Abbreviated Name

Hollingsworth 2012

Lead Investigator

Hollingsworth, Michael — University of Nebraska Medical Center

Coordinating Investigator

No coordinating investigator

Involved Investigators

• Hollingsworth, Michael — University of Nebraska Medical Center

Abstract

Study Overview. We will examine DNA extracted from FFPE sections from approximately 200 different surgically resected primary pancreatic tumors from the UNMC Department of Pathology and Microbiology. DNA will be purified from those sections and subjected to deep sequencing for the entire TP53 locus. Expected Outcomes We expect to find a difference in the p53 mutation status between tumor samples from patients that ultimately experienced tumor recurrence (more aggressive) compared to those that did not. Parallel studies to develop ICP will enable us to rapidly develop a low cost platform to extend these studies to larger patient populations for future validation studies. Future Studies The experiments proposed in this application represent a state-of-the-art approach to identify molecular markers that will help clinicians to ascertain the tumor recurrence risk for their pancreatic cancer patients who have undergone a Whipple procedure. If our initial studies support the hypothesis that p53 mutations are associated with early metastasis of pancreatic cancer, these studies would be extended to other cohorts of patient samples that are available at other major centers that see pancreatic cancer patients. Development of ICE COLD-PCR platforms to screen for these mutations will facilitate a reliable, rapid and low cost method for predicting tumor aggressiveness in these patients, which will be deployed in future studies should the hypothesis be supported.

Aims

Specific Aim 1. Deep sequencing of DNA samples from surgically resected primary pancreatic tumors, and correlation of TP53 mutation status with clinical outcomes data. The surgical oncologists at the University of NE Medical Center currently perform approximately 25 Whipple procedures annually. Each sample is fixed in formalin, embedded in paraffin (FFPE) and catalogued for future use. Presently there are at least 200 FFPE samples available. Representative sections from each tumor block will be examined by our pathologist Dr Lazenby and selected for having cellular content of tumor greater than 20% (well above the minimum needed for detecting mutations using greater than 80x coverage in deep sequencing). An appropriate number of sections (8-20 depending on cellular content) will be prepared from each block by the UNMC Tissue Sciences Facility. The tissue on those sections will be used as the source material for DNA isolation. DNA isolation will be performed utilizing commercially-available reagents (Qiagen, Inc) according to the manufacturer’s instructions. The TP53 gene encompassing all exons will be amplified by PCR, the products purified and subjected to library generation (Illumina Inc.) for next generation sequencing (NGS) utilizing the Illumina Hi-Seq 2000 instrument in the UNMC Next Generation Sequencing Core Facility. NGS platforms enable the identification of mutant sequences from heterogeneous samples containing both tumor cells and normal cells, even when the mutant sequence only comprises ~5% of the total sequence (19). This is due to the extensive coverage of NGS methods and the independent sequence data output for each molecule sequenced. De-identified clinical outcome data for all samples will be used for correlative studies of outcome. For each sample, tumor recurrence status and overall survival will be determined. The results will allow us to ascertain if TP53 mutation status in the primary tumor can be used as an accurate predictor of tumor aggressiveness. Data from this analysis will be provided to and analyzed by the DMCC for performance characteristics in predicting tumor recurrence among surgically resected patients. Although we do not currently know the potential power of this analysis, this represents the largest set of clinical samples that are currently available to us to undertake this analysis and the study should be adequately powered to address the simple hypothesis that is under investigation. Specific Aim 2. Development of ICE COLD-PCR reagents and protocols for detecting TP53 mutations in human samples. While NGS methods provide high quality data, the cost per sample makes them unsuitable for general large-scale patient screening purposes. An alternative and cheaper method to identify mutant sequences in a pool of normal sequence is ICE COLD-PCR (ICP; Improved and Complete Enrichment Co-amplification at Lower Denaturation Temperature-PCR). COLD-PCR is a novel form of PCR that allows

Analytic Method

Together with the experts at Transgenomic, Inc., we will design primary and nested amplification primers for each of the p53 mutation hotspots that are detected in our analysis of pancreatic cancer samples, as well as RS-oligos for each that are complementary to the wild type sequence. Plasmids containing wild type and mutant sequences will be used to empirically determine the appropriate reaction conditions for each site. Mixtures of wild type and mutant plasmids will be prepared (eg-10:1, 100:1 and 1000:1, wild type:mutant) and used to test the limits of detection for each. Finally, clinical samples will be tested independently on FFPE samples and the data compared to the deep sequencing data obtained in Specific Aim 1.

Outcome

Study Overview. We will examine DNA extracted from FFPE sections from approximately 200 different surgically resected primary pancreatic tumors from the UNMC Department of Pathology and Microbiology. DNA will be purified from those sections and subjected to deep sequencing for the entire TP53 locus. Expected Outcomes We expect to find a difference in the p53 mutation status between tumor samples from patients that ultimately experienced tumor recurrence (more aggressive) compared to those that did not. Parallel studies to develop ICP will enable us to rapidly develop a low cost platform to extend these studies to larger patient populations for future validation studies. Future Studies The experiments proposed in this application represent a state-of-the-art approach to identify molecular markers that will help clinicians to ascertain the tumor recurrence risk for their pancreatic cancer patients who have undergone a Whipple procedure. If our initial studies support the hypothesis that p53 mutations are associated with early metastasis of pancreatic cancer, these studies would be extended to other cohorts of patient samples that are available at other major centers that see pancreatic cancer patients. Development of ICE COLD-PCR platforms to screen for these mutations will facilitate a reliable, rapid and low cost method for predicting tumor aggressiveness in these patients, which will be deployed in future studies should the hypothesis be supported.

Publications

• No publications available at this time for this protocol.

Biomarkers

• No biomarkers available at this time for this protocol.

Data Collections

• No data collections available at this time for this protocol.

 Team Project

Start Date

Jul 1 2012

Estimated Finish Date

Jun 30 2013

Finish Date

Jun 2 2023

Protocol ID

357

Protocol Type

Single

Fields of Research

• UNKNOWN

Collaborative Group

G.I. and Other Associated Cancers Research Group

Cancer Types

• (No cancer types specified)