The role of protein localization along the apical-basal axis of polarized cells is difficult to investigate in vivo, partially due to lack of suitable tools. Here, we present the GrabFP system, a collection of four nanobody-based GFP-traps that localize to defined positions along the apical-basal axis. We show that the localization preference of the GrabFP traps can impose a novel localization on GFP-tagged target proteins and results in their controlled mislocalization. These new tools were used to mislocalize transmembrane and cytoplasmic GFP fusion proteins in the Drosophila wing disc epithelium and to investigate the effect of protein mislocalization. Furthermore, we used the GrabFP system as a tool to study the extracellular dispersal of the Decapentaplegic (Dpp) protein and show that the Dpp gradient forming in the lateral plane of the Drosophila wing disc epithelium is essential for patterning of the wing imaginal disc.DOI: http://dx.doi.org/10.7554/eLife.22549.001

The field of cancer diagnostics has recently been impacted by new and exciting developments in the area of liquid biopsy. A liquid biopsy is a minimally invasive alternative to surgical biopsies of solid tissues, typically achieved through the withdrawal of a blood sample or other body fluids, allowing the interrogation of tumor-derived material including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) fragments that are present at a given time point. In this short review, we discuss a few studies that summarize the state-of-the-art in the liquid biopsy field from a diagnostic perspective, and speculate on current challenges and expectations of implementing liquid biopsy testing for cancer diagnosis and monitoring in the clinical setting.

In non-small cell lung cancer (NSCLC), immune checkpoint inhibitors (ICIs) significantly improve overall survival (OS). Tumor mutational burden (TMB) has emerged as a predictive biomarker for patients treated with ICIs. Here, we evaluated the predictive power of TMB measured by the Oncomine™ Tumor Mutational Load targeted sequencing assay in 76 NSCLC patients treated with ICIs. TMB was assessed retrospectively in 76 NSCLC patients receiving ICI therapy. Clinical data (RECIST 1.1) were collected and patients were classified as having either durable clinical benefit (DCB) or no durable benefit (NDB). Additionally, genetic alterations and PD-L1 expression were assessed and compared with TMB and response rate. TMB was significantly higher in patients with DCB than in patients with NDB (median TMB = 8.5 versus 6.0 mutations/Mb, Mann-Whitney p = 0.0244). 64% of patients with high TMB (cut-off = third tertile, TMB ≥ 9) were responders (DCB) compared to 33% and 29% of patients with intermediate and low TMB, respectively (cut-off = second and first tertile, TMB = 5-9 and TMB ≤ 4, respectively). TMB-high patients showed significantly longer progression-free survival (PFS) and OS (log-rank test p = 0.0014 for PFS and 0.0197 for OS). While identifying different subgroups of patients, combining PD-L1 expression and TMB increased the predictive power (from AUC 0.63 to AUC 0.65). Our results show that the TML panel is an effective tool to stratify patients for ICI treatment. A combination of biomarkers might maximize the predictive precision for patient stratification. Our study supports TMB evaluation through targeted NGS in NSCLC patient samples as a tool to predict response to ICI therapy. We offer recommendations for a reliable and cost-effective assessment of TMB in a routine diagnostic setting.

Pre-symptomatic screening of genetic alterations might help identify subpopulations of individuals that could enter into early access prevention programs. Since liquid biopsy is minimally invasive it can be used for longitudinal studies in healthy volunteers to monitor events of progression from normal tissue to pre-cancerous and cancerous condition. Yet, cell-free DNA (cfDNA) analysis in healthy individuals comes with substantial challenges such as the lack of large cohort studies addressing the impact of mutations in healthy individuals or the low abundance of cfDNA in plasma. In this study, we aimed to investigate the technical feasibility of cfDNA analysis in a collection of 114 clinically healthy individuals. We first addressed the impact of pre-analytical factors such as cfDNA yield and quality on sequencing performance and compared healthy to cancer donor samples. We then confirmed the validity of our testing strategy by evaluating the mutational status concordance in matched tissue and plasma specimens collected from cancer patients. Finally, we screened our group of healthy donors for genetic alterations, comparing individuals who did not develop any tumor to patients who developed either a benign neoplasm or cancer during 1–10 years of follow-up time. To conclude, we have established a rapid and reliable liquid biopsy workflow that allowed us to study genomic alterations with a limit of detection as low as 0.08% of variant allelic frequency in healthy individuals. We detected pathogenic cancer mutations in four healthy donors that later developed a benign neoplasm or invasive breast cancer up to 10 years after blood collection. Even though larger prospective studies are needed to address the specificity and sensitivity of liquid biopsy as a clinical tool for early cancer detection, systematic screening of healthy individuals will help understanding early events of tumor formation.

Sox6 belongs to the Sry (sex-determining region Y)-related high-mobility-groupbox family of transcription factors, which control cell-fate specification of many cell types. Here, we explored the role of Sox6 in human erythropoiesis by its overexpression both in the erythroleukemic K562 cell line and in primary erythroid cultures from human cord blood CD34 ؉ cells. Sox6 induced significant erythroid differentiation in both models. K562 cells underwent hemoglobinization and, despite their leukemic origin, died within 9 days after transduction; primary erythroid cultures accelerated their kinetics of erythroid maturation and increased the number of cells that reached the final enucleation step. Searching for direct Sox6 targets, we found SOCS3 (suppressor of cytokine signaling-3), a known mediator of cytokine response. Sox6 was bound in vitro and in vivo to an evolutionarily conserved regulatory SOCS3 element, which induced transcriptional activation. SOCS3 overexpression in K562 cells and in primary erythroid cells recapitulated the growth inhibition induced by Sox6, which demonstrates that SOCS3 is a relevant Sox6 effector. (Blood. 2011;117(13):3669-3679) IntroductionSox proteins are important transcriptional regulators of different developmental processes in which they control the specification and differentiation of many cell types. [1][2][3] In particular, Sox6, originally isolated from adult mouse testis, 4 is required for the development of the central nervous system, 5-7 for chondrogenesis, 8 and for cardiac and skeletal muscle formation. 9,10 Recently, Sox6 has been demonstrated to be crucial for definitive erythropoiesis, 11-15 a process in which committed progenitors progressively differentiate into burst-forming-unit erythroid cells and colonyforming-unit (CFU) erythroid cells, which in turn give rise to proerythroblasts and erythroblasts and finally to mature, enucleated red blood cells. These differentiation stages are accompanied by profound maturational changes: Within few cell divisions, in parallel with the accumulation of erythroid-specific markers (membrane proteins, enzymes required for the heme biosynthesis pathway, and globins), cells undergo chromatin condensation and enucleate. 16,17 This complex spectrum of maturational steps is controlled at the molecular level by the integration of extrinsic (growth factors; oxygen and iron availability) and intrinsic (growth factor receptors, signaling mediators, transcription factors) signals.Several transcription factors are essential for erythroid commitment and for differential globin gene expression during development; their absence is associated with a wide spectrum of phenotypes ranging from mild perturbation to death because of a complete failure of erythropoiesis. 18,19 Among them, Sox6 recently has been shown to stimulate erythroid cell survival, proliferation, and terminal maturation during definitive murine erythropoiesis. 11,12 Sox6-null mouse fetuses and pups are anemic and have defective red blood cells. Recently, Sox6 has been implicated...

The origin recognition complex (ORC) marks chromosomal sites as replication origins and is essential for replication initiation. In yeast, ORC also binds to DNA elements called silencers, where its primary function is to recruit silent information regulator (SIR) proteins to establish transcriptional silencing. Indeed, silencers function poorly as chromosomal origins. Several genetic, molecular, and biochemical studies of HMR-E have led to a model proposing that when ORC becomes limiting in the cell (such as in the orc2-1 mutant) only sites that bind ORC tightly (such as HMR-E) remain fully occupied by ORC, while lower affinity sites, including many origins, lose ORC occupancy. Since HMR-E possessed a unique non-replication function, we reasoned that other tight sites might reveal novel functions for ORC on chromosomes. Therefore, we comprehensively determined ORC “affinity” genome-wide by performing an ORC ChIP–on–chip in ORC2 and orc2-1 strains. Here we describe a novel group of orc2-1–resistant ORC–interacting chromosomal sites (ORF–ORC sites) that did not function as replication origins or silencers. Instead, ORF–ORC sites were comprised of protein-coding regions of highly transcribed metabolic genes. In contrast to the ORC–silencer paradigm, transcriptional activation promoted ORC association with these genes. Remarkably, ORF–ORC genes were enriched in proximity to origins of replication and, in several instances, were transcriptionally regulated by these origins. Taken together, these results suggest a surprising connection among ORC, replication origins, and cellular metabolism.

Background: Tumor mutational burden (TMB) is an increasingly important biomarker for immune checkpoint inhibitors. Recent publications have described strong association between high TMB and objective response to mono-and combination immunotherapies in several cancer types. Existing methods to estimate TMB require large amount of input DNA, which may not always be available.Methods: In this study, we develop a method to estimate TMB using the Oncomine Tumor Mutation Load (TML) Assay with 20 ng of DNA, and we characterize the performance of this method on various formalinfixed, paraffin-embedded (FFPE) research samples of several cancer types. We measure the analytical performance of TML workflow through comparison with control samples with known truth, and we compare performance with an orthogonal method which uses matched normal sample to remove germline variants. We perform whole exome sequencing (WES) on a batch of FFPE samples and compare the WES TMB values with TMB estimates by the TML assay. Results:In-silico analyses demonstrated the Oncomine TML panel has sufficient genomic coverage to estimate somatic mutations with a strong correlation (r 2 =0.986) to WES. Further, in silico prediction using WES data from three separate cohorts and comparing with a subset of the WES overlapping with the TML panel, confirmed the ability to stratify responders and non-responders to immune checkpoint inhibitors with high statistical significance. We found the rate of somatic mutations with the TML assay on cell lines and control samples were similar to the known truth. We verified the performance of germline filtering using only a tumor sample in comparison to a matched tumor-normal experimental design to remove germline variants. We compared TMB estimates by the TML assay with that from WES on a batch of FFPE research samples and found high correlation (r 2 =0.83). We found biologically interesting tumorigenesis signatures on FFPE research samples of colorectal cancer (CRC), lung, and melanoma origin. Further, we assessed TMB on a cohort of FFPE research samples including lung, colon, and melanoma tumors to discover the biologically relevant range of TMB values.Conclusions: These results show that the TML assay targeting a 1.7-Mb genomic footprint can accurately predict TMB values that are comparable to the WES. The TML assay workflow incorporates a simple workflow using the Ion GeneStudio S5 System. Further, the AmpliSeq chemistry allows the use of low input DNA to estimate mutational burden from FFPE samples. This TMB assay enables scalable, robust research into immuno-oncology biomarkers with scarce samples.

No abstract