David Dow

Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients.

Next-Generation Gene Therapy Few disciplines in contemporary clinical research have experienced the high expectations directed at the gene therapy field. However, gene therapy has been challenging to translate to the clinic, often because the therapeutic gene is expressed at insufficient levels in the patient or because the gene delivery vector integrates near protooncogenes, which can cause leukemia (see the Perspective by Verma ). Biffi et al. ( 1233158 , published online 11 July) and Aiuti et al. ( 1233151 ; published online 11 July) report progress on both fronts in gene therapy trials of three patients with metachromatic leukodystrophy (MLD), a neurodegenerative disorder, and three patients with Wiskott-Aldrich syndrome (WAS), an immunodeficiency disorder. Optimized lentiviral vectors were used to introduce functional MLD or WAS genes into the patients' hematopoietic stem cells (HSCs) ex vivo, and the transduced cells were then infused back into the patients, who were then monitored for up to 2 years. In both trials, the patients showed stable engraftment of the transduced HSC and high expression levels of functional MLD or WAS genes. Encouragingly, there was no evidence of lentiviral vector integration near proto-oncogenes, and the gene therapy treatment halted disease progression in most patients. A longer follow-up period will be needed to further validate efficacy and safety.

Functional genomics approaches can overcome limitations-such as the lack of identification of robust targets and poor clinical efficacy-that hamper cancer drug development. Here we performed genome-scale CRISPR-Cas9 screens in 324 human cancer cell lines from 30 cancer types and developed a data-driven framework to prioritize candidates for cancer therapeutics. We integrated cell fitness effects with genomic biomarkers and target tractability for drug development to systematically prioritize new targets in defined tissues and genotypes. We verified one of our most promising dependencies, the Werner syndrome ATP-dependent helicase, as a synthetic lethal target in tumours from multiple cancer types with microsatellite instability. Our analysis provides a resource of cancer dependencies, generates a framework to prioritize cancer drug targets and suggests specific new targets. The principles described in this study can inform the initial stages of drug development by contributing to a new, diverse and more effective portfolio of cancer drug targets.

Hypersensitivity to abacavir affects about 4% of patients who receive the drug for HIV-1 infection. We did a retrospective, case-control study to identify multiple markers in the vicinity of HLA-B associated with hypersensitivity reactions. HLA-B57 was present in 39 (46%) of 84 patients versus four (4%) of 113 controls (p<0 small middle dot0001). However, because of low numbers of women and other ethnic groups enrolled, these findings relate largely to white men. The lower sensitivity of HLA-B57 for predicting hypersensitivity to abacavir identified in this study compared with a previous report highlights that predictive values for markers will vary across populations. Clinical monitoring and management of hypersensitivity reactions among patients receiving abacavir must remain unchanged.

Hematopoietic stem/progenitor cells (HSPCs) are capable of supporting the lifelong production of blood cells exerting a wide spectrum of functions. Lentiviral vector HSPC gene therapy generates a human hematopoietic system stably marked at the clonal level by vector integration sites (ISs). Using IS analysis, we longitudinally tracked >89,000 clones from 15 distinct bone marrow and peripheral blood lineages purified up to 4 years after transplant in four Wiskott-Aldrich syndrome patients treated with HSPC gene therapy. We measured at the clonal level repopulating waves, populations' sizes and dynamics, activity of distinct HSPC subtypes, contribution of various progenitor classes during the early and late post-transplant phases, and hierarchical relationships among lineages. We discovered that in-vitro-manipulated HSPCs retain the ability to return to latency after transplant and can be physiologically reactivated, sustaining a stable hematopoietic output. This study constitutes in vivo comprehensive tracking in humans of hematopoietic clonal dynamics during the early and late post-transplant phases.

The expansion of a tandemly repeated trinucleotide sequence, CAG, is the mutational mechanism for several human genetic diseases. We present a generally applicable PCR amplification method using a fluorescently labelled locus specific primer flanking the CAG repeat together with paired primers amplifying from multiple priming sites within the CAG repeat. Triplet repeat primed PCR (TP PCR) gives a characteristic ladder on the fluorescence trace enabling the rapid identification of large pathogenetic CAG repeats that cannot be amplified using flanking primers. We used our method to test a cohort of 183 people from myotonic dystrophy families including unaffected subjects and spouses. Eighty five clinically affected subjects with expanded alleles on Southern blot analysis were all correctly identified by TP PCR. This method is applicable for any human diseases involving CAG repeat expansions.

