The use of conventional methods (immunohistochemistry, pentaplex PCR) for detecting microsatellite instability (MSI), a predictive biomarker of immunotherapy efficacy, is debated for cancers with low MSI prevalence, such as breast cancer (BC). We developed two multiplex drop-off droplet digital PCR (ddPCR) assays targeting four microsatellites, initially identified from public BC whole-genome sequencing dataset. Performances of the assays were investigated and 352 tumor DNA and 28 circulating cell-free DNA from BC patients, with unknown MSI status were blindly screened. Cross-validation of ddPCR MSI status with other MSI detection methods was performed. We then monitored circulating tumor DNA (ctDNA) dynamics before and during pembrolizumab immunotherapy in one patient with MSI-high (MSI-H) metastatic BC. The assays showed high analytical specificity and sensitivity (limit of detection = 0.16%). Among N = 380 samples, seven (1.8%) were found as MSI-H by ddPCR with six of them confirmed by next-generation sequencing (NGS). Specificity was 100% in N = 133 microsatellite stable BC submitted to NGS. In the patient with MSI-H metastatic BC, ctDNA monitoring revealed an early decrease of microsatellite mutant allelic frequencies during immunotherapy. These results demonstrated MSI detection by ddPCR, a non-invasive, fast and cost-effective approach, allowing for large pre-screening of BC patients who may benefit from immunotherapy.

中文翻译：

使用常规方法（免疫组织化学、五重 PCR）检测微卫星不稳定性 (MSI) 是一种免疫治疗效果的预测性生物标志物，对于 MSI 患病率低的癌症（例如乳腺癌 (BC)）存在争议。我们开发了两种针对四个微卫星的多重滴落式数字 PCR (ddPCR) 检测，这些微卫星最初是从公共 BC 全基因组测序数据集中识别出来的。对检测的性能进行了研究，并对来自 BC 患者且 MSI 状态未知的 352 种肿瘤 DNA 和 28 种循环游离 DNA 进行了盲法筛选。对 ddPCR MSI 状态与其他 MSI 检测方法进行了交叉验证。然后，我们监测了一名 MSI-high (MSI-H) 转移性 BC 患者在派姆单抗免疫治疗之前和期间的循环肿瘤 DNA (ctDNA) 动态。该测定显示出高分析特异性和灵敏度（检测限 = 0.16%）。之中N  = 380 个样本，其中 7 个 (1.8%) 通过 ddPCR 发现为 MSI-H，其中 6 个通过下一代测序 (NGS) 确认。提交给 NGS 的N = 133 个微卫星稳定 BC的特异性为 100%  。在 MSI-H 转移性 BC 患者中，ctDNA 监测显示免疫治疗期间微卫星突变等位基因频率的早期降低。这些结果证明了通过 ddPCR 检测 MSI，这是一种非侵入性、快速且具有成本效益的方法，可以对可能受益于免疫疗法的 BC 患者进行大规模预筛查。