Abstract 796: Molecular characterization of circulating tumor cells using fluidigm biomark dynamic array

Abstract

Abstract Circulating tumor cells (CTCs) are cancer cells derived from a primary tumor and/or its metastases that circulate in the peripheral blood. The number of CTCs in whole blood of cancer patients has been shown to have clinical relevance with respect to patient prognosis. There is, in addition to CTC enumeration, great interest in the molecular characterization of isolated CTCs. However, there are challenges for molecular characterization of CTCs with commonly used methodologies. The number of CTCs is extremely low and despite the depletion of leukocytes during cell enrichment, the CTC-enriched fractions still include numerous leukocytes which strongly impact CTC-specific gene expression profiling. CellSearch™ CTC test is a FDA approved diagnostic test for enumeration of CTCs. The fixation reagents in the CellSave tube allow processing of samples for several days post-collection, making analysis of clinical samples more practical compared to near-same day processing required for samples collected in EDTA tubes. However, the fixation decreases the mRNA signal and assay sensitivity. We have established a protocol which overcomes these hurdles and allows the profiling of select transcripts of interest in CTCs. By applying a proteinase K treatment and Trizol LS lysis prior to RNA isolation, we were able to overcome the impact of fixation on RNA isolation. RNA was isolated from the treated lysate using a commercially available kit applicable to low cell numbers. Use of this kit allowed us to utilize all of the isolated RNA in the cDNA step. Superscript III cDNA synthesis was then carried out according to the manufacturer's protocol. Target genes of interest were then pre-amplified using the Life Technologies established methodology and reagents. The resulting volume of pre-amplified cDNA was concentrated to a set volume to maximize the amount of transcript profiled. Genes of interest were profiled on the Fluidigm Biomark 48.48 Array. mRNA expression analysis was performed on genes which are not expressed in a pure leukocyte population to ensure that any signal detected was from the CTCs, and not from the background leukocyte population. Using this methodology, we succeeded in performing quantitative gene expression analysis on genes such as PDGFRα on as few as five tumors cells (MG63) fixed in CellSave tubes and spiked into a background environment of contaminating leukocytes (typically 1000 or more) pulled down from normal blood using the CellSearch system. These methods could potentially be used not only for greater understanding of the biology and metastatic potentialof CTCs Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 796.