PO.CL01.13 · 临床研究
利用空间蛋白质组学评估免疫聚集体作为阑尾腺癌（AA）免疫治疗反应潜在生物标志物的作用
Assessing the role of immune aggregates as potential biomarkers of immunotherapy response in appendiceal adenocarcinoma (AA) using spatial proteomics
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
阑尾腺癌是一种罕见肿瘤，占所有胃肠道恶性肿瘤的比例不到1%。不幸的是，超过50%的AA患者就诊时已为IV期疾病，许多患者无法手术切除，且系统性治疗选择有限。近期一项临床试验的数据提示，阿替利珠单抗（atezolizumab）和贝伐珠单抗（bevacizumab）联合治疗可改善不可切除的转移性AA患者的总生存期（NCT03074513）。我们此前使用空间转录组学研究了这些患者中一个亚组的总体免疫效应，提示三级淋巴结构（TLS）等免疫聚集体可能是AA中重要的反应生物标志物。在此，我们旨在使用单细胞空间蛋白质组学来刻画免疫聚集体的格局，以进一步理解AA中免疫治疗反应的潜在生物标志物。使用nanoString的CosMx空间分子成像仪（SMI）搭配64重人类IO蛋白组合，对8名试验参与者的治疗前FFPE活检样本进行了单细胞空间蛋白质组学分析。数据处理使用nanoString的AtoMx平台完成，并导出至R包Seurat进行分析。细胞类型注释使用CELESTA获得。免疫聚集体使用CosMx染色图像和Napari手动识别并标记。所分析的试验队列（n=8）按临床反应分组，即以患者在试验随访时存活（n=4）或死亡（n=4）来衡量。使用来自AtoMx和Napari的CD45染色图像，在所有患者中手动标注了瘤周免疫聚集体，并为细胞添加标签。总体而言，我们在所分析的8例患者样本中的7例中检测到75个独特的免疫聚集体。在组织层面，我们发现死亡患者相比存活患者具有更高的Treg密度（p=0.085，t检验）。在免疫聚集体内的细胞中，存活患者的聚集体中B细胞（p=0.14）和成纤维细胞（p=0.16）密度呈现更高的趋势。在这些结构内进行的邻域分析揭示了9个细胞邻域，代表了三级淋巴结构的诸多组分。根据细胞邻域组成对免疫聚集体进行的无监督聚类，将含有较多生发中心B细胞的聚集体与含有较多CD4+和CD8+ T细胞的其他聚集体区分开来。鉴于AA手术标本在组织采集和测序方面存在挑战，空间蛋白质组学为研究此类组织中的肿瘤微环境和治疗反应提供了有意义的框架。AA肿瘤中成熟免疫聚集体的存在提示其在驱动免疫治疗反应中可能发挥作用，进一步刻画这些聚集体以最佳预测未来AA患者对免疫治疗反应的工作仍在进行中。
查看英文原文 English abstract
Appendiceal adenocarcinoma is a rare tumor representing less than 1% of all gastrointestinal malignancies. Unfortunately, over 50% of AA patients present with stage IV disease with many patients being unresectable and with few systemic therapy options. Data from a recent clinical trial suggests improved overall survival on atezolizumab and bevacizumab combination therapy for patients with unresectable metastatic AA (NCT03074513). We previously studied general immune effects in a subset of these patients using spatial transcriptomics, suggesting immune aggregates like tertiary lymphoid structures (TLS) could be important biomarkers of response in AA. Here, we aim to characterize the landscape of immune aggregates using single-cell spatial proteomics to further understand potential biomarkers of immunotherapy response in AA. Single-cell spatial proteomics was performed on pre-treatment FFPE biopsies from eight trial participants using nanoString's CosMx Spatial Molecular Imager (SMI) with the 64-plex human IO protein panel. Data processing was done using nanoString's AtoMx platform and exported to the R package Seurat for analysis. Cell type annotations were obtained using CELESTA. Immune aggregates were manually identified and labeled using CosMx staining images and Napari. The profiled trial cohort (n=8) was grouped by clinical response, measured as whether a patient was alive (n=4) or deceased (n=4) at trial follow-up. Peritumoral immune aggregates were manually annotated across all patients using the CD45 staining images from AtoMx and Napari to add labels to cells. Overall, we detected 75 unique immune aggregates across 7 of the 8 patient samples profiled. At the tissue level, we identified higher Treg density (p=0.085, t-test) in deceased patients compared to living patients. Among cells in immune aggregates, there was a trend towards higher B cell (p=0.14) and fibroblast (p=0.16) density in aggregates from living patients. Neighborhood analysis performed within these structures uncovered 9 cellular neighborhoods, representing many components of tertiary lymphoid structures. Unsupervised clustering of immune aggregates according to cellular neighborhood composition resulted in the separation of aggregates with more germinal center B cells from others with more CD4+ and CD8+ T cells. With challenges in tissue collection and sequencing of AA surgical specimens, spatial proteomics provides a meaningful framework for studying the tumor microenvironment and treatment response in such tissues. The presence of mature immune aggregates in AA tumors suggests a potential role in driving response to immunotherapy, and work remains ongoing to further characterize these aggregates to best predict response to immunotherapy in future AA patients.
利益披露 Disclosure
M. Lastrapes, None..
V. Orellana, None.