Abstract
Circulating tumor cells (CTCs) are shed into the bloodstream by invasive cancers, but the difficulty inherent in identifying these rare cells by microscopy has precluded their routine use in monitoring or screening for cancer. We recently described a high-throughput microfluidic CTC-iChip, which efficiently depletes hematopoietic cells from blood specimens and enriches for CTCs with well-preserved RNA. Application of RNA-based digital PCR to detect CTC-derived signatures may thus enable highly accurate tissue lineage-based cancer detection in blood specimens. As proof of principle, we examined hepatocellular carcinoma (HCC), a cancer that is derived from liver cells bearing a unique gene expression profile. After identifying a digital signature of 10 liver-specific transcripts, we used a cross-validated logistic regression model to identify the presence of HCC-derived CTCs in nine of 16 (56%) untreated patients with HCC versus one of 31 (3%) patients with nonmalignant liver disease at risk for developing HCC (P < 0.0001). Positive CTC scores declined in treated patients: Nine of 32 (28%) patients receiving therapy and only one of 15 (7%) patients who had undergone curative-intent ablation, surgery, or liver transplantation were positive. RNA-based digital CTC scoring was not correlated with the standard HCC serum protein marker alpha fetoprotein (P = 0.57). Modeling the sequential use of these two orthogonal markers for liver cancer screening in patients with high-risk cirrhosis generates positive and negative predictive values of 80% and 86%, respectively. Thus, digital RNA quantitation constitutes a sensitive and specific CTC readout, enabling high-throughput clinical applications, such as noninvasive screening for HCC in populations where viral hepatitis and cirrhosis are prevalent.
Highlights
Circulating tumor cells (CTCs) are shed into the bloodstream by invasive cancers, but the difficulty inherent in identifying these rare cells by microscopy has precluded their routine use in monitoring or screening for cancer
Of the 20,000 genes measured from publicly available microarray datasets (15), the 100 most highly expressed genes in hepatocellular carcinoma (HCC) were screened against expression profiles of hematopoietic cells and other normal tissues (16)
These genes were validated against a separate RNA-sequencing dataset, comparing 10 primary HCCs (17) with white blood cells (WBCs) from eight healthy donor blood samples processed through the CTC-iChip (Fig. 1B)
Summary
Circulating tumor cells (CTCs) are shed into the bloodstream by invasive cancers, but the difficulty inherent in identifying these rare cells by microscopy has precluded their routine use in monitoring or screening for cancer. Emerging microfluidic CTC isolation technologies that effectively deplete leukocytes without manipulating tumor cells (5) preserve cell viability and ensure high-quality RNA content, as demonstrated by single-cell RNA sequencing studies (6–8) These CTC isolation platforms enable the application of powerful RNA-based digital PCR (dPCR) technologies to score molecular signatures of cancer cells, providing a potentially robust and high-throughput readout for the presence of CTCs within blood specimens. To test the feasibility of RNA-derived digital scoring of CTC-enriched cell populations, we applied this strategy to hepatocellular carcinoma (HCC), a cancer that lacks defining gene mutations but originates in liver cells with unique tissue-specific expression profiles. By combining microfluidic depletion of hematopoietic cells from blood specimens with absolute quantification of lineage-derived transcripts, we demonstrate the highly specific detection of circulating tumor cells, enabling noninvasive detection and clinical monitoring of HCC
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