CLIA validation of a novel HER2 FISH test involving image-based cell-sorting to recover pure tumor cell populations from formalin fixed paraffin embedded tissue.

Abstract

e12519 Background: The use of the DEPArray™ system to prepare pure tumor cell populations for more reliable and accurate downstream molecular sequence analysis has been previously demonstrated. To formally evaluate the utility of the DEPArray™ for sample preparation prior to molecular testing, we conducted a CLIA validation study to investigate the analytical performance of the instrument as well as accuracy in determining HER2 status in FFPE tumor specimens using a standard FISH assay. Methods: An initial cohort consisting of 93 FFPE samples (68 from Breast and 25 from Stomach) were selected based on defined inclusion criteria (tumor type and tumor content). For each sample, a single 50µm FFPE scroll was dissociated and then stained using fluorescently labeled Vimentin and Cytokeratin markers to distinguish between putative stromal and tumor populations, respectively. Following separation of these populations on the DEPArray™, a minimum of 100 single cells from each population was recovered and used for subsequent HER2 FISH testing. In addition, an H&E of each sample was evaluated by a Pathologist to confirm the presence of tumor content. Single-cell HER2-FISH analysis was then performed on the DEPArray™ processed samples to assess the number of signals present for each of the chromosome 17 and HER2 loci. Results were compared to the conventional tissue section FISH score. Results: Of the 93 specimens, 80 samples met pre-analytical acceptability criteria that were also confirmed by conventional methods to be either HER2-positive (n = 43) or HER2-negative (n = 37). Overall, a 95% concordance between HER2 results derived from the conventional as compared to the DEPArray™ method was observed. In addition, the instrument performance in terms of reproducibility and reliability was reported as 100%. Conclusions: DEPArray™ for preparation of FFPE-derived tumor cells was analytically validated and shown to yield high confidence in performing HER2-FISH analysis on recovered pure tumor cells. Current strategies to establish clinical utility and efficacy of this approach are underway for cases characterized as equivocal for HER2 or indeterminate by FISH.