Pancreatic Cancer Malignancy Assessment By KRAS Mutation Detection In Circulating DNA

Pancreatic cancer(PC)is one of the most lethal malignancies in the world.The poor prognosis is partly due to the fact that PC is usually diagnosed at advanced stages,subject to relapse and resistant to therapy.There is an urgent need for convenient and effective biomarkers to improve the earlier diagnosis and monitoring of cancer progress.Liquid biopsy has allowed for minimally invasive detection on somatic genomic alterations in tumors.Recently,the research on circulating DNA including circulating tumor DNA(ctDNA)and exosomal DNA has revealed the diagnostic and monitoring value of KRAS mutation detection in PC.Although the detection rate of KRAS mutation in ctDNA was relatively consistent with that in tumor tissue,whether the KRAS mutant allele fraction(MAF)differed was still not reported.Besides,according to the recent reports,exosomal DNA was found to be a high-quality material for KRAS mutation detection in PC.Unfortunately,the techniques for exosomal DNA extraction and mutation detection are still lack of methodological standardization.So far,the clinical application of circulating DNA detection in PC remains inconclusive.More and larger clinical trials and methodological research are still needed.In the first part of this study,the detection of KRAS mutation in ctDNA was performed using droplet digital PCR(dd PCR)and compared with that in matched tumor tissue.We assessed the utility of KRAS MAFs in ctDNA and tissue for pancreatic malignancy assessment.We found that KRAS MAF in ctDNA was significantly different from that in matched tissue.KRAS MAF in ctDNA of PC patients was obviously associated with tumor staging and distant metastasis while KRAS MAF in tumor tissue was not.In addition,mutant KRAS ctDNA combined with carbohydrate antigen 19-9(CA19-9)could increase the sensitivity rate of early-stage PC prediction.In the second part,we took PC as an example and explored whether types of blood samples,exosome isolation method,exosome suspension buffer and testing method of dd PCR could influence KRAS mutation detection on exosomal DNA.The methodological experiments showed that concentration of exosomal DNA extracted from serum was much higher than that from plasma,whereas the MAF of KRAS in serum exosomal DNA was obviously lower.Membrane-based method for exosomes isolation showed no obvious difference on DNA yield and KRAS MAF from the classical ultracentrifugation method.DNase I pretreatment on exosomes could remove DNA outside of exosomes and increase the MAF of KRAS.As for the suspending buffer,PBS could reduce DNA solubility and interfere with the DNase I digestion.Finally,the denaturation-enhanced dd PCR could effectively improve the total KRAS copy number and mutation detection rate.In conclusion,we proved for the first time that KRAS MAF in ctDNA of PC patients was obviously associated with tumor staging while KRAS MAF in tumor tissue was not.The MAF of KRAS in ctDNA could improve the sensitivity in early diagnosis of PC as a complement to CA19-9.Our study also optimized the protocol of mutation detection on exosomal DNA,and provided methodology evidence for the reproducibility and comparability of experimental results from different laboratories.Our study could benefit future research on ciculating DNA in PC patients and facilitate their translation to clinical practice.