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (THPP) and N-benzyl-6',7'-dihydrospiro[piperidine-4,4'-thieno[3,2-c]pyran] (Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This 'genetic phenotype' was further confirmed by a 'chemical phenotype', whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice.

Abstract Many gene fusions are reported in tumours and for most their role remains unknown. As fusions are used for diagnostic and prognostic purposes, and are targets for treatment, it is crucial to assess their function in cancer. To systematically investigate the role of fusions in tumour cell fitness, we utilized RNA-sequencing data from 1011 human cancer cell lines to functionally link 8354 fusion events with genomic data, sensitivity to &gt;350 anti-cancer drugs and CRISPR-Cas9 loss-of-fitness effects. Established clinically-relevant fusions were identified. Overall, detection of functional fusions was rare, including those involving cancer driver genes, suggesting that many fusions are dispensable for tumour fitness. Therapeutically actionable fusions involving RAF1 , BRD4 and ROS1 were verified in new histologies. In addition, recurrent YAP1-MAML2 fusions were identified as activators of Hippo-pathway signaling in multiple cancer types. Our approach discriminates functional fusions, identifying new drivers of carcinogenesis and fusions that could have clinical implications.

Next-generation sequencing (NGS) technologies represent a paradigm shift in sequencing capability. The technology has already been extensively applied to biological research, resulting in significant and remarkable insights into the molecular biology of cells. In this review, we focus on current and potential applications of the technology as applied to the drug discovery and development process. Early applications have focused on the oncology and infectious disease therapeutic areas, with emerging use in biopharmaceutical development and vaccine production in evidence. Although this technology has great potential, significant challenges remain, particularly around the storage, transfer and analysis of the substantial data sets generated.

We previously reported an association with a putative functional variant in the ADAMTSL3 gene, just below genome-wide significance in a genome-wide association study of schizophrenia. As variants impacting the function of ADAMTSL3 (a disintegrin-like and metalloprotease domain with thrombospondin type I motifs-like-3) could illuminate a novel disease mechanism and a potentially specific target, we have used complementary approaches to further evaluate the association. We imputed genotypes and performed high density association analysis using data from the HapMap and 1000 genomes projects. To review all variants that could potentially cause the association, and to identify additional possible pathogenic rare variants, we sequenced ADAMTSL3 in 92 schizophrenics. A total of 71 ADAMTSL3 variants were identified by sequencing, many were also seen in the 1000 genomes data, but 26 were novel. None of the variants identified by re-sequencing was in strong linkage disequilibrium (LD) with the associated markers. Imputation analysis refined association between ADAMTSL3 and schizophrenia, and highlighted additional common variants with similar levels of association. We evaluated the functional consequences of all variants identified by sequencing, or showing direct or imputed association. The strongest evidence for function remained with the originally associated variant, rs950169, suggesting that this variant may be causal of the association. Rare variants were also identified with possible functional impact. Our study confirms ADAMTSL3 as a candidate for further investigation in schizophrenia, using the variants identified here. The utility of imputation analysis is demonstrated, and we recommend wider use of this method to re-evaluate the existing canon of suggestive schizophrenia associations.

The field of cell and gene therapy (GT) is expanding rapidly and there is undoubtedly a wave of enthusiasm and anticipation for what these treatments could achieve next. Here we assessed the worldwide landscape of GT assets currently in early clinical development (clinical trial phase 1/2 or about to enter clinical trial). We included all gene therapies, i.e., strategies that modify an individual's protein make-up by introducing exogenous nucleic acid or nucleic acid modifiers, regardless of delivery. Unmodified cell therapies, oncology therapies (reviewed elsewhere), and vaccine programs (distinct therapeutic strategy) were not included. Using a December 31, 2018 cutoff date, we identified 336 gene therapies being developed for 138 different indications covering 165 genetic targets. In all, we found that the early clinical GT landscape comprises a very disparate group of drug candidates in terms of indications, organizations, and delivery methods. We also highlight interesting trends, revealing the evolution of the field toward <i>in vivo</i> therapies and adeno-associated virus vector-based delivery systems. It will be interesting to witness what proportion of this current list effectively translates into new medicines.

