A Tumor Sorting Protocol that Enables Enrichment of Pancreatic Adenocarcinoma Cells and Facilitation of Genetic Analyses Zachary S. Boyd, Rajiv Raja,

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A Tumor Sorting Protocol that Enables Enrichment of Pancreatic Adenocarcinoma Cells and Facilitation of Genetic Analyses Zachary S. Boyd, Rajiv Raja, Stephanie Johnson, David A. Eberhard, Mark R. Lackner The Journal of Molecular Diagnostics Volume 11, Issue 4, Pages 290-297 (July 2009) DOI: 10.2353/jmoldx.2009.080124 Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Analysis of tumor content in a range of solid tumor malignancies. A: Histopathological review was conducted on at least 10 samples from a range of tumor types and the percentage of tumors with at least 80% tumor cell content is shown. Gastric and pancreatic cancers were found to have substantially lower tumor content than other tumor types. H&E-stained sections showing relatively high (B) and low (C) tumor cell content sections. Samples similar to that shown in C were used for sorting experiments described in this study. The Journal of Molecular Diagnostics 2009 11, 290-297DOI: (10.2353/jmoldx.2009.080124) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Fluorescence-activated cell sorting tumor sorting data. A: Example of a solid tumor section stained with DAPI (blue) and anti-pan-cytokeratin antibody (C-11) (green) in the left panel and after mechanical dissociation in the right panel. B: Representative flow cytometric sorting data from a pancreatic adenocarcinoma sample. The y axis indicates DAPI staining intensity and the x axis anti-pan-cytokeratin staining. Boxes indicate subpopulations enriched for stromal or tumor cells, as indicated. The Journal of Molecular Diagnostics 2009 11, 290-297DOI: (10.2353/jmoldx.2009.080124) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Genome-wide LOH analysis from a single pancreatic adenocarcinoma sample. Heatmap showing regions of normal heterozygosity (red, black) or regions of LOH (yellow) as determined by SNP array analysis. Colors represent the probability that a stretch of homozygous SNPs could arise by chance as indicated in the key. Columns represent data from genomic DNA from unsorted tissue and sorted stromal and tumor (ie, neoplastic) fractions and chromosomal location is indicated to the right. The approximate locations of key tumor suppressor loci in pancreatic cancer are indicated to the right. Widespread LOH is observed specifically in the tumor fraction but not unsorted or stromal fractions. The Journal of Molecular Diagnostics 2009 11, 290-297DOI: (10.2353/jmoldx.2009.080124) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Specific examples of LOH in pancreatic adenocarcinoma samples. Top: Chromosomal region of chromosome 10 that harbors the PTEN tumor gene (coordinates indicated to the right) in unsorted and sorted fractions from three separate tumors. Heatmap colors represent the probability that a stretch of homozygous SNPs could arise by chance as indicated in the key. Bottom: Region of chromosome 9 that harbors the CDK2NA locus. Tumor 2 shows LOH at the PTEN and CDK2NA loci specifically in the neoplastic fraction but not in unsorted tissue or the stromal fraction. The Journal of Molecular Diagnostics 2009 11, 290-297DOI: (10.2353/jmoldx.2009.080124) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Copy number alterations in sorted tumor and stromal fractions from pancreatic adenocarcinomas. A: Genome-wide patterns of chromosomal gains (red) and losses (green) in sorted stromal (S) and tumor (T) fractions from three tumor samples. The key at the bottom indicates copy number in the heatmap chromosomal location is indicated to the right of the heatmap. The location of several important tumor suppressors in pancreatic cancer are indicated by arrows. B: Analysis of the region on chromosome 3 harboring the MITF transcription factor, with focal amplification apparent in tumor fraction 1 and broader amplification of the region apparent in tumor fraction 2. Chromosomal coordinates are given to the right of the figure. C: Analysis of the region on chromosome 19 harboring CCNE1, with high-level amplification apparent specifically in tumor fraction 1. The Journal of Molecular Diagnostics 2009 11, 290-297DOI: (10.2353/jmoldx.2009.080124) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 6 Sequence analysis of the KRAS locus in sorted pancreatic adenocarcinoma tissue. Upper sequence traces show analysis of a region of exon 2 in KRAS with nucleotide change predicted to cause a G12D amino acid substitution apparent in the neoplastic fraction but not the stromal fraction. Lower sequence traces show analysis of a region of exon 3 in KRAS with nucleotide change predicted to cause a Q61R amino acid substitution apparent in the tumor and stromal fractions but substantially enriched in the tumor fraction. The Journal of Molecular Diagnostics 2009 11, 290-297DOI: (10.2353/jmoldx.2009.080124) Copyright © 2009 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions