CLIA validation workflow of a novel tumor cell isolation platform.

Abstract

e23161 Background: DEPArray™ technology is based on Dielectrophoresis (DEP). High quality image-based cell selection enables users to identify, isolate and recover intact specific individual rare cells of interest from complex, heterogeneous tissues such as live or fixed cell suspensions. Here we demonstrate a validation workflow for application of molecular sequencing technologies and FISH downstream of rare cell recovery. Methods: FFPE scrolls (n = 93) were dissociated and stained with markers to distinguish putative stromal and tumor populations. DNA integrity was tested using a qPCR technique to predict the amount of DNA required for a successful downstream sequencing metrics. DEPArray™ recovered stromal and tumor cells underwent NGS analysis on an Illumina platform based DEPArray™ OncoSeek Panel. Separately, recovered stromal and tumor cells were also utilized for targeted HER2 FISH. CellSearch® cartridges (19 Breast and 5 Bladder Cancers) were processed through the DEPArray to isolate WBCs (CK-/CD45+/DAPI+; n = 30), CTCs (CK+/CD45-/DAPI+; n = 33) and atypical (CK-/CD45-/DAPI+; n = 47) cells. Recovered cells underwent whole-genome amplification with Ampli1™ WGA and quality controlled by Ampli1 QC. Ampli1 LowPass kit was then used to prepare NGS libraries for absolute CNA profiling by low-pass WGS. Results: Reproducibility and reliability was reported as 100% for instrument performance. For FFPE, the OncoSeek panel simultaneously detected SNPs, indels and CNAs of 63actionable and oncology relevant genes. For FISH, 95% concordance with conventional HER2 results was observed. For CTCs, Ampli1 LowPass detected copy number gains/losses confirming tumor origin of the CK+ cells. A portion of non-conventional cells also demonstrated copy number alterations consistent with tumor origin. Conclusions: The isolation of pure single or pooled tumor cells with the DEPArray™ technology can be used as a method to improve downstream MDx analysis using different techniques, thus to inform treatment decisions and provide valuable prognostic information.