背景与目的：体细胞突变在整个肿瘤进化过程中逐步积累。肿瘤基因组表现出特定突变模式，称为突变特征，其反映了外源性和内源性突变过程的印迹。载脂蛋白B mRNA编辑酶催化多肽（apolipoprotein B mRNA editing enzyme catalytic polypeptide, APOBEC）相关突变特征，即SBS2和SBS13，在至少22种类型的肿瘤中存在。据报道SBS2和SBS13在食管鳞癌中几乎普遍存在，这表明APOBEC突变模式在食管鳞癌中是一个必不可少的强制性过程。此外，SBS2和SBS13存在于大多数食管鳞癌样本中，占总突变负荷的25%，揭示了APOBEC突变模式是食管鳞癌进化过程中的关键一步。APOBEC突变模式以TCW序列中C>T或C>G的改变（其中W指A或T）为特点。APOBEC3亚家族成员可催化胞嘧啶脱氨基为尿嘧啶，对于APOBEC突变模式的形成具有重要作用，但是其中哪一个成员在食管鳞癌中作为APOBEC突变模式的主要来源目前仍然是未知的。免疫治疗已在食管鳞癌中取得较大进展，已成为局部晚期或转移性患者的一线治疗方案。肿瘤免疫微环境的特点是患者免疫治疗疗效分层的先决条件。APOBEC突变模式的生物学功能在不同类型肿瘤中差异较大，但它在食管鳞癌中对肿瘤免疫原性和免疫治疗应答中的作用及其机制尚未得到充分了解。

方法：在本研究中，我们从三个已发表的队列中收集了169例食管鳞癌患者的配对的多组学数据，包括体细胞突变数据、bulk转录组数据和单细胞转录组数据。为了剖析APOBEC突变模式对肿瘤免疫浸润的影响，我们使用体细胞突变数据进行突变富集分析，利用ESTIMATE和TMEscore算法对bulk RNA测序（RNA sequencing, RNA-seq）数据进行肿瘤微环境反卷积计算，并使用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）数据和多重免疫荧光进行验证。更重要的是，我们通过T>C和T>G的突变在突变碱基上下游20bp的碱基序列以及APOBEC3亚家族各个成员的表达情况确定了食管鳞癌中介导APOBEC突变模式的主要突变来源，阐明了食管鳞癌中APOBEC突变模式激活免疫的潜在分子机制。

结果：我们发现APOBEC突变模式的富集可使食管鳞癌患者的总生存时间（overall survival, OS）延长，造成这种结果的原因可能是由于较高的抗肿瘤免疫细胞浸润，免疫检查点表达和抗肿瘤免疫相关通路的富集，包括干扰素（interferon, IFN）信号通路，固有免疫系统和适应性免疫系统的活化。A3A的活性升高对APOBEC突变模式至关重要。从机制上讲，A3A的表达上调会加剧胞质中双链DNA（double-stranded DNA, dsDNA）的积累，从而激活cGAS-STING通路。同时，使用肿瘤免疫功能异常和免疫逃避（Tumor Immune Dysfunction and Exclusion, TIDE)算法预测A3A的表达水平与免疫治疗应答之间的关系，发现A3A高表达的患者免疫治疗效果好。另外我们在一个临床队列中验证了这个结论。这些结果系统阐明了APOBEC突变模式在食管鳞癌中的临床相关性、免疫学特征、免疫治疗的预后价值以及潜在机制，在临床应用上具有促进临床决策的巨大潜力。

结论：总之，我们使用全基因组/全外显子组数据（whole genome/exome sequencing, WGS/WES），bulk转录组和单细胞转录组数据以及功能实验阐明了APOBEC突变模式是否激活免疫及其潜在的具体机制。同时，我们揭示了A3A作为一种新的生物标志物，用于预测食管鳞癌免疫治疗效果。

Background and objective: Somatic mutations accumulate throughout the process of tumor evolution. The cancer genome exhibits certain patterns of mutation, known as mutational signatures, reflecting the footprints of exogenous and endogenous mutational processes. The apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC)-related signatures, SBS2 and SBS13, are detected in at least 22 different cancer types and reported to be near-universally exhibited in esophageal squamous cell carcinoma (ESCC), indicating that APOBEC mutagenesis is an essential and mandatory process due to its prevalence in ESCC. Moreover, it is reported that SBS2 and SBS13 present in majority of ESCC samples and account for about 25% of the mutation burden, revealing that APOBEC mutagenesis is a crucial step during the evolutionary history in ESCC. The APOBEC signatures are characterized by C-to-T or C-to-G changes at TCW motifs (where W refers to A or T). APOBEC3 subfamily members, which catalyze the deamination of cytosine to uracil, are responsible for the vital mutational process but which one predominantly contribute to APOBEC mutagenesis remains unknown in ESCC. Immunotherapy has become a promising strategy for ESCC, and the characteristics of the tumor immune microenvironment are prerequisites for patient stratification. The biological functions of APOBEC mutagenesis are diverse in different types of cancer, but its functional roles and mechanisms in tumor immunogenicity and immunotherapy response have not been fully understood in ESCC.  

Methods: In the present study, we collect matched multi-omics data of 169 patients with ESCC from three previously published cohorts. To dissect the effects of APOBEC mutagenesis on tumor immune infiltration, we perform mutation enrichment analysis based on the somatic mutation data and employ CIBERSORT, ESTIMATE and TMEscore to conduct tumor microenvironment deconvolution using bulk RNA sequencing (RNA-seq) data, which is verified using single-cell RNA sequencing (scRNA-seq) data and multiplex immunofluorescence. What’s more, we identify which member of APOBEC3 subfamily is the dominant mutator through the 20 bp upstream and downstream base sequence of the mutated base and the expression of the APOBEC3 subfamily members, and illustrate the underlying molecular mechanisms of immune activation for APOBEC mutagenesis in ESCC by conducting a set of functional assays.

Results: We find that APOBEC mutagenesis prolongs overall survival (OS) of ESCC patients. The reason for this outcome is probably due to high anti-tumor immune infiltration, immune checkpoints expression and immune related pathway enrichment, such as interferon (IFN) signaling, innate and adaptive immune system. The elevated AOBEC3A (A3A) activity paramountly contributes to the footprints of APOBEC mutagenesis. Mechanistically, upregulated A3A exacerbates cytosolic double-stranded DNA (dsDNA) accumulation, thus stimulating cGAS-STING pathway. Simultaneously, A3A is associated with immunotherapy response which is predicted by Tumor Immune Dysfunction and Exclusion (TIDE) algorithm and validated in a clinical cohort. These findings systematically elucidate the clinical relevance, immunological characteristics, prognostic value for immunotherapy and underlying mechanisms of APOBEC mutagenesis in ESCC, which demonstrate great potential in clinical utility to facilitate clinical decisions.

Conclusion: In conclusion, we illustrated whether and how APOBEC mutagenesis activate immune response using whole genome/exome sequencing (WGS/WES), bulk RNA-seq, scRNA-seq data and functional assays. These findings uncovered a new biomarker to accelerate the clinical application and improve immunotherapy effects in ESCC.