Genomic instability appears to cooperate with Darwinian selection to promote cancer formation through a process in which genomic aberrations occur at accelerated rates, and those alterations that provide a selective growth advantage lead to clonal evolution and expansion. Consequently the patterns of genomic aberrations in cancers can vary extensively, even in tumors arising in the same organ site. A fundamental hypothesis of current cancer genome efforts is that tumor cells become differentially resistant or sensitive to available clinical interventions according to selected aberrations present in each sample and the pathways that they target. Thus the identification of selected aberrations in patient samples will help develop novel therapeutic targets that can be advanced for improved more personalized approach to the treatment of cancer. Recent advances in genomic technologies provide highly detailed analyses of samples of interest. For example oligonucleotide CGH arrays can distinguish single copy changes across an entire genome at intragenic mapping resolution with probe error rates of <5%. A challenge in studying complex tissues is the presence of admixtures of cells and the polyclonal nature of human neoplasias. Furthermore, in addition to masking critical genomic aberrations, the presence of clonal mixtures of neoplastic cell populations in biopsies makes it prohibitively difficult to discern which genomic aberrations occur concurrently and to comprehensively define the genomic contexts of patient samples. Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of clinically annotated samples with patient follow-up data including diagnostic and therapeutic outcomes. As such, these samples represent highly desirable and informative materials for the application of high definition genomics that could improve patient management and provide the molecular basis for the selection of personalized therapeutics. However a major limitation to the use of these samples for high resolution genomic analyses to date is the highly variable quality of the DNA extracted from samples of interest. Flow cytometry has been used to identify and isolate neoplastic clones from primary biopsies in a variety of tissues using objective quantifiable markers. Once identified individual populations can be flow purified to greater than 95% purity for subsequent molecular analyses. We have recently developed methods that adapt single parameter flow cytometry of fresh frozen samples to high definition clonal aCGH analyses of pancreatic cancer. The overall objective of this application is to extend these methods by developing and validating multiparameter flow cytometry assays that are compatible with high definition array CGH analyses and next generation sequencing of clinical samples. These will include diploid and aneuploid cell populations from formulin fixed paraffin embedded (FFPE) samples of pancreatic adenocarcinomas.
Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of clinically annotated samples with patient follow-up data including diagnostic and therapeutic outcomes. However a major limitation to the use of these samples is the heterogeneity of tumors and the highly variable quality of the DNA extracted from samples of interest. In this proposal we will: 1) develop highly sensitive flow cytometry assays that can objectively identify and purify clonal populations of pancreatic adenocarcinoma cells from these samples, 2) develop methods that will enable the application of high definition genomics, including CGH microarrays and next generation sequencing to these samples.
|Sinha, Arkadeep; Cherba, David; Bartlam, Heather et al. (2014) Mesenchymal-like pancreatic cancer cells harbor specific genomic alterations more frequently than their epithelial-like counterparts. Mol Oncol 8:1253-65|
|Holley, Tara; Lenkiewicz, Elizabeth; Evers, Lisa et al. (2012) Deep clonal profiling of formalin fixed paraffin embedded clinical samples. PLoS One 7:e50586|