Mazurek Agnieszka M, Fiszer-Kierzkowska A, Rutkowski T, Składowski K, Pierzyna M, Scieglińska D, Woźniak G, Głowacki G, Kawczyński R, Małusecka E
Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
I Radiotherapy Clinic, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
Cancer Biomark. 2013;13(5):385-94. doi: 10.3233/CBM-130371.
The precise analysis of tumour markers in blood such as circulating cell-free DNA (cfDNA) could have a significant impact in facilitating monitoring of patients after initial therapy. Although high levels of total cfDNA in plasma of cancer patients are consistently demonstrated, a low sensitivity of DNA alterations is reported.
The major question regards the recovery of tumour-specific cfDNA such as KRAS mutated DNA and cancer-associated type 16 of human papillomavirus (HPV16).
TaqMan technology was used for detection of KRAS mutation, HPV16 and to quantify cfDNA in blood plasma.
Comparison of four different column-based commercial kits shows that the cfDNA purification carried out by the Genomic Mini AX Body Fluids kit and the QIAamp Circulating Nucleic Acid kit gave us the possibility to improve the sensitivity of detection of KRAS mutation and HPV16. The optimized method was used to follow the reduction in cancer-specific cfDNA after therapy. We found that large volume extractions with low volume of DNA eluate enabled trace amounts of tumour-specific cfDNA from cancer patients to be effectively identified.
Data presented in this study facilitate detection of tumour-specific cfDNA and improve standards needed for the implementation of cfDNA technology into routine clinical practice.
对血液中的肿瘤标志物进行精确分析，如循环游离DNA（cfDNA），可能对促进初始治疗后患者的监测产生重大影响。尽管癌症患者血浆中总cfDNA水平一直很高，但DNA改变的敏感性较低。
主要问题在于肿瘤特异性cfDNA的回收，如KRAS突变DNA和人乳头瘤病毒16型（HPV16）相关癌症。
采用TaqMan技术检测KRAS突变、HPV16并定量血浆中的cfDNA。
对四种不同的基于柱的商业试剂盒进行比较，结果表明，使用基因组迷你AX体液试剂盒和QIAamp循环核酸试剂盒进行cfDNA纯化，使我们有可能提高KRAS突变和HPV16检测的敏感性。优化后的方法用于跟踪治疗后癌症特异性cfDNA的减少情况。我们发现，大量提取并减少DNA洗脱液体积能够有效识别癌症患者中痕量的肿瘤特异性cfDNA。
本研究提供的数据有助于检测肿瘤特异性cfDNA，并提高将cfDNA技术应用于常规临床实践所需的标准。
