Abstract 5254: RNA extraction and gene expression profiling in circulating tumor cells (CTC) using a sized-based method for CTC capture
Authors: Williams Anthony , Hahn Noah , Galsky Matthew , Theodorescu Dan , Datar Ram , Tai YC , Cote Richard 
Published: June 2012

Abstract
Background: Circulating tumor cell (CTC) quantitation has demonstrated prognostic significance in multiple tumor types. Experience utilizing CTC to study tumor gene expression is limited and unvalidated. We explored our ability to perform RNA extraction from CTC's using a novel membrane microfilter device (MD). Our objectives were to evaluate (a) the feasibility to extract RNA from cells on the MD, (b) the detection of CTC-specific gene expression on the MD above basal levels of normal cells, and (c) the ability to perform quantitative gene expression analysis in clinical blood samples using the MD.
Methods: RNA was extracted from MDA-MB-231 breast cancer cells as follows: (1) fresh, unfixed cells as a control, (2) cells fixed in 1% formalin for 10 min at room temperature, (3) cells resuspended into 5ml PBS, fixed in 1% formalin for 10 min at room temperature, processed by the MD, and either (3.1) removed from the microfilter by LCM for RNA recovery, or (3.2) lysed from the entire filtration area for RNA recovery. We compared respective expression levels by qRT-PCR of 44 genes with known relevance in metastasis from each of the 3 sources described above to the expression levels of the cells from the control source. We also evaluated RNA integrity from each source by 260/280 ratio. Additionally, varying dilutions of tumor cells were spiked into blood from normal healthy donors, processed by the MD, and the expression levels of 92 genes by qRT-PCR with known relevance in metastasis were compared to expression levels from a healthy donor blood sample. In clinical samples, 10ml blood was drawn from 4 patients with metastatic bladder cancer, processed by the MD, RNA recovered, and the expression levels of 92 genes by qRT-PCR with known relevance in metastasis were compared to the expression levels from a healthy donor blood sample. All cDNA samples prepared from the microfilters were amplified using custom designed gene pools by PCR prior to qPCR analysis.
Results: Our data indicate an ability to extract RNA with good integrity from tumor cells (by 260/280 ratio) captured by the MD despite pre-incubation of samples in a mild fixative, that RNA can be extracted from tumor cells that have been removed from the MD by LCM without any significant degradation, and that low yields of RNA can be reverse transcribed and amplified prior to qPCR analysis. In both spiked and clinical blood samples, we detected differential expression in numerous genes as compared to healthy donor blood.
Conclusion: While the enumeration of CTC's has been the primary means to collect clinically useful data, molecular characterization of CTC is yet to be widely accomplished. Our data demonstrate potential to quantitatively characterize relative gene expression levels in CTC, which will enhance our understanding of the metastatic process, and improve prediction of therapeutic response.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5254. doi:10.1158/1538-7445.AM2011-5254