Multiplex assays, involving the simultaneous use of multiple circulating tumor DNA (ctDNA) markers, can improve the performance of liquid biopsies so that they are highly predictive of cancer recurrence. We have developed a single-tube methylation-specific quantitative PCR assay (mqMSP) that uses 10 different methylation markers and is capable of quantitative analysis of plasma samples with as little as 0.05% tumor DNA. In a cohort of 179 plasma samples from colorectal cancer (CRC) patients, adenoma patients, and healthy controls, the sensitivity and specificity of the mqMSP assay were 84.9% and 83.3%, respectively. In a head-to-head comparative study, the mqMSP assay also performed better for detecting early-stage (stage I and II) and premalignant polyps than a published SEPT9 assay. In an independent longitudinal cohort of 182 plasma samples (preoperative, postoperative, and follow-up) from 82 CRC patients, the mqMSP assay detected ctDNA in 73 (89.0%) of the preoperative plasma samples. Postoperative detection of ctDNA (within 2 wk of surgery) identified 11 of the 20 recurrence patients and was associated with poorer recurrence-free survival (hazard ratio, 4.20; P = 0.0005). With subsequent longitudinal monitoring, 14 patients (70%) had detectable ctDNA before recurrence, with a median lead time of 8.0 mo earlier than seen with radiologic imaging. The mqMSP assay is cost-effective and easily implementable for routine clinical monitoring of CRC recurrence, which can lead to better patient management after surgery.

Lens transparency is established by abundant accumulation of crystallin proteins and loss of organelles in the fiber cells. It requires an efficient translation of lens messenger RNAs (mRNAs) to overcome the progressively reduced transcriptional activity that results from denucleation. Inappropriate regulation of this process impairs lens differentiation and causes cataract formation. However, the regulatory mechanism promoting protein synthesis from lens-expressed mRNAs remains unclear. Here we show that in zebrafish, the RNA-binding protein Rbm24 is critically required for the accumulation of crystallin proteins and terminal differentiation of lens fiber cells. In the developing lens, Rbm24 binds to a wide spectrum of lens-specific mRNAs through the RNA recognition motif and interacts with cytoplasmic polyadenylation element-binding protein (Cpeb1b) and cytoplasmic poly(A)-binding protein (Pabpc1l) through the C-terminal region. Loss of Rbm24 reduces the stability of a subset of lens mRNAs encoding heat shock proteins and shortens the poly(A) tail length of crystallin mRNAs encoding lens structural components, thereby preventing their translation into functional proteins. This severely impairs lens transparency and results in blindness. Consistent with its highly conserved expression in differentiating lens fiber cells, the findings suggest that vertebrate Rbm24 represents a key regulator of cytoplasmic polyadenylation and plays an essential role in the posttranscriptional control of lens development.