Our flow assays can efficiently sort sufficient numbers this website of nuclei to provide those inputs. However sample availability, the quality of the FFPE preparation, and the cellular heterogeneity of the tumor frequently limit the number of samples that can be analyzed. Our direct comparison of aCGH data using template prepared from cell line genomic DNA,phi29 amplified FF DNA, and SPIA-amplified DNA from sorted FFPE samples validates the linearity of this amplification method (Figure 4, Figures S9, S10). Our subsequent analysis of sorted FFPE samples for NGS exploited a PDA cell line whose exome has been extensively studied as a control with known somatic mutations. The primary FF tissue from which the cell line was derived and the corresponding FFPE blocks provided a unique sample set for validating our sorting-based analyses.

The overlap of unique reads and the detection of known mutations across the 3 independent sample preparations demonstrate that sorted FFPE samples can be used for NGS. Thus, the linear whole genome amplification of sorted FFPE samples is an efficient method to extend both aCGH and NGS to these highly informative clinical tissues. In contrast to the cell line and the matching 3.0N population the total diploid sorted fractions from the PDA tissues were non-aberrant by aCGH analysis. However a low (<5�C10%) number of reads for some mutations present in the aneuploid fraction (e.g. KRAS) were observed in the NGS data for the total diploid fraction in both the amplified and unamplified samples (Figure S12).

The total diploid peaks in DNA content based flow sorted tumor samples may contain admixtures of neoplastic and non-neoplastic cell types. To determine whether these low frequency mutation reads represent subpopulations of neoplastic cells we used a DAPI/cytokeratin 19 and a DAPI/vimentin flow assay to resort the biopsy. The cytokeratin 19+ and the vimentin+ diploid populations each had the heterozygous KRAS mutation detected of the aneuploid population and cell line (Fi
The small GTPase K-RAS is frequently mutated in human cancers, with mutations occurring in 90% of non neuro-endocrine pancreatic tumors [1]. The presence of these mutations locks the protein in a constitutively activated form, which in turn results in enhanced stimulation of proliferative pathways, thus conferring a growth advantage on the cancer cell [2].

A number of genetic studies have shown that such AV-951 activating K-RAS mutations are necessary for the onset of pancreatic cancer [3]�C[5]. An inducible pancreas-specific expression system was used recently to show that K-RASG12D expression is also required for tumor maintenance [6]. This renders K-RAS a highly validated target for which specific inhibitors are expected to lead to antitumor efficacy. Unfortunately, all attempts to develop such molecular entities have failed so far, placing this target in the so-called difficult-to-drug target category [7]�C[8].