Abstract 1144: High-throughput genetic and expression analysis of circulating tumor cells
Authors: Talasaz AmirAli H. , Cann Gordon , Gulzar Zulfiqar , Chen Jing , April Craig , Klotzle Brandy , Tat Mai , Li Robin , Liu Shujun , Schroth Gary , Brooks James D. , Fan Jian-Bing , Ronaghi Mostafa 
Published: February 2011

Abstract
We are developing novel sample preparation technologies for genomic analysis of circulating tumor cells (CTCs): a new cell-sorting device for purification of single CTCs from a tube of blood; and highly sensitive and quantitative genomic technologies for single cell analysis using Illumina's microarray and next-generation sequencing platforms.
We have prototyped a new ultra-rare cell enrichment device that produces cell samples compatible with Illumina's nucleic-acid analysis platforms. Sorting is performed on whole blood samples with no pre-fractionation, to minimize the genetic alteration of CTCs. To validate the performance of our cell sorting device, we have monitored the recovery of LNCaP prostate cancer cells spiked into 7.5ml normal blood samples at concentration of 10 cells/7.5ml. For ease of analysis, the cancer cells were labeled with cell tracker dye and leukocyte nuclei in blood were labeled with Hoechst 33342 dye. Cancer cells were immunomagnetically labeled in blood with EpCAM beads and then isolated by our cell sorter. We found that the capturing efficiency of our platform was 60% ± 24% for 10 cells/7.5ml samples. The purity of cancer cells among contaminating white blood cells was 91% ± 6%, after a second round of extraction of individual cancer cells. The entire purification protocol of CTCs from 7.5ml blood samples takes 2 hours. This platform has been earlier validated for CTC isolation from blood samples of breast cancer patients and is currently under evaluation for prostate and ovarian cancer patients.
We are also developing new genomic technologies for whole-genome expression profiling, somatic mutation analysis, and transcriptome sequencing of purified CTCs: a) using a Multiple Displacement Amplification (MDA)-based protocol and a 300K-SNP chip readout, we were able to obtain 88.3% and 93.9% call rate, and 97.4% and 99.9% call accuracy with direct cell lysis from 1 and 5 LNCaP cells, respectively. We also detected the chromosomal amplification and loss-of-heterozygosity; b) with our current RNA amplification protocol, we were able to generate reproducible expression profiles, R2 = 0.37 and 0.75, from 1 and 10 cell inputs, respectively. In addition, the expression profiles correlated well with those obtained with standard 100 ng total RNA input, R2 = 0.36 and 0.72, respectively; c) we have also developed next-generation sequencing protocols to profile single-cell transciptomes.
We have introduced a new platform for isolation of small numbers of CTCs from patient blood samples that are compatible with Illumina genomic assays. Our gDNA and RNA amplification protocols work with direct cell lysates; no need to extract DNA or RNA from single cells. The protocols are compatible with Illumina microarray and next-gen sequencing platforms.
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 1144.