Abundance of Tumor Related Mutations in High Molecular Weight cf-DNA Isolated from PDAC Cancer Patient Plasma Samples
Michael Heller,
Distinguished Scientist - Knight Cancer Institute, Center for Cancer Early Detection and Research (CEDAR),
Oregon Health & Science University (OHSU)
The availability of enough tumor related cell free (cf) DNA in patient blood samples could be a limitation for viable early cancer detection. Presently, next generation sequencing techniques focus on the detection of mutations in the smaller (<200bp) cancer related apoptotic cf-DNA fragments. We now have considerable evidence that cancer related mutations are also present in the higher molecular weight (>200bp) cf-DNA, which is often found at elevated levels in cancer patient blood samples. Use of a new electrokinetic chip device allows isolation and multi-omics analysis of the exosome protein biomarkers, as well as the high molecular weight (hmw) cf-DNA present in the “same” 50 µL blood, plasma or serum sample. The exosome protein biomarkers are first detected directly by on-chip immunofluorescent analysis. Subsequently, the hmw cf-DNA is eluted from the chip, whereupon digital PCR (dd-PCR), PCR and sequencing analysis is conducted to identify cancer-related point mutations. In a blinded pancreatic ductal adenocarcinoma cancer (PDAC) patient study, we were able to show correlation of elevated levels of glypican-1 and cf-DNA in PDAC positive samples, with the presence of KRAS mutations in hmw cf-DNA as determined by digital PCR and Sanger sequencing. These results provide support that a relatively large percentage of the hmw cf-DNA in the patient blood/plasma is coming from the tumors. Since low copy number of tumor related cf-DNA may be a limitation for early detection, the fact that the mutations exist in the hmw cf-DNA could be very important. This would of course have considerable implications for NGS, which preferentially tends to focus on mutation detection in the smaller cancer related apoptotic cf-DNA fragments. We now envision a future strategy for Multi-Omic analysis with on-chip exosome biomarker detection and PCR amplification, with option to do dd-PCR, Sanger sequencing and/or more encompassing NGS mutation and epigenetic analysis. The goal is to provide seamless sample-to-answer point-of-care liquid biopsy diagnostics, therapy monitoring and ultimately early cancer detection from a small sample of patient blood.
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