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HPB

HPB Parallel Session 1

48: Highly Sensitive Low Input Methylated Digital Droplet PCR Assay for Pancreatic Adenocarcinoma Molecular Subtyping

Thursday, March 21, 2024
3:02pm – 3:12pm ET
Location: C101
Disclosure(s):
Hannah Trembath, MD: No financial relationships to disclose
Jen Jen Yeh, MD: Focal Medical: Ownership Interest (Ongoing)
Introduction: Pancreatic adenocarcinoma (PDAC) has the highest mortality rate of all major cancers. The two molecular subtypes of PDAC, basal and classical, are predictive of survival and response to FOLFIRINOX, with basal subtype exhibiting worse outcomes and increased FOLFIRINOX resistance. We have identified discriminatory methylation changes between the subtypes that have been validated in independent datasets. We hypothesize that we can utilize the epigenetic changes to develop a circulating tumor DNA assay for patients.


Methods:

We developed a single sample methylation-based classifier, MYSTIC, using 20 predictive methylation probe sites between the subtypes. To be clinically usable, we converted this first to a quantitative PCR (qPCR) platform using patient derived xenograft (PDX) tumors which recapitulate molecular subtypes. We then moved to development of a digital droplet PCR (ddPCR) assay.


Results:

We identified methylation patterns between basal and classical subtypes in TCGA PAAD (n=150) and selected the top 20 differentially methylated sites. We constructed a model to predict subtype that was validated on two independent cohorts (n= 82, n=21). Primers designed for the top 20 probe sites were first tested and validated on qPCR. 24 of 25 PDX samples were predicted in near perfect concordance with their known RNA sequencing subtypes (κ = 0.911, p</em>< 0.001). Using ddPCR with only 14 nanograms of input DNA, we evaluated 6 PDX tumors (3 basal, 3 classical). Running the model with the ddPCR data predicted 6 of 6 molecular subtypes with perfect concordance. Figure 1 depicts ddPCR droplets of the 6 samples at both a basal and classical methylation site with the basal samples showing more droplets at the basal methylation site and vice versa.


Conclusions:

Circulating tumor DNA exists within the blood of PDAC patients. ddPCR is a powerful, high sensitivity, lower cost technology offering clinical promise for the early detection and disease monitoring for many cancers, and it is already FDA approved for CML treatment response monitoring. Here we show our ability to predict subtype using only 14 nanograms of DNA on a ddPCR platform, >10 fold less required than Nanostring and >70 fold less than RNA sequencing. Our results reflect significant progress towards our goal for liquid biopsy subtyping. Development of a noninvasive blood-based classifier has the potential to change clinical practice, providing noninvasive diagnostics and longitudinal monitoring.

Learning Objectives:

  • Describe the benefits of digital droplet PCR compared to quantitative PCR.
  • Define the two molecular subtypes of pancreatic adenocarcinoma and their association with prognosis and treatment response.
  • Demonstrate an understanding of the clinical impact of a noninvasive diagnostic tool for molecular pancreatic adenocarcinoma subtyping.