Data Sheet 1_Unraveling the indolence of papillary thyroid carcinoma: an exploratory study on B-cell subsets based on genetic predisposition and tumor immunity.zip

BackgroundActive surveillance for low-risk papillary thyroid carcinoma (PTC) is hampered by the lack of reliable biomarkers to distinguish indolent from progressive tumors. While our previous single-cell analysis identified tumor-infiltrating B cells as key determinants of indolent PTC, their clinical utility remains constrained by low abundance and peripheral undetectability. We therefore employed Mendelian randomization (MR) to investigate this causal relationship and assess the potential of peripheral B-cell profiling as a non-invasive strategy for distinguishing indolent PTC.
MethodsWe integrated MR, flow cytometry, single-cell transcriptomics, and clinical validation. A two-sample MR framework was used to assessed causal relationships between immunophenotypes and thyroid cancer risk. Findings were exploratorily investigated by flow cytometric comparison of B-cell subsets between indolent and progressive PTC patients, further characterized using single-cell RNA sequencing data, and evaluated for prognostic significance in the TCGA-THCA cohort.
ResultsMultivariable MR identified CD20+ IgD+ CD38- naïve B cells, CD27+ unswitched memory B cells and CD3 on activated CD4 regulatory T cells as independent protective factors thyroid cancer susceptibility (OR<1, P < 0.05), supporting a potential link between thyroid cancer-susceptible B-cell phenotypes and indolent tumor behavior. Flow cytometry confirmed a significantly higher proportion of peripheral naïve B-cell in indolent compared progressive PTC (70.8% vs. 60.9%, P = 0.032). scRNA-seq revealed these subsets as the predominant tumor-infiltrating B populations in indolent PTC. In the TCGA cohort, high enrichment scores for these B-cell subsets were associated with improved T stage. Furthermore, among patients ≥55 years, high naïve B-cell scores correlated with improved disease-free survival (DFS) (HR = 0.233, P < 0.001) and overall survival (OS) (HR = 0.292, P = 0.0111), while high CD27+ memory B-cell levels were associated with better DFS (HR = 0.212, P < 0.001) and OS (HR = 0.346, P = 0.0326).
ConclusionThis study provides exploratory genetic and clinical evidence supporting a causal, protective role for specific peripheral and tumor-infiltrating B-cell subsets in PTC. Naïve B cells and CD27+ unswitched memory B cells are linked to indolent tumor behavior and favorable prognosis, highlighting their potential as biomarkers for risk stratification and non-invasive monitoring in PTC management.

# 研究背景
低风险甲状腺乳头状癌（papillary thyroid carcinoma, PTC）的主动监测受限于缺乏可靠的生物标志物以区分惰性肿瘤与进展性肿瘤。尽管本团队此前的单细胞分析已证实肿瘤浸润B细胞是惰性PTC的关键决定因素，但因其丰度较低且在外周血中难以检测，其临床应用仍受到限制。为此，我们采用孟德尔随机化（Mendelian randomization, MR）方法探究这一因果关系，并评估外周血B细胞谱作为区分惰性PTC的非侵入性策略的潜力。

# 研究方法
本研究整合了孟德尔随机化、流式细胞术（flow cytometry）、单细胞转录组学（single-cell transcriptomics）及临床验证方案。采用两样本孟德尔随机化框架评估免疫表型与甲状腺癌发病风险之间的因果关系。通过流式细胞术对比惰性与进展性PTC患者的B细胞亚群以开展探索性分析，进一步借助单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）数据对其进行特征解析，并在癌症基因组图谱-甲状腺癌队列（TCGA-THCA cohort）中评估其预后价值。

# 研究结果
多变量孟德尔随机化分析显示，CD20+ IgD+ CD38-初始B细胞（naïve B cells）、CD27+未转换记忆B细胞（unswitched memory B cells）以及活化CD4+调节性T细胞表面的CD3分子，均为甲状腺癌易感性的独立保护因素（比值比<1，P<0.05），这支持了甲状腺癌易感B细胞表型与惰性肿瘤行为之间存在潜在关联的结论。流式细胞术证实，相较于进展性PTC患者，惰性PTC患者外周血中初始B细胞的占比显著更高（70.8% vs. 60.9%，P=0.032）。单细胞RNA测序结果显示，上述细胞亚群是惰性PTC中主要的肿瘤浸润B细胞群体。在TCGA队列中，这些B细胞亚群的高富集得分与更良好的T分期相关。此外，在年龄≥55岁的患者中，高初始B细胞富集得分与更好的无病生存期（disease-free survival, DFS）（风险比=0.233，P<0.001）及总生存期（overall survival, OS）（风险比=0.292，P=0.0111）相关；而高CD27+记忆B细胞水平则与更佳的DFS（风险比=0.212，P<0.001）及OS（风险比=0.346，P=0.0326）相关。

# 研究结论
本研究提供了探索性的遗传学与临床证据，支持特定外周血及肿瘤浸润B细胞亚群在PTC中发挥因果性保护作用。初始B细胞与CD27+未转换记忆B细胞与惰性肿瘤行为及良好预后相关，这凸显了其作为PTC诊疗中风险分层与非侵入性监测生物标志物的潜在价值。