Abstract 3615: Comparison of whole genome amplification methods on single and pooled cells for comparative genomic hybridization array analysis
Authors: Needham Rachel H.V. , Ramirez Arturo B. , Kishawi Iman , Stilwell Jackie L. , Kaldjian Eric P. 
Published: July 2016

Background: Because blood can be easily sampled repeatedly over the course of disease, circulating tumor cells (CTCs) offer an opportunity to measure phenotypic changes during treatment permitting adjustments to therapy as cancer evolves and/or develops resistance. Since cancer therapeutics are often selected based upon molecular information, reliable, high quality methods for whole genome amplification (WGA) are required for single and small pools of CTCs. Here we compared REPLI-g (QIAGEN) and PicoPLEX (Rubicon) WGA methods using picked model CTCs (SKBR3 cells) employing the AccuCyte-CyteFinder-CytePicker system (RareCyte) and subsequent molecular analysis by array comparative genomic hybridization (aCGH).

Methods: We compared aCGH performed on WGA products from single and pooled SKBR3 cells processed with the typical AccuCyte protocol, or an alternative protocol using live cells in suspension. For the typical AccuCyte protocol, isolated buffy coat was spread and dried onto microscope slides, fixed and antibody stained (EpCAM, cytokeratin, CD45) on an automated instrument. Alternatively, pure, live SKBR3 cells were stained in suspension for EpCAM and Her2, and then placed into well slides. Slides were imaged using the CyteFinder digital fluorescence scanning microscope. Fixed SKBR cells were identified by positive nuclear, EpCAM, and CK staining, and negative CD45 staining. Live cells were identified by positive EpCAM/Her2 staining. Cells were picked into PCR tubes using the CytePicker module. DNA from individual or pools of cells (2, 3, 5, or 10) was amplified using either the REPLI-g or PicoPLEX WGA kits and processed for aCGH using Genetisure arrays (Agilent). WGA products were hybridized against matched WGA controls from normal male genomic DNA.

Results: aCGH profiles for pools of 10 cells using both WGA methods were very similar to unamplified SKBR3 genomic DNA. Signal to noise (S:N) was significantly higher with the REPLI-g amplified DNA compared to PicoPLEX, but single cells did not show similar profiles of genomic aberrations to unamplified genomic DNA until at least 3 cells were pooled before amplification. In contrast, single cells amplified using PicoPLEX demonstrated genomic aberrations consistent with unamplified genomic DNA. Fixation did not appear to adversely affect PicoPLEX WGA performance, but DNA from fixed cells could not be amplified with REPLI-g.

Conclusions: Diagnostic decisions can potentially be made from molecular data derived from small numbers of CTCs requiring WGA methods that provide accurate results in downstream analysis. WGA methods should be selected based upon the molecular data required. In this study, higher S:N indicate that REPLI-g may be more sensitive for finding smaller genomic changes than PicoPLEX, however PicoPLEX generates more consistent data with single fixed cells.

Citation Format: Rachel H.V. Needham, Arturo B. Ramirez, Iman Kishawi, Jackie L. Stilwell, Eric P. Kaldjian. Comparison of whole genome amplification methods on single and pooled cells for comparative genomic hybridization array analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3615.