cDNA clones from a human adult testis cDNA library were isolated and sequenced as part of a programme to produce expressed sequence tags (ESTs). ESTs were used routinely to search DNA and protein sequence databases. One clone (142) showed 60% identity to the Bacillus subtilis glycerol kinase gene at both the DNA and amino acid sequence levels. Analysis of DNA from somatic cell hybrids carrying deleted X chromosomes, has shown that clone 142 detects homologous sequences between Xp21.2–p22.1 (the interval containing the locus responsible for glycerol kinase deficiency—GKD). These sequences are deleted in two patients with GKD. Clone 142 also detects homologous sequences on Xq and at several autosomal loci. The sequences of clone 142 and two further cDNA clones isolated from a human foetal brain cDNA library are presented.

Introduction: The analysis of pharmaceutical industry data indicates that the major reason for drug candidates failing in late stage clinical development is lack of efficacy, with a high proportion of these due to erroneous hypotheses about target to disease linkage. More than ever, there is a requirement to better understand potential new drug targets and their role in disease biology in order to reduce attrition in drug development. Genome editing technology enables precise modification of individual protein coding genes, as well as noncoding regulatory sequences, enabling the elucidation of functional effects in human disease relevant cellular systems.Areas covered: This article outlines applications of CRISPR genome editing technology in target identification and target validation studies.Expert opinion: Applications of CRISPR technology in target validation studies are in evidence and gaining momentum. Whilst technical challenges remain, we are on the cusp of CRISPR being applied in complex cell systems such as iPS derived differentiated cells and stem cell derived organoids. In the meantime, our experience to date suggests that precise genome editing of putative targets in primary cell systems is possible, offering more human disease relevant systems than conventional cell lines.

The folate-sensitive fragile site FRAXE is located in proximal Xq28 of the human X chromosome and lies approximately 600 kb distal to the fragile X syndrome (FRAXA) fragile site at Xq27.3. The cytogenetic expression of FRAXE is thought to be associated with mental handicap, but this is usually mild compared to that of the more common fragile X syndrome that is associated with the expression of the FRAXA fragile site. The exact incidence of FRAXE mental retardation is uncertain. We describe here the results of a U.K. survey designed to assess the frequency of FRAXE in a population of individuals referred for fragile X syndrome testing and found to be negative for expansion events at the FRAXA locus. No FRAXE expansion events were found in 362 cytogenetically negative males studied, and one expansion event was identified in a sample of 534 males for whom cytogenetic analyses were either unrecorded or not performed. Further FRAXE expansion events were detected in two related females known to be cytogenetically positive for a fragile site in Xq27.3-28. To gain insight into the FRAXE phenotype, the clinical details of the identified FRAXE male plus three other FRAXE individuals identified through previous referrals for fragile X syndrome testing are presented. For the population studied, we conclude that FRAXE mental retardation is a relatively rare but significant form of mental retardation for which genetic diagnosis would be appropriate.

We report the ultrasound detection of cranial abnormalities at 14 weeks' gestation in a fetus subsequently confirmed as having tuberous sclerosis using DNA linkage analysis within the affected family. The presence of asymmetrical ventricular enlargement persisted antenatally. Magnetic resonance imaging at 26 weeks indicated the possibility of poor gyral formation consistent with a neuronal migration disorder. Cardiac rhabdomyomata were not visualized on ultrasound scan until 30 weeks' gestation. Postnatal cranial ultrasound confirmed the significant neuropathology which was manifested by severe developmental delay and intractable fits in the child. The potential benefits of earlier diagnosis of tuberous sclerosis by cranial imaging are discussed, although in this patient the routine booking scan resulted in a path of prenatal diagnosis being undertaken which had originally been declined. A mechanism is proposed to explain the variable expression of tuberous sclerosis within this family based on altered TSC2 activity affecting neuronal migration.

Background GW320659, a highly selective neuronal norