1. Novel Biomarker for Early Detection of Pancreatic Cancer
Daniel P. Regan, Michelle J. Veite, Michael A. Kennedy, Ph.D.
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio.
regandp@miamioh.edu, http://chemistry.muohio.edu/kennedy/
Over the course of the past fifteen years, pancreatic cancer has risen to be the
fourth leading cause of cancer-related deaths. With no accurate clinical
screenings available, the late detection of pancreatic cancer has contributed to
the 95% fatality rate after five years of prognosis[1]. The objective of this study is
to find a novel biomarker that will allow for a non-invasive diagnostic test to
identify precancerous lesions known as pancreatic intraepithelial neoplasia. By
utilizing a mouse model, PDAC (pancreatic ductal adenocarcinoma) will be
induced by activating the Kras oncogene, which is the same mutation that occurs
in human oncolytic pathways. The tissues of this sample will be put through
histological analysis for PanIN development and tissue activity. Samples from the
blood, fecal matter, and urine will then be analyzed with NMR spectroscopy. The
aim of this project is to combine the metabonomic data from the NMR and the
PanIN development from the histology to form a statistically specific and
sensitive correlation between a biomarker and known levels of PanIN
development[2]. This biomarker can then be used to perform clinical trials,
allowing for pre-cancerous detection of pancreatic cancer in patients. The
methodology of this project may also give rise to future investigations of
biomarker detection in many other diseases.
Abstract
Methodology
 This study was conducted by using a mouse model. In order for the
development of pancreatic cancer, Ptf1a-Cre and Kras G12D mouse lines were
bred to form the study sample. After being genotyped by southern blot,
tissue collection was set up to be completed from a range of 5-16 months of
age. Sample size included 24 study mice per sex, as well as per age.
 Within 24 hours of the mouse’s sac date, the mouse was transferred to a
metabolite cage for fecal and urine samples. Upon dissection, a serum
sample, collection of the vital organs and GI tract, and collection of the
pancreas and spleen are performed. Metabolic samples are stored a -80
freezer and organs are transferred to 70% EtOH solution an placed in cold
storage.
 Pancreatic tissues are then processed and embedded into wax cassettes.
Protocol for H&E staining is conducted and histological analysis is then
conducted. Tissues are graded based on PanIN development. Remaining
tissue is saved for IHC analysis, targeting p53 among other metabolic activity.
 NMR Spectroscopy is then conducted on the fecal, serum, and urine samples.
Samples will be prepared in 1 mL tubes for the 600 MHz Bruker Spectrometer.
Extensive metabonomic activity will be conducted at this level.
 All data compiled into the projects database will be analyzed for a novel
biomarker to pancreatic cancer. This will be conducted by analyzing the
metabonomic data and comparing it against the PanIN development. Trends
in age, PanIN thresholds, and sex will be sought out.
 The findings of our data will be published and give suggestion to conduct a
clinical experiment in which our biomarker is sought out using our non-
invasive methodology.
This project is made possible by the financial support of the National Institutes of Health, the First Year Research
Experience, Undergraduate Research Award, and Undergraduate Summer Scholars. Collaboration with Dr. David
Klimstra, Director of Pathology at Memorial Sloan Kettering Cancer Center, provides for expert analysis of the
histology. For information and publications related to this research and The HFMR Laboratory visit our website:
http://chemistry.muohio.edu/kennedy/
Acknowledgements
Discussion
The lab is currently in its fourth year of research on this study. At this point in the
study, the lab is collecting the NMR-Spectra and analyzing the histology of the
pancreata. Once all of the bio-fluids and tissues have been analyzed, our lab will have
compiled one of the most extensive molecular study of pancreatic cancer in terms of
sample size and correlation factors. Reviewing previous works and the data that has
been collected thus far, we have reason to believe that there will be a significant
biomarker that correlates to PanIN-2 and PanIN-3 Development[3]. Further
correlations to age, gender, and protein expression will also be analyzed. The goal of
this study is not only to introduce a novel biomarker for pancreatic cancer, but to
open up the door for others to use metabonomic research to find other biomarkers in
the developmental stages of other cancerous models.
Objectives
 Create a database with all of the H&E and IHC slides, including notations
 Discover trends in age, gender, or protein expression from histology
 Understand the mutations caused by pancreatic cancer on a molecular level
 Find dissimilarities in the NMR Spectra between control and study mice
 Establish a biomarker that correlates to specific PanIN development
 Suggest methodology for non-invasive screening for development of
pancreatic cancer in a clinical trial
 Provide methodology of metabolomics research of biomarkers for other
cancer models
Results
Figure 1: The photos above are histology slides of the pancreatic tissue from the study exhibiting
varying PanIN development. Normal pancreas, Figure 1A. PanIN 1, Figure 1B. PanIN 2 &3,
Figure 1C. PanIN 3 & PDAC, Figure 1D.
Figure 2: This NMR-Sectra is from a urine sample of an 11 month study mouse. These spectra
will be analyzed against the control samples for variance in peak intensities and up-regulation or
down-regulation of specific metabonomics.
References
[1] Hidalgo, M. (2010). Pancreatic cancer. The New England Journal of Medicine, 362(17), 1605–17.
doi:10.1056/NEJMra0901557
[2]Goodpaster, A. M., Romick-Rosendale, L. E., & Kennedy, M. a. (2010). Statistical significance analysis of nuclear
magnetic resonance- based metabonomics data. Analytical Biochemistry, 401(1), 134–43.
doi:10.1016/j.ab.2010.02.005
[3]Hruban, R. H., Maitra, A., & Goggins, M. (2008). Update on pancreatic intraepithelial neoplasia. International
Journal of Clinical and Experimental Pathology, 1, 306–316.
A B
C D