Part1:Multiregion Whole-exome Sequencing Of Intraductal Papillary Mucinous Neoplasms Reveals Frequent Somatic KLF4 Mutations Predominantly In Low-grade Regions Part2:Simultaneous Profiling Of Mutations And Methylation Alterations From Cell Free DNA And It

Objective Intraductal papillary mucinous neoplasms(IPMNs)are non-invasive precursor lesions that can progress to invasive pancreatic cancer and are classified as low-grade or high-grade based on the morphology of the neoplastic epithelium.We aimed to compare genetic alterations in low-grade and high-grade regions of the same IPMN in order to identify molecular alterations underlying neoplastic progression.Design We performed multiregion whole exome sequencing on tissue samples from 17 IPMNs with both low-grade and high-grade dysplasia(76 IPMN regions,including 49 from low-grade dysplasia and 27 from high-grade dysplasia).We reconstructed the phylogeny for each case,and we assessed mutations in a novel driver gene in an independent cohort of 63 IPMN cyst fluid samples.Results Our multiregion whole exome sequencing identified KLF4,a previously unreported genetic driver of IPMN tumorigenesis,with hotspot mutations in one of two codons identified in>50%of the analyzed IPMNs.Mutations in KLF4 were significantly more prevalent in low-grade regions in our sequenced cases.Phylogenetic analyses of whole exome sequencing data demonstrated diverse patterns of IPMN initiation and progression.Hotspot mutations in KLF4 were also identified in an independent cohort of IPMN cyst fluid samples,again with a significantly higher prevalence in low-grade IPMNs.Conclusion Hotspot mutations in KLF4 occur at high prevalence in IPMNs.Unique among pancreatic driver genes,KLF4 mutations are enriched in low-grade IPMNs.These data highlight distinct molecular features of low-grade and high-grade dysplasia and suggest diverse pathways to high-grade dysplasia via the IPMN pathway.BACKGROUND Cell free DNA(cfDNA)-based biomarkers,especially mutations and methylation changes,have shown promising results in the liquid biopsy of cancer.However,most existing cfDNA profiling methods detect only one type of biomarker in one test,and the limited yield of cfDNA from one blood draw is sufficient to support only one test in most cases.Here,we developed a technology,Mutation Capsule(MC),to achieve simultaneous mutation/methylation detection and multiple tests on a single cfDNA sample.METHODS Methylation sensitive restriction enzyme digestion on cfDNA was used to transfer methylation status to a format detectable by high-throughput sequencing.The status of mutations and methylations were preserved and amplified in a whole genome library(MC library),which can be further used in multiple tests targeting mutation and methylation sites.We applied the MC platform to detect genetic and epigenetic alterations in clinical samples.cfDNA was isolated from plasma samples from HCC patients(n=85),high-risk individuals with HBV infection(n=100).RESULTS The technology can simultaneously detect up to 0.02%mutation and 0.25%methylation alterations in one test.Furthermore,an aliquot(10%)of the amplified MC library is sufficient to detect mutations and methylations with high sensitivity comparable to the direct detection on the original cfDNA sample.This feature,together with the special design of amplification,leads to higher conversion efficiency from cfDNA molecules to sequencing constructs so that more original cfDNA molecules can be detected.In this study,the genetic mutations and methylation changes showed complementary patterns in cfDNA samples from HCC patients.Profiling the two types of biomarkers could cover more HCC cases compared with detecting only one type of biomarker.CONCLUSION The Mutation Capsule technology enables multiple tests on one cfDNA sample,with mutations and methylation sites simultaneously profiled in each test.The multiplex test feature also enables more sensitive detection in low yield cfDNA samples.