The Problem Pancreatic ductal adenocarcinoma (PDA) has an abysmal 5-year survival rate of <5% and is projected to be leading cause of cancer death by 2030. Poor clinical outcomes are driven by nonspecific symptoms, aggressive tumor biology, and early metastatic dissemination. These characteristics result in late stage diagnosis and limited treatment options. Limited understanding of the molecular underpinnings of PDA progression hinders the development of better treatment strategies. Though PDA is very genetically heterogeneous, there are a small number of well characterized and highly prevalent variants, such as KRAS mutations which are present in over 90% of PDA patient. Prior work has shown that cancer-specific molecular signatures can be detected in urine, peripheral blood, precursor cyst lesion fluid, as well as other body fluids. These liquid biopsy alternatives can be accessed non-invasively or acquired via endoscopic ultrasound (EUS) during clinical staging. However, clinical biospecimens are often limited, and the fraction of tumor-derived material can be lower than 0.1%.
The Approach I employed microfluidic digital droplet PCR (ddPCR) for blood-based detection of oncogenic mutations. ddPCR is a nimble and cost-effective alternative to next-generation sequencing when interrogating a small number of genetic targets. I showed that partitioning single cell-free DNA (cfDNA) molecules into microfluidic reaction volumes eliminates PCR amplification bias against rare alleles and hard-to-amplify sequences. I demonstrated this assay had the capacity to accurately quantify single mutant DNA fragments in total cfDNA inputs as low as 5 ng . In a follow-up study, I showed that assay sensitivity remained high in localized PDA, where ctDNA levels can be ten-fold lower of than metastatic . I retrospectively analyzed PDA plasma samples and found low ctDNA allelic frequency (<0.3%) correlated with a two-fold increase in overall and progression-free survival. I employed a similar strategy for rare genetic variant detection in other clinical specimens , using digital PCR and next-generation sequencing.
Project Funding Cancer Biology Training Grant (CA967622, 2015), Mistletoe Foundation Unfettered Research Grant (2019)
Relevant Publications & Presentations
1. Pratt, E.D., Zhen, D.B., Cowan, R.W., Manning, S.L., Qiao, E., Cameron, H., Schradle, K., Simeone, D., and D, A. (2019). Multiplex enrichment and detection of rare KRAS mutations in liquid biopsy samples using digital droplet pre-amplification. Analytical Chemistry 91, 7516–7523.
2. Pratt, E.D., Zhen, D.B., Manning, S.L., Cameron, H., Schradle, K., Gunchick, V., Sahai, V., Simeone, D.M., and Rhim, A.D. (2018). Multiplex enrichment and detection of KRAS mutations in liquid biopsy samples using digital droplet pre-amplification. In AACR special conference on pancreatic cancer: Advances in science and clinical care.
3. Cowan, R., Pratt, E.D., Wilhelm, J., Abdulla, M., Qiao, E., Brennan, L., Ulintz, P., Bellin, M., and Rhim, A. (2016). Patients with severe chronic pancreatitis contain widespread and numerous pancreatic cancer-related somatic variants as detected by picodroplet-enabled targeted sequencing. In AACR special conference on pancreatic cancer: Advances in science and clinical care.
4. Pratt, E.D., Londono, A., Qiao, E., Brennan, L., Simeone, D., Ulintz, P., Samuels, M., and Andrew (2016). Prediction of histologic grade of precancerous cystic lesions using picodroplet PCR-enabled targeted sequencing. In AACR special conference on pancreatic cancer: Advances in science and clinical care.
5. Farrell, A.S., Joly, M.M., Allen-Petersen, B.L., Worth, P.J., Lanciault, C., Sauer, D., Link, J., Pelz, C., Heiser, L.M., and Morton, J.P. et al. (2017). MYC regulates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma associated with poor outcome and chemoresistance. Nature Communications 8, 1728.