Changes in Circulating Tumor DNA Reflect Clinical Benefit Across Multiple Studies of Patients With Non-Small-Cell Lung Cancer Treated With Immune Checkpoint Inhibitors.

PURPOSE: As immune checkpoint inhibitors (ICI) become increasingly used in frontline settings, identifying early indicators of response is needed. Recent studies suggest a role for circulating tumor DNA (ctDNA) in monitoring response to ICI, but uncertainty exists in the generalizability of these studies. Here, the role of ctDNA for monitoring response to ICI is assessed through a standardized approach by assessing clinical trial data from five independent studies. PATIENTS AND METHODS: Patient-level clinical and ctDNA data were pooled and harmonized from 200 patients across five independent clinical trials investigating the treatment of patients with non-small-cell lung cancer with programmed cell death-1 (PD-1)/programmed death ligand-1 (PD-L1)-directed monotherapy or in combination with chemotherapy. CtDNA levels were measured using different ctDNA assays across the studies. Maximum variant allele frequencies were calculated using all somatic tumor-derived variants in each unique patient sample to correlate ctDNA changes with overall survival (OS) and progression-free survival (PFS). RESULTS: We observed strong associations between reductions in ctDNA levels from on-treatment liquid biopsies with improved OS (OS; hazard ratio, 2.28; 95% CI, 1.62 to 3.20; P < .001) and PFS (PFS; hazard ratio 1.76; 95% CI, 1.31 to 2.36; P < .001). Changes in the maximum variant allele frequencies ctDNA values showed strong association across different outcomes. CONCLUSION: In this pooled analysis of five independent clinical trials, consistent and robust associations between reductions in ctDNA and outcomes were found across multiple end points assessed in patients with non-small-cell lung cancer treated with an ICI. Additional tumor types, stages, and drug classes should be included in future analyses to further validate this. CtDNA may serve as an important tool in clinical development and an early indicator of treatment benefit.

Whole-genome and targeted sequencing of drug-resistant Mycobacterium tuberculosis on the iSeq100 and MiSeq: A performance, ease-of-use, and cost evaluation.

BACKGROUND: Accurate, comprehensive, and timely detection of drug-resistant tuberculosis (TB) is essential to inform patient treatment and enable public health surveillance. This is crucial for effective control of TB globally. Whole-genome sequencing (WGS) and targeted next-generation sequencing (NGS) approaches have potential as rapid in vitro diagnostics (IVDs), but the complexity of workflows, interpretation of results, high costs, and vulnerability of instrumentation have been barriers to broad uptake outside of reference laboratories, especially in low- and middle-income countries. A new, solid-state, tabletop sequencing instrument, Illumina iSeq100, has the potential to decentralize NGS for individual patient care. METHODS AND FINDINGS: In this study, we evaluated WGS and targeted NGS for TB on both the new iSeq100 and the widely used MiSeq (both manufactured by Illumina) and compared sequencing performance, costs, and usability. We utilized DNA libraries produced from Mycobacterium tuberculosis clinical isolates for the evaluation. We conducted WGS on three strains and observed equivalent uniform genome coverage with both platforms and found the depth of coverage obtained was consistent with the expected data output. Utilizing the standardized, cloud-based ReSeqTB bioinformatics pipeline for variant analysis, we found the two platforms to have 94.0% (CI 93.1%-94.8%) agreement, in comparison to 97.6% (CI 97%-98.1%) agreement for the same libraries on two MiSeq instruments. For the targeted NGS approach, 46 M. tuberculosis-specific amplicon libraries had 99.6% (CI 98.0%-99.9%) agreement between the iSeq100 and MiSeq data sets in drug resistance-associated SNPs. The upfront capital costs are almost 5-fold lower for the iSeq100 ($19,900 USD) platform in comparison to the MiSeq ($99,000 USD); however, because of difference in the batching capabilities, the price per sample for WGS was higher on the iSeq100. For WGS of M. tuberculosis at the minimum depth of coverage of 30x, the cost per sample on the iSeq100 was $69.44 USD versus $28.21 USD on the MiSeq, assuming a 2 × 150 bp run on a v3 kit. In terms of ease of use, the sequencing workflow of iSeq100 has been optimized to only require 27 minutes total of hands-on time pre- and post-run, and the maintenance is simplified by a single-use cartridge-based fluidic system. As these are the first sequencing attempts on the iSeq100 for M. tuberculosis, the sequencing pool loading concentration still needs optimization, which will affect sequencing error and depth of coverage. Additionally, the costs are based on current equipment and reagent costs, which are subject to change. CONCLUSIONS: The iSeq100 instrument is capable of running existing TB WGS and targeted NGS library preparations with comparable accuracy to the MiSeq. The iSeq100 has reduced sequencing workflow hands-on time and is able to deliver sequencing results in <24 hours. Reduced capital and maintenance costs and lower-throughput capabilities also give the iSeq100 an advantage over MiSeq in settings of individualized care but not in high-throughput settings such as reference laboratories, where sample batching can be optimized to minimize cost at the expense of workflow complexity and time.

