Deep Clonal Profiling of Formalin Fixed Paraffin Embedded Clinical Samples

<div><p>Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of annotated clinical samples. As such, they represent highly desirable and informative materials for the application of high definition genomics for improved patient management and to advance the development of personalized therapeutics. However, a limitation of FFPE tissues is the variable quality of DNA extracted for analyses. Furthermore, admixtures of non-tumor and polyclonal neoplastic cell populations limit the number of biopsies that can be studied and make it difficult to define cancer genomes in patient samples. To exploit these valuable tissues we applied flow cytometry-based methods to isolate pure populations of tumor cell nuclei from FFPE tissues and developed a methodology compatible with oligonucleotide array CGH and whole exome sequencing analyses. These were used to profile a variety of tumors (breast, brain, bladder, ovarian and pancreas) including the genomes and exomes of matching fresh frozen and FFPE pancreatic adenocarcinoma samples.</p> </div

Combined aCGH and whole exome analysis.

<p>Whole exome sequencing of sorted fresh frozen (FF) and formalin fixed paraffin embedded (FFPE) pancreatic ductal adenocarcinoma (PDA) tissue. A) Flow sorted 3.0N tumor population from PDA tissue. B) Homozygous <i>TP53</i> mutation in sorted FF and FFPE tissues, and matching cell line. C) Chromosome 17 CGH plot of 3.0N population from sorted FF sample. D) Heterozygous <i>KRAS</i> mutation in sorted FF and FFPE tissues, and matching cell line. E) Chromosome 12 aCGH plot of 3.0N population from sorted FF sample.</p

Whole exome sequencing of matching cell line and sorted FF and FFPE PDA tissues.

<p>A) Flow cytometry histograms and the detection of a 3.0N tumor population in FF (A10-46) and FFPE (A10_AT) tissues. B) Exomic sequencing coverage for 3.0N PDA genome from flow sorted FF (A10-46) and FFPE (A10_AT) tissues, and the matched tumor derived cell line A10_74. Left <u>></u>20×coverage, right <u>></u>30×coverage. The % coverage is based on Agilent SureSelect target regions.</p

Whole genome comparison of aCGH results with matching sorted FFPE and FF samples.

<p>Flow sorting and aCGH analysis of matching fresh frozen (FF) and formalin fixed paraffin embedded (FFPE) samples from a pancreatic ductal adenocarcinoma. Flow sorting histogram and detection of 3.2N tumor population in A) FF tissue 11164 and B) FFPE tissue B3733. C–D) Whole genome aCGH plots of 3.2N population sorted from each tissue. Red arrow denotes highly aberrant region on chromosome 2. Black arrow denotes highly aberrant region on chromosome 9. Light blue shaded areas in lower left of A and B show sample and cell debris in flow data.</p

Gene-specific comparison of aCGH results with matching sorted FFPE and FF samples.

<p>Gene view comparison of ADM2 calls in matched fresh frozen (FF) and formalin fixed paraffin embedded (FFPE) pancreatic ductal adenocarcinoma sample. A) Chromosome 2p14 region includes a focal amplicon with the <i>MEIS1</i> gene (arrow). B) Chromosome 9p22.2 region includes a homozygous deletion of <i>CDKN2A</i> (black arrow) and a focal amplicon of <i>SH3GL2</i> (blue arrow). Shaded areas denote ADM2 defined copy number aberrant region.</p

Whole genome aCGH plots of matching cell line and sorted aneuploid pancreatic ductal adenocarcinoma samples.

<p>A) Cell line A10_74 genomic DNA. B) Flow sorted 3.0N tumor population from fresh frozen tissue phi29 amplified DNA. C) Flow sorted 3.0N tumor population from formalin fixed paraffin embedded tissue (FFPE) and genomic DNA. D) Flow sorted 3.0N tumor population from FFPE tissue SPIA amplified DNA.</p

Sorted nuclei from FFPE samples required for aCGH.

<p>Signal intensity histograms (left), gene and whole genome aCGH plots (middle), and the derivative log ratio spreads (DLRS) (right) for hybridizations done with varying inputs from a sorted FFPE triple negative breast cancer sample PS02 1557 E3. A) 50,000 sorted nuclei input for DNA extraction and Cy-5 labeling (red channel in histogram). B) 25,000 sorted nuclei input for DNA extraction and Cy-5 labeling (red channel in histogram). C) 10,000 sorted nuclei input for DNA extraction and Cy-5 labeling (red channel in histogram). Shaded areas in aCGH plots denote ADM2-defined copy number aberrant regions. The gene view shows a focal amplicon that disrupts the <i>USP25</i> locus. A pooled 46,XX sample was used as a Cy-3 labeled (green channel in histogram) reference for each hybridization.</p