Background/Objectives: Lymph node metastases (LNM) undetected by standard hematoxylin and eosin (H&E) have been associated with unfavorable prognosis in colorectal cancer. The One-Step Nucleic Acid Amplification (OSNA) assay has demonstrated superior sensitivity in detecting LNM compared to H&E. We aimed to assess the performance of OSNA in detecting LNM, as well as its prognostic value in rectal cancer (RC) patients. Methods: Lymph nodes (LNs) of patients from 15 centers were analyzed by both H&E and OSNA. The total tumor load (TTL) was defined as the sum of cytokeratin 19 mRNA copies/µL in all LNs from a surgical specimen, using a threshold of 250 copies/μL for OSNA positivity. Cox proportional hazard regression was used to assess the effect of TTL ≥ 250 or 6000 copies/μL on cancer-specific survival (CSS) and recurrence-free survival (RFS), with Firth’s method applied to account for low event rate. Results: A total of 97 RC patients were included. Of these, 84 patients were eligible for survival analysis. The sensitivity and specificity of OSNA, compared to H&E, were 91.7% and 84.7%, respectively. TTL ≥ 6000 versus <6000 copies/μL was related to worse CSS and RFS. When dividing TTL into three groups: ≤250, 250–6000, and >6000 copies/μL, only TTL ≥ 6000 copies/μL was significantly associated with worse CSS and RFS. Conclusions: The OSNA assay is highly sensitive for detecting LNM in RC patients. A TTL of ≥6000 copies/μL could identify a subset of RC patients with worse CSS and RFS who might benefit from adjuvant treatment or intensive surveillance.
背景/目的：标准苏木精-伊红染色未能检出的淋巴结转移与结直肠癌不良预后相关。一步核酸扩增法在检测淋巴结转移方面显示出优于H&E染色的敏感性。本研究旨在评估OSNA法检测直肠癌患者淋巴结转移的效能及其预后价值。方法：收集15个医疗中心的患者淋巴结样本，分别采用H&E染色和OSNA法进行检测。总肿瘤负荷定义为手术标本所有淋巴结中细胞角蛋白19 mRNA拷贝数/μL的总和，OSNA阳性阈值设定为250拷贝/μL。采用Cox比例风险回归模型评估TTL≥250或6000拷贝/μL对癌症特异性生存期和无复发生存期的影响，并应用Firth方法校正低事件率。结果：共纳入97例直肠癌患者，其中84例符合生存分析条件。与H&E染色相比，OSNA法的敏感性和特异性分别为91.7%和84.7%。TTL≥6000拷贝/μL与较差的CSS和RFS显著相关。将TTL分为三组（≤250、250-6000、>6000拷贝/μL）时，仅TTL≥6000拷贝/μL与较差的CSS和RFS存在显著关联。结论：OSNA法对直肠癌患者淋巴结转移具有高检测敏感性。TTL≥6000拷贝/μL可识别出CSS和RFS较差的直肠癌患者亚群，该群体可能从辅助治疗或强化监测中获益。
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