探测轻微晚期的乳腺癌生物标志物仍面临挑战。通过循环游离DNA（cfDNA）分析，可以检测特定异常，选择靶向治疗，预测肿瘤预后并监测治疗效果。本研究将通过测序癌症相关基因组（MGM455-Oncotrack Ultima），包括56个治疗性基因（SNV和小INDEL），检测女性乳腺癌患者的血浆cfDNA中的特定基因异常。我们首先使用PredictSNP、iStable、Align-GVGD和ConSurf服务器确定观察到的突变的致病性。第二步，利用分子动力学（MD）确定SMAD4突变（V465M）的功能重要性。最后，利用Cytoscape插件GeneMANIA检查突变基因之间的关联关系。利用ClueGO，我们确定基因的功能富集和综合分析。通过MD模拟分析SMAD4 V465M蛋白的结构特征进一步证明了这种突变的有害性。模拟表明，原始结构更受SMAD4（V465M）突变的影响。我们的发现表明，SMAD4 V465M突变可能与乳腺癌显著相关，其他患者发现的突变（AKT1-E17K和TP53-R175H）在SMAD4转位到核酸酶的过程中具有协同作用，从而影响靶基因翻译。因此，这种基因突变组合可能会改变BC中的TGF-β信号通路。我们进一步提出，SMAD4蛋白损失可能通过抑制TGF-β信号通路促进侵袭性表型。因此，乳腺癌的SMAD4（V465M）突变可能增加其侵袭性和转移能力。

Breast cancer biomarkers that detect marginally advanced stages are still challenging. The detection of specific abnormalities, targeted therapy selection, prognosis, and monitoring of treatment effectiveness over time are all made possible by circulating free DNA (cfDNA) analysis. The proposed study will detect specific genetic abnormalities from the plasma cfDNA of a female breast cancer patient by sequencing a cancer-related gene panel (MGM455 - Oncotrack Ultima), including 56 theranostic genes (SNVs and small INDELs). Initially, we determined the pathogenicity of the observed mutations using PredictSNP, iStable, Align-GVGD, and ConSurf servers. As a next step, molecular dynamics (MD) was implemented to determine the functional significance of SMAD4 mutation (V465M). Lastly, the mutant gene relationships were examined using the Cytoscape plug-in GeneMANIA. Using ClueGO, we determined the gene's functional enrichment and integrative analysis. The structural characteristics of SMAD4 V465M protein by MD simulation analysis further demonstrated that the mutation was deleterious. The simulation showed that the native structure was more significantly altered by the SMAD4 (V465M) mutation. Our findings suggest that SMAD4 V465M mutation might be significantly associated with breast cancer, and other patient-found mutations (AKT1-E17K and TP53-R175H) are synergistically involved in the process of SMAD4 translocate to nuclease, which affects the target gene translation. Therefore, this combination of gene mutations could alter the TGF-β signaling pathway in BC. We further proposed that the SMAD4 protein loss may contribute to an aggressive phenotype by inhibiting the TGF-β signaling pathway. Thus, breast cancer's SMAD4 (V465M) mutation might increase their invasive and metastatic capabilities.Communicated by Ramaswamy H. Sarma.