Whole-exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer

The purpose of this study was to determine the accuracy, precision, and linearity of the CellSearch system and evaluate the number of circulating tumor cells (CTCs) per 7.5 mL of blood in healthy subjects, patients with nonmalignant diseases, and patients with a variety of metastatic carcinomas. The CellSearch system was used to enumerate CTCs in 7.5 mL of blood. Blood samples spiked with cells from tumor cell lines were used to establish analytical accuracy, reproducibility, and linearity. Prevalence of CTCs was determined in blood from 199 patients with nonmalignant diseases, 964 patients with metastatic carcinomas, and 145 healthy donors. Enumeration of spiked tumor cells was linear over the range of 5 to 1,142 cells, with an average recovery of >/=85% at each spike level. Only 1 of the 344 (0.3%) healthy and nonmalignant disease subjects had >/=2 CTCs per 7.5 mL of blood. In 2,183 blood samples from 964 metastatic carcinoma patients, CTCs ranged from 0 to 23,618 CTCs per 7.5 mL (mean, 60 +/- 693 CTCs per 7.5 mL), and 36% (781 of 2,183) of the specimens had >/=2 CTCs. Detection of >/=2 CTCs occurred at the following rates: 57% (107 of 188) of prostate cancers, 37% (489 of 1,316) of breast cancers, 37% (20 of 53) of ovarian cancers, 30% (99 of 333) of colorectal cancers, 20% (34 of 168) of lung cancers, and 26% (32 of 125) of other cancers. The CellSearch system can be standardized across multiple laboratories and may be used to determine the clinical utility of CTCs. CTCs are extremely rare in healthy subjects and patients with nonmalignant diseases but present in various metastatic carcinomas with a wide range of frequencies.

Recent molecular studies have revealed that, even when derived from a seemingly homogenous population, individual cells can exhibit substantial differences in gene expression, protein levels, and phenotypic output 1–5 , with important functional consequences 4,5 . Existing studies of cellular heterogeneity, however, have typically measured only a few pre-selected RNAs 1,2 or proteins 5,6 simultaneously because genomic profiling methods 3 could not be applied to single cells until very recently 7–10 . Here, we use single-cell RNA-Seq to investigate heterogeneity in the response of bone marrow derived dendritic cells (BMDCs) to lipopolysaccharide (LPS). We find extensive, and previously unobserved, bimodal variation in mRNA abundance and splicing patterns, which we validate by RNA-fluorescence in situ hybridization (RNA-FISH) for select transcripts. In particular, hundreds of key immune genes are bimodally expressed across cells, surprisingly even for genes that are very highly expressed at the population average. Moreover, splicing patterns demonstrate previously unobserved levels of heterogeneity between cells. Some of the observed bimodality can be attributed to closely related, yet distinct, known maturity states of BMDCs; other portions reflect differences in the usage of key regulatory circuits. For example, we identify a module of 137 highly variable, yet co-regulated, antiviral response genes. Using cells from knockout mice, we show that variability in this module may be propagated through an interferon feedback circuit involving the transcriptional regulators Stat2 and Irf7. Our study demonstrates the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering cell states and circuits.

The use of tyrosine kinase inhibitors to target the epidermal growth factor receptor gene (EGFR) in patients with non-small-cell lung cancer is effective but limited by the emergence of drug-resistance mutations. Molecular characterization of circulating tumor cells may provide a strategy for noninvasive serial monitoring of tumor genotypes during treatment. We captured highly purified circulating tumor cells from the blood of patients with non-small-cell lung cancer using a microfluidic device containing microposts coated with antibodies against epithelial cells. We performed EGFR mutational analysis on DNA recovered from circulating tumor cells using allele-specific polymerase-chain-reaction amplification and compared the results with those from concurrently isolated free plasma DNA and from the original tumor-biopsy specimens. We isolated circulating tumor cells from 27 patients with metastatic non-small-cell lung cancer (median number, 74 cells per milliliter). We identified the expected EGFR activating mutation in circulating tumor cells from 11 of 12 patients (92%) and in matched free plasma DNA from 4 of 12 patients (33%) (P=0.009). We detected the T790M mutation, which confers drug resistance, in circulating tumor cells collected from patients with EGFR mutations who had received tyrosine kinase inhibitors. When T790M was detectable in pretreatment tumor-biopsy specimens, the presence of the mutation correlated with reduced progression-free survival (7.7 months vs. 16.5 months, P<0.001). Serial analysis of circulating tumor cells showed that a reduction in the number of captured cells was associated with a radiographic tumor response; an increase in the number of cells was associated with tumor progression, with the emergence of additional EGFR mutations in some cases. Molecular analysis of circulating tumor cells from the blood of patients with lung cancer offers the possibility of monitoring changes in epithelial tumor genotypes during the course of treatment. 2008 Massachusetts Medical Society