Clonal Analysis Of Aneuploid Pancreatic Ductal Adenocarcinoma Genomes In Patient Biopsies Using Agilent Oligonucleotide Technologies And Pacific Biosciences Smrt (Tm) Sequencing System

One of the challenges for the application of emerging genome technologies to the study of human cancer in patients in vivo is the presence of admixtures of normal cells and stroma in patient samples. Furthermore, tumor samples frequently contain multiple neoplastic populations even in small clinical biopsies. Consequently, it is difficult to accurately detect the multiple genomic aberrations typically present in a biopsy of interest and to distinguish whether they arise concurrently or are present in distinct cell populations in the same sample. Recent advances in flow cytometry technology provide high throughput flow rates and the detection of relatively rare events in dilute samples. Coupling this technology with robust methods to label cells and advanced data acquisition and analyses methods enables the application of flow cytometry to high definition cancer genomics in patients. In our current studies of pancreatic ductal adenocarcinoma (PDA) we developed methods that enable whole genome analyses of clonal populations of sorted aneuploid and diploid nuclei from patient biopsies with as little as 1–5% tumor cell content. Our aCGH studies using Agilent 400k oligonucleotide arrays and flow sorted samples identified 19q13.2 as a region that is recurrently targeted by gene amplification in PDA genomes. Significantly, we identified this amplicon in two distinct aneuploid populations representing 19.8% and 17.4% of the total cellular fraction of a PDA biopsy. We mapped the amplicon to an interval spanning from 43,526,781-cen to 46,171,091-tel, a region targeting 2,644,310 bases in each of the aneuploid populations. This region of interest is relatively gene rich and contains a series of cancer associated genes of interest (e.g. PAK4, AKT2, HIPK4, MAP3K10, RAB4B) to PDA. In addition, our data suggests this region contains variable internal regions of copy number changes suggesting structural rearrangements within the amplicon and its immediate boundaries. To further study this clonally selected region we used the Agilent Sure Select Target Enrichment System to design capture oligonucleotides to sample the 19q13.2 amplicon and its boundaries in the aneuploid PDA genome. These sequences were then interrogated with the PacBio RS system. The integration of these data provides a high resolution clonal analysis of genomic aberrations including point mutations, inversions and insertions that are present in the 19q13.2 amplicon, its immediate boundaries, and the genomes in which it arose. These data provide a highly unique and valuable proof of concept for the application of our integrated clonal genomics methods to the advancement of personalized medicine for patients with PDA and other malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-266. doi:10.1158/1538-7445.AM2011-LB-266