Transcriptome analysis of neural progenitor cells by a genetic dual reporter strategy.

Global analysis of stem/progenitor cells promises new insight into mechanisms that govern self-renewal and cellular potential, an unresolved question of stem/progenitor cell biology. Despite rapid advance of genome-wide profiling methods, the difficulty in cell purification remains a major challenge for global analysis of somatic stem/progenitor cells. Genetic tagging with a reporter provides a powerful tool for identification and isolation of a specific mature cell type; however, for stem/progenitor cells, reporter retention by progeny may be a concern for impurity. Here, we describe a genetic system combining a progenitor cell specific label with a second tag for marking differentiation. We present evidence that differential labeling of neural progenitor cells and their progeny enables prospective purification of these two cell types, whereas isolation based on a single marker compromises the purity of the intended progenitor population. Comparative expression profiling between the purified progenitors and progeny documents a neural progenitor cell transcriptome and uncovers an important role of Tyro3/Axl/Mer receptor tyrosine kinases in the maintenance of neural progenitor cells. This study establishes a general strategy for isolation of somatic stem/progenitor cells and provides a transcriptome database of neural progenitor cells useful for identification of causal factors of neural progenitor cell state, global dissection of epigenetic control of cellular potential, as well as for developing biomarkers or targets of brain cancer stem/initiating cells for therapeutic interventions.

Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs.

MicroRNAs (miRNAs) are 19-22-nucleotide noncoding RNAs that post-transcriptionally regulate mRNA targets. We have identified endogenous miRNA binding sites in mouse embryonic stem cells (mESCs), by performing photo-cross-linking immunoprecipitation using antibodies to Argonaute (Ago2) followed by deep sequencing of RNAs (CLIP-seq). We also performed CLIP-seq in Dicer⁻/⁻ mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA dependent. A significantly enriched motif, GCACUU, was identified only in wild-type mESCs in 3' untranslated and coding regions. This motif matches the seed of a miRNA family that constitutes ~68% of the mESC miRNA population. Unexpectedly, a G-rich motif was enriched in sequences cross-linked to Ago2 in both the presence and absence of miRNAs. Expression analysis and reporter assays confirmed that the seed-related motif confers miRNA-directed regulation on host mRNAs and that the G-rich motif can modulate this regulation.

Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters.

miR-17 approximately 92, miR-106b approximately 25, and miR-106a approximately 363 belong to a family of highly conserved miRNA clusters. Amplification and overexpression of miR-1792 is observed in human cancers, and its oncogenic properties have been confirmed in a mouse model of B cell lymphoma. Here we show that mice deficient for miR-17 approximately 92 die shortly after birth with lung hypoplasia and a ventricular septal defect. The miR-17 approximately 92 cluster is also essential for B cell development. Absence of miR-17 approximately 92 leads to increased levels of the proapoptotic protein Bim and inhibits B cell development at the pro-B to pre-B transition. Furthermore, while ablation of miR-106b approximately 25 or miR-106a approximately 363 has no obvious phenotypic consequences, compound mutant embryos lacking both miR-106b approximately 25 and miR-17 approximately 92 die at midgestation. These results provide key insights into the physiologic functions of this family of microRNAs and suggest a link between the oncogenic properties of miR-17 approximately 92 and its functions during B lymphopoiesis and lung development.