM+ memory B, naïve B, and CD20+ B cells to PDAC cells also strongly correlated with patient survival. PDAC patients with P-TLSs or In-TLSs are more likely to benefit from ACT.

Conclusions: The clinical significance of spatial profiling of B cells and their related TLSs were identified which may potentially provide novel insights into tumor immunobiology and facilitate the design of more effective personalized treatments for patients with PDAC.

Keywords: Pancreatic ductal adenocarcinoma; B cell; tertiary lymphoid structures; PD-L1

Project Ⅴ Intratumoral neutrophil extracellular traps are associated with unfavorable clinical outcomes and immunogenic context in pancreatic ductal adenocarcinoma

Background: Extracellular traps (ETs) and tumor-infiltrating immune cells play crucial roles in tumor progression. However, little is known about the clinical significance of tumor-infiltrating neutrophils and macrophages and the related ETs in pancreatic ductal adenocarcinoma (PDAC). This study investigates the associations between neutrophil or macrophage infiltration or ET formation and the clinicopathological features, molecular characteristics, immune checkpoint molecules, clinical outcomes, and response to adjuvant chemotherapy (ACT) in PDAC.

Methods: We performed multiplex immunofluorescence staining to detect ET formation by neutrophils or macrophages using tissue microarrays obtained from 205 patients, and analyzed the immunohistochemistry data for PD-L1, PD-L2, B7-H3, and B7-H4.

Results: The ET expression rates in macrophages and neutrophils were 23.9% and 45.4%, respectively. Patients with a high density of neutrophils or positive expression of neutrophil ETs exhibited poorer progression-free survival (PFS) and disease-specific survival (DSS), whereas macrophage ETs were not related to PFS and DSS. Neutrophil infiltration and ET formation were identified as independent prognostic predictors of DSS using univariate and multivariate Cox analyses. Patients with PDAC with lower neutrophil infiltration or negative staining for neutrophil ETs are more likely to benefit from ACT. Patients with PDAC were more accurately stratified based on the infiltration of neutrophils and presence of neutrophil ETs, and patients with low neutrophil infiltration or negative staining for neutrophil ETs showed the best survival. Patients with positive neutrophil ETs demonstrated inferior DSS compared to those with negative neutrophil ETs in the PD-L1 tumor proportion score (TPS) < 1% and PD-L1 IC < 1% subgroups. However, the positive expression of neutrophil ETs was not related to DSS in the PD-L1 TPS ≥ 1% or PD-L1 IC ≥ 1% subgroup.

Conclusions: Our findings emphasize the potential of neutrophil infiltration and ETs as prognostic markers that could guide the formulation of more effective personalized treatments for PDAC.

Key Words: Pancreatic ductal adenocarcinoma; neutrophil; neutrophil extracellular traps; PD-L1