Hullar Meredith A J, Kahsai Orsalem J, Hill Courtney, Levy Lisa, Malen Rachel C, Curtis Keith R, Ammar Hamza, Sillah Arthur, Reedy Adriana M, Lampe Johanna W, Ogino Shuji, Potter John D, Newcomb Polly A, Phipps Amanda I
Division of Public Health Sciences, Fred Hutchinson Cancer Center, Seattle, Washington.
Department of Epidemiology, University of Washington, Seattle, Washington.
Cancer Epidemiol Biomarkers Prev. 2025 Aug 1;34(8):1377-1385. doi: 10.1158/1055-9965.EPI-24-1020.
Fusobacterium nucleatum (Fn) has been associated with the risk of colorectal cancer, poor colorectal cancer survival, and tumor attributes. Accurate and sensitive detection of Fn in tumor tissue is critical for evaluating their role in colorectal cancer.
We developed a droplet digital PCR (ddPCR) assay for detecting Fn using the transcription termination/antitermination gene (nusG) normalized for host tissue (solute carrier organic anion transporter family member 2A1). We assayed Fn(nusG) in matched tumor and normal tissues for 613 participants in the Seattle site of the Colon Cancer Family Registry. We used logistic regression to determine the odds of Fn enrichment in tumor tissue according to the tumor site and stage, adjusting for age, sex, and body mass index.
The limit of quantitation for Fn(nusG) was 4.1 copies/10 ng host tissue. Detection of Fn was quenched and poor at low levels in formalin-fixed, paraffin-embedded tissues using qPCR. There was a low agreement between qPCR and ddPCR (Cohen's kappa = 0.46). Fn(nusG) was detected in tumor (21%) and normal (10%) tissues and was enriched in 19% of tumors. Individuals with tumors enriched in Fn were more likely to be female (59% vs. 48%, respectively; P = 0.04) with proximal colon tumors (57% vs. 43%; P = 0.026). In multivariable-adjusted analyses, proximal colon tumors were significantly associated with Fn enrichment (OR vs. rectal tumors: 1.86; 95% confidence interval, 1.11-3.24).
We established a sensitive and specific method to detect Fn enrichment in human tissues.
ddPCR enhanced detection of Fn(nusG) for studies targeting tumor-associated bacteria.
具核梭杆菌（Fn）与结直肠癌风险、结直肠癌患者较差的生存率及肿瘤特征相关。准确、灵敏地检测肿瘤组织中的Fn对于评估其在结直肠癌中的作用至关重要。
我们开发了一种液滴数字PCR（ddPCR）检测法，用于检测Fn，该方法使用针对宿主组织（溶质载体有机阴离子转运体家族成员2A1）标准化的转录终止/抗终止基因（nusG）。我们对结肠癌家族登记处西雅图站点的613名参与者的配对肿瘤组织和正常组织进行了Fn（nusG）检测。我们使用逻辑回归来确定根据肿瘤部位和分期，肿瘤组织中Fn富集的比值比，并对年龄、性别和体重指数进行了调整。
Fn（nusG）的定量下限为4.1拷贝/10 ng宿主组织。使用定量PCR（qPCR）检测福尔马林固定、石蜡包埋组织中的Fn时，检测受到抑制且低水平时效果不佳。qPCR与ddPCR之间的一致性较低（科恩kappa系数=0.46）。在肿瘤组织（21%）和正常组织（10%）中均检测到Fn（nusG），且在19%的肿瘤中呈富集状态。Fn富集的肿瘤患者更可能为女性（分别为59%和48%；P = 0.04），且肿瘤位于近端结肠（57%和43%；P = 0.026）。在多变量调整分析中，近端结肠肿瘤与Fn富集显著相关（与直肠肿瘤相比的比值比：1.86；95%置信区间，1.11 - 3.24）。
我们建立了一种灵敏且特异的方法来检测人体组织中Fn的富集情况。
ddPCR增强了针对肿瘤相关细菌研究中Fn（nusG）的检测。
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