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.
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.
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.
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
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
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.
There are currently no biomarkers annotated for this protocol.
No datasets are currently associated with this protocol.
Thank you to everyone who helped make the 27th EDRN Steering Committee Meeting a success. We look forward to seeing everyone at the 9th EDRN Scientific Workshop from September 8-11, 2014 in Washington D.C.