Abstract 2730: Image-based single cell-sorting to separate and recover distinct cell populations from complex heterogeneous mixed tissue: precise sample preparation upstream of FISH
Authors: Gerber Amanda , Khurana Aditi , Koenig Lisa , Strotoman Lindsay , Millner Lori , Sero Valeria , Bolognesi Chiara , Kasimir-bauer Sabine , Medoro Gianni , Moore Matthew , Cotter Philip , Manaresi Nicolo , Bischoff Farideh 
Published: July 2017

Abstract
Fluorescent in Situ Hybridization (FISH) is commonly used for assessment of chromosomal alterations. Guidelines for determining FISH-based classification of clinical biomarkers exist but are based on pre-analytical factors, including fixation/sectioning/thickness/age, that can greatly influence biomarker status determination. Here, we use single-cell image-based cell sorting by DEPArrayTM for the separation and recovery of pure distinct cell populations prior to FISH.
Methods: A multi-center study to evaluate HER2-FISH based analysis on FFPE with and without DEPArrayTM pre-processing was conducted using breast tumors classified as infiltrating ductal carcinoma (n=12), metastatic (n=1) and ductal carcinoma (n=1). From each block, four 50-micron thick curls were sectioned. One curl from each sample was sent to each of four centers (3 US; 1 EU). Each site performed disassociation of curls to generate a single cell suspension. Cells were then stained and sorted using the DEPArrayTM platform for recovery of tumor (cytokeratin+/vimentin-/DAPI+) and stromal (cytokeratin-/vimentin+/DAPI+) cells. Dual-probe FISH for HER2 and centromere 17 was performed on the sorted cells and compared with conventional tissue section FISH.
Results: Overall, ≥ 90% concordance between the sorted tumor cells and the conventional HER2 FISH result was observed. Among the 7 HER2+ cases, HER2 ratio scores for the sorted tumor cells ranged slightly higher, from 2.60 to 8.95, as compared to the conventional method (from 2.10 to 5.14). In all cases in which stromal cells were also recovered, an expected normal ratio was observed, thus verifying that the populations were efficiently separated. Discordance can be attributed to intra-tumoral heterogeneity and the fact that conventional FISH on FFPE requires only a 4-micron section for analysis.
Conclusion: Today, a percentage of patients are likely misclassified for the biomarker of interest as result of pre-analytical factors. We demonstrate here the ability to overcome these pre-analytic factors and ultimately improve the accuracy in determining biomarker status using the DEPArrayTM
Note: This abstract was not presented at the meeting.
Citation Format: Amanda Gerber, Aditi Khurana, Lisa Koenig, Lindsay Strotoman, Lori Millner, Valeria Sero, Chiara Bolognesi, Sabine Kasimir-bauer, Gianni Medoro, Matthew Moore, Philip Cotter, Nicolo Manaresi, Farideh Bischoff. Image-based single cell-sorting to separate and recover distinct cell populations from complex heterogeneous mixed tissue: precise sample preparation upstream of FISH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2730. doi:10.1158/1538-7445.AM2017-2730