Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA.

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy.

Use of Spiked Normalizers to More Precisely Quantify Tumor Markers and Viral Genomes by Massive Parallel Sequencing of Plasma DNA.

A problematic aspect of massive parallel sequencing is that somatic mutations and viral loads are typically quantified as a fraction relative to wild-type human DNA, yet wild-type levels vary with diverse biologic and preanalytic interferences. A novel strategy was devised to quantify target analytes in copies per mL of plasma after normalizing for read counts of spiked DNAs. Five synthetic DNAs (called EndoGenus spikes) were added to plasma before library preparation (modified ArcherDX LiquidPlex 28). By normalizing to the fractional recovery of EndoGenus spike reads, numerical values for each disease marker were reportable in units of copies per mL. To show how well this system operates, replicate assays were performed on 40 mock plasmas having 23 engineered mutations and on 21 natural plasmas. Reads for all five EndoGenus spikes were recovered (means, 313 and 376 copies/mL in mock and natural plasmas, respectively). Normalizing read counts for the proportional recovery of spikes helped control for variables in the multistep protocol, reducing the CV in replicate tests from 34% to 22% for mutations and from 25% to 7% for viral loads. In conclusion, the EndoGenus system is useful for evaluating efficiency of the total test system and for precisely quantifying target molecules. This system may benefit patients being monitored for disease burden while also tracking emerging subclones.

Multilaboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements.

We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele frequencies (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, perhaps because of differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multilaboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and to ensure their quality assurance.

Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers.

Histone deacetylase inhibitors (HDACIs) are known to alter gene expression by both up- and down-regulation of protein-coding genes in normal and cancer cells. However, the exact regulatory mechanisms of action remain uncharacterized. Here we investigated genome wide dose-dependent epigenetic and transcriptome changes in response to HDACI largazole in a transformed and a non-transformed cell line. Exposure to low nanomolar largazole concentrations (

Widespread predicted nonsense-mediated mRNA decay of alternatively-spliced transcripts of human normal and disease genes.

We have recently shown that a third of reliably-inferred alternative mRNA isoforms are candidates for nonsense-mediated mRNA decay (NMD), an mRNA surveillance system (Lewis et al., 2003; PROC: Natl Acad. Sci. USA, 100, 189-192). Rather than being translated to yield protein, these transcripts are expected to be degraded and may be subject to regulated unproductive splicing and translation (RUST). Our initial experimental studies are consistent with these predictions and suggest an unappreciated role for NMD in several human diseases.