5mC- and 5hmC-based Biomarkers for Non-invasive and Early Detection of Pancreatic Cancer
LABS-seq and nano-hmC-Seal represent two highly sensitive techniques for epigenetic profiling in clinical biospecimens: Circulating cell-free DNA (cfDNA) contains epigenetic information, including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). 5mC and 5hmC regulate chromatin states and gene expression, and their deposition, oxidation, and removal are all tightly governed in the cell. 5mC is a major negative marker for gene expression. In contrast, 5hmC is closely associated with open chromatin, reflecting positive gene expression 5mC and 5hmC are sensitive and powerful analytes for biomarker development, as they are chemically stable, abundant in the genome, and closely associated with disease states. There are several advantages to using genomewide 5mC and 5hmC profiling for biomarker development, including
(1) they are abundant in the genome, chemically stable, and closely associated with disease states;
(2) aberrant DNA methylation occurs early in neoplastic development and progression;
(3) the genomic nature of epigenetic alterations provides a rich dataset (as opposed to mutations, which are often unique);
(4) changes 5mC and 5hmC track with disease burden;
(5) 5mC and 5hmC modifications provide information about the underlying transcriptional suppression or activation, respectively, and reveal genomic elements driving malignant progression.
Furthermore, 5mC modifications likely represent early attempts of cancer cells to avoid apoptosis, cell cycle arrest, and other tumor suppressor mechanisms. Subsequently, 5hmC modifications provide cancer cells with the autonomy to increase the expression level of their genes of interest. Because 5hmC modifications need a preliminary step (a 5mC modification), 5mC modifications occur early and are therefore sensitive, while later occurring 5hmC modifications are more specific. Therefore, a combination of the two approaches, one contributing sensitivity (5mC) and the other contributing specificity (5hmC) is desirable, innovative, and even necessary if one truly wishes to optimize the early detection of PDAC
The overarching theme of this proposal is to develop robust biomarkers for detecting PDAC through comprehensive 5mC and 5hmC-based biomarker discovery (with LABS-seq, and nano-hmC-Seal, respectively) and validation approaches in large, independent, and multicenter patient cohorts
In this proposal, our goal is to identify diagnostic 5mC/5hmC biomarkers from cfDNA, informed by 5mC and 5hmC profiles from tumor tissue, for the development of sensitive, specific, minimally invasive, and clinically feasible blood tests for early PDAC detection. Improved early diagnosis of PDAC represents the best opportunity to select patients for resection before their cancer progresses to invasive PDAC. To achieve this goal, we propose three Specific Aims:
# SPECIFIC AIM 1: Identify differentially (hydroxy-)methylated loci potentially diagnostic for PDAC with 5mC and 5hmC genomic profiling in circulating cfDNA and matching tissue.
* Aim 1A: Conduct genome-wide 5mC and 5hmC profiling using LABS-seq and nano-5hmC-Seal of plasma-derived DNA from 150 PDAC patients, 150 healthy controls, and matching tissue samples.
* Aim 1B: Perform statistical and bioinformatics analyses to identify the differentially methylated and hydroxymethylated genomic regions.
# SPECIFIC AIM 2: Develop mC and 5hmC-based diagnostic biomarker signatures that discriminate patients with PDAC from controls.
* Aim 2a: Perform plasma DNA qPCR for the candidate 5mC- and 5hmC-differentially-enriched loci.
* Aim 2b: Develop biomarker signatures diagnostic of PDAC from qPCR results using multiple advanced machine learning and statistical approaches.
# SPECIFIC AIM 3: Validate our clinic-friendly, PCR-based biomarker panels for PDAC detection in two independent cohorts, both cross-sectionally and retrospectively.
* Aim 3a: Validate the 5mC/hmC panels using PCR in a large, cross-sectional cohort of biobanked samples from patients with PDAC vs. controls.
* Aim 3b: Independently validate the performance characteristics of the 5mC/5hmnC panels by PCR analysis in plasma from prospectively collected samples in the PLCO study to evaluate the earliest possible signal.
Impact: We expect the successful completion of these Aims to result in the identification of sensitive, specific, and clinically feasible biomarkers for identifying patients at substantial risk of PDAC. We have unique access to specimen collections from worldwide sources and a team of accomplished collaborators with complementary talents who will converge on our group’s proven pipeline of biomarker discovery. For lethal diseases like PDAC, early detection offers the best opportunity for improving clinical outcomes.