Detection of EpCAM-Negative and Cytokeratin-Negative Circulating Tumor Cells in Peripheral Blood

Enrichment of rare circulating tumor cells (CTCs) in blood is typically achieved using antibodies to epithelial cell adhesion molecule (EpCAM), with detection using cytokeratin (CK) antibodies. However, EpCAM and CK are not expressed in some tumors and can be downregulated during epithelial-to-mesenchymal transition. A micro-fluidic system, not limited to EpCAM or CK, was developed to use multiple antibodies for capture followed by detection using CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to CTCs. Higher recovery of CTCs was demonstrated using antibody mixtures compared to anti-EpCAM. In addition, CK-positive breast cancer cells were found in 15 of 24 samples (63%; range 1–60 CTCs), while all samples contained additional CE-positive cells (range 1–41; median = 11; P = .02). Thus, antibody mixtures against a range of cell surface antigens enables capture of more CTCs than anti-EpCAM alone and CE staining enables the detection of CK-negative CTCs

Hematogenous Metastasis of Ovarian Cancer: Rethinking Mode of Spread

SummaryOvarian cancer has a clear predilection for metastasis to the omentum, but the underlying mechanisms involved in ovarian cancer spread are not well understood. Here, we used a parabiosis model that demonstrates preferential hematogenous metastasis of ovarian cancer to the omentum. Our studies revealed that the ErbB3-neuregulin 1 (NRG1) axis is a dominant pathway responsible for hematogenous omental metastasis. Elevated levels of ErbB3 in ovarian cancer cells and NRG1 in the omentum allowed for tumor cell localization and growth in the omentum. Depletion of ErbB3 in ovarian cancer impaired omental metastasis. Our results highlight hematogenous metastasis as an important mode of ovarian cancer metastasis. These findings have implications for designing alternative strategies aimed at preventing and treating ovarian cancer metastasis

Improving Enrichment of Circulating Fetal DNA for Genetic Testing: Size Fractionation Followed by Whole Gene Amplification

OBJECTIVE: Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma. METHODS: Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100–300, 500–700 and 1,500–2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of β-globin and DYS1 were measured. RESULTS: Distribution of β-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both β-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100–300 bp) accounted for nearly 50% (39.76 ± 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 ± 6.49%) of β-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100–300 bp fragments as template. CONCLUSIONS: Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA