Abstract 63: Incorporation of flanking probes reduces truncation losses for fluorescence in situ hybridization analysis of recurrent genomic deletions in tumor sections.
Authors: Yoshimoto Maisa , Ludkovski Olga , Good Jennifer , Gooding Robert J. , McGowan-Jordan Jean , Boag Alexander , Evans Andrew , Tsao Ming-Sound , Nuin Paulo , Squire Jeremy A. 
Published: August 2014

Abstract
Fluorescence in situ hybridization (FISH) is a robust technique when used with appropriate interpretative guidelines, and the assay can yield consistent results that provide diagnostically and prognostically useful information to help guide patient therapy. The establishment of quality control standards in clinical laboratories using routine FISH analysis of formalin fixed paraffin embedded (FFPE) sections in different tumor types for fusion and break-apart strategies and gene amplifications have been developed for both analysis and reporting, but at the present time there are no comprehensive guidelines for the analysis of genomic deletions using FFPE sections. The use of FISH on archival FFPE samples is technically demanding and becomes more challenging when applied to paraffin-embedded tissue microarrays. The evaluation of FISH signals in interphase nuclei of FFPE sections is affected by truncation and overlapping of the nuclei due to varying cell density, tissue architecture differences and histological forms. In this study we report a generalizable four-color deletion FISH approach to assist interpretation problems arising when evaluating FISH signals. The guidelines will help address interpretative dilemmas associated with overlapping and truncated nuclei in FFPE prostate cancer sections. The four-color FISH approach was developed using the PTEN tumor suppressor gene deletion model in prostate cancer. The PTEN assay was based on a robust bioinformatics analysis of 311 published human genome array datasets and comprises a centromeric “chromosome enumeration” probe, a specific PTEN gene probe, and control flanking probes either side of the target probe. The sensitivity and specificity parameters of the four-color PTEN probe set were further characterized using a large number of well-characterized tumors and stringent scoring criteria. The incorporation of flanking control probes allowed the analysts to determine if the chromosomal region was subject to truncation loss. A minimum threshold for apparent deletion frequency was set to address the heterogeneous and homogeneous nature of tumor histology. In addition the approach facilitated analysis of genotypic heterogeneity and varying clonality within different foci of tumor in the prostate. Overall the approach provided robust and highly reproducible results that minimized inter- and intra-assay variability. The four-color FISH deletion assay reduced the frequency of misinterpretation and improved both the quality and throughput of FISH analyses using clinical samples. Moreover the use of established controls and conservative cut-offs for assigning deletions will facilitate more coherent approach to developing reporting standards for deletion assays as more tumor suppressor genes of clinical importance are discovered by next generation sequencing methods.
Citation Format: Maisa Yoshimoto, Olga Ludkovski, Jennifer Good, Robert J. Gooding, Jean McGowan-Jordan, Alexander Boag, Andrew Evans, Ming-Sound Tsao, Paulo Nuin, Jeremy A. Squire. Incorporation of flanking probes reduces truncation losses for fluorescence in situ hybridization analysis of recurrent genomic deletions in tumor sections. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 63. doi:10.1158/1538-7445.AM2013-63