摘要
目的探讨荧光PCR-毛细管电泳法(fluorescent PCR-capillary electrophoresis,PCR/CE)检测子宫内膜癌(endometrial carcinoma,EC)DNA聚合酶ε核酸外切酶域体细胞突变(somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon,POLE-exo*)的临床应用价值,并与Sanger测序法进行比较,以评估其检测优势与临床应用潜力。方法收集北京大学第三医院病理科2022年12月至2023年12月确诊的EC病例共计280例。排除了其中10例已经通过下一代测序(next-generation sequencing,NGS)确定为POLE致病性突变的EC病例。对剩余的270例POLE基因状态未知的EC石蜡组织样本,采用PCR/CE和Sanger测序法进行平行测序,以检测POLE第9、11、13及14号外显子区域内的11个已知致病性突变位点。针对PCR/CE法和/或Sanger测序检测结果显示携带POLE-exo*的EC病例,进一步使用NGS进行验证。结果在270例EC样本中,使用Sanger测序法在4例(4/270,1.5%)中检测到了POLE-exo*。相比之下,使用PCR/CE则在12例(12/270,4.4%)中检测出了POLE-exo*。值得注意的是,所有通过Sanger测序法检测出POLE-exo*的病例,在使用PCR/CE时也均被成功检出(4/4,即100%的检出率),并且这些结果到了NGS的验证。此外,在Sanger测序法未能检测出POLE-exo*的266例EC样本中,PCR/CE额外检测出了8例(8/266,3.0%)存在POLE-exo*。这8例样本中,有4例通过NGS技术成功得到了验证,这些病例的肿瘤POLE-exo*突变丰度均低于10%,但肿瘤突变负荷均大于10个突变/Mb。由于肿瘤组织样本量不足,剩余4例PCR/CE阳性而Sanger测序法阴性的病例未能进行NGS验证。结论PCR/CE在检测EC样本中的POLE-exo*时表现出了比Sanger测序法更高的灵敏度和检测能力,尤其是在识别低丰度突变方面。
Objective To investigate the clinical values of fluorescent PCR-capillary electrophoresis(PCR/CE)for detecting somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon(POLE-exo*)in endometrial carcinomas(EC),as compared with Sanger sequencing.Methods A total of 280 EC cases diagnosed at the Department of Pathology at Peking University Third Hospital,Beijing,China from December 2022 to December 2023 were collected.Ten cases,which had previously been confirmed to harbor POLE pathogenic mutations through next-generation sequencing(NGS),were excluded.Subsequently,parallel sequencing using both PCR/CE and Sanger sequencing methods was conducted on the remaining 270 EC samples without prior POLE testing,aiming to examine 11 known pathogenic mutation-sites located within exons 9,11,13,and 14 of the POLE gene.NGS was then carried out on the EC cases in which the PCR/CE and/or Sanger sequencing results indicated the presence of POLE-exo*.Results Among the 270 EC samples,POLE-exo*was detected in 4 cases(4/270,1.5%)using Sanger sequencing.In contrast,the PCR/CE identified POLE-exo*in 12 cases(12/270,4.4%).It was noteworthy that all cases in which POLE-exo*was detected through Sanger sequencing were also successfully identified using PCR/CE(4/4,with a detection rate of 100%).These results were further verified by NGS.The PCR/CE also uncovered an additional 8 cases(8/266,3.0%)of POLE-exo*in the 266 samples that were negative for POLE mutations per Sanger sequencing.Of these 8 cases,4 were validated using NGS,exhibiting variant allele frequency(VAF)below 10%,but tumor mutation burdens exceeding 10 mutations per megabase.However,due to small tumor sizes,NGS verification could not be performed on the remaining 4 PCR/CE-positive but Sanger-negative cases.Conclusion The PCR/CE exhibits better sensitivity and detection capabilities than the Sanger sequencing in identifying POLE-exo*in EC samples,particularly in detecting low VAF.
作者
胡阿锦
刘岩
刘从容
Hu Ajin;Liu Yan;Liu Congrong(Department of Pathology,Peking University Third Hospital/School of Basic Medical Sciences,Peking University,Beijing 100191,China)
出处
《中华病理学杂志》
北大核心
2025年第12期1324-1329,共6页
Chinese Journal of Pathology
基金
国家自然科学基金(82473150)
北京大学第三医院雏鹰计划立项项目(BYSYCY2024001)
北京大学第三医院临床重点项目孵育项目A类(BYSYZD2024020)。
