Table_2_Bile is a reliable and valuable source to study cfDNA in biliary tract cancers.xlsx

ObjectiveThe aim of this study is to determine the clinical efficacy of bile-derived liquid biopsy compared with plasma and tumor tissue biopsy in patients with biliary tract carcinoma (BTC).
MethodsA total of 13 patients with BTC were enrolled in this cohort. Tumor tissue, bile, and plasma samples were obtained and analyzed using next-generation sequencing for genomic profiling.
ResultsBile and plasma samples were collected from all 13 patients, and 11 patients also had matched tumor tissues available. The cell-free DNA (cfDNA) concentration was significantly higher in the bile supernatant than in plasma (median: 1918 vs. 63.1 ng/ml, p = 0.0017). The bile supernatant and pellet had a significantly higher mean mutation allele frequency (MF) than plasma (median: 3.84% vs. 4.22% vs. 0.16%; p < 0.001). Genomic alterations were predominantly missense. Both bile supernatant and pellet had significantly more genomic alterations than plasma (average: 9.3 vs. 7.2 vs. 2.3 alterations per sample; p < 0.01). Among the top 10 most frequent genomic alterations, the consistency between bile supernatant and tumor tissue was 90.00% (18/20), that between bile pellet and tumor tissue was 85.00% (17/20), and that between the plasma and tissue was only 35.00% (7/20). MAF of both bile supernatant and pellet was positively correlated with that in tissue samples (ρ < 0.0001, spearman r = 0.777, and ρ < 0.0001, spearman r = 0.787, respectively), but no significant correlation with tissue was found in the plasma (ρ = 0.966, spearman r = 0.008). Furthermore, additional genomic alterations could be detected in bile supernatant and pellet than in tissue. Potential targets for targeted therapy were identified in bile supernatant and pellet. Regarding copy number variation (CNV) and chromosomal instability (CIN) detection, four additional CNVs from two patients were detected in the bile supernatant that was not detected in tissues (i.e., amplification of TERC, IL7R, RICTOR, and TERT). CIN was significantly higher in tumor tissue than in plasma. The CIN of the bile was also significantly higher than that of plasma. There was no significant difference in CIN between the tissue and the bile supernatant.
ConclusionThe consistency of all genomic alterations and tumor tissue-determined genomic alteration in the bile supernatant/pellet was significantly higher than in plasma. Bile supernatants/pellets are better for genetic sequencing and may also have potential clinical value to guide targeted therapy and evaluate prognosis. Bile cfDNA may be a feasible substitute for tumor tissue in the genetic testing of patients with BTC.

研究目标：本研究旨在探讨胆道癌（biliary tract carcinoma, BTC）患者采用胆汁来源液体活检与血浆、肿瘤组织活检的临床疗效差异。
方法：本队列研究共纳入13例胆道癌患者。收集肿瘤组织、胆汁及血浆样本，通过下一代测序（next-generation sequencing, NGS）进行基因组谱分析。
结果：本研究收集了全部13例患者的胆汁与血浆样本，其中11例患者同时匹配有可用的肿瘤组织样本。胆汁上清液中的游离DNA（cell-free DNA, cfDNA）浓度显著高于血浆（中位数：1918 vs 63.1 ng/ml，p=0.0017）。胆汁上清液与沉淀的平均突变等位基因频率（MF）均显著高于血浆（中位数：3.84% vs 4.22% vs 0.16%；p<0.001）。基因组变异以错义突变为主。胆汁上清液与沉淀的基因组变异数量均显著多于血浆（平均每样本分别含9.3、7.2与2.3个变异；p<0.01）。在排名前10的高频基因组变异中，胆汁上清液与肿瘤组织的一致性为90.00%（18/20），胆汁沉淀与肿瘤组织的一致性为85.00%（17/20），而血浆与肿瘤组织的一致性仅为35.00%（7/20）。胆汁上清液与沉淀的MF均与组织样本中的MF呈显著正相关（ρ<0.0001，斯皮尔曼相关系数r=0.777；ρ<0.0001，斯皮尔曼相关系数r=0.787），但血浆MF与组织样本未发现显著相关性（ρ=0.966，r=0.008）。此外，胆汁上清液与沉淀可检测到较肿瘤组织更多的基因组变异。在胆汁上清液与沉淀中可识别出靶向治疗的潜在靶点。在拷贝数变异（copy number variation, CNV）与染色体不稳定性（chromosomal instability, CIN）检测方面，2例患者的胆汁上清液中检测到4个肿瘤组织未检出的CNV（即TERC、IL7R、RICTOR与TERT的扩增）。肿瘤组织的CIN显著高于血浆，胆汁样本的CIN也显著高于血浆，而肿瘤组织与胆汁上清液的CIN无显著差异。
结论：胆汁上清液/沉淀中的所有基因组变异与肿瘤组织确定的基因组变异的一致性均显著高于血浆。胆汁上清液/沉淀更适用于基因组测序，或可在指导靶向治疗与评估预后方面具备潜在临床价值。胆道癌患者的基因检测中，胆汁cfDNA或可作为肿瘤组织的可行替代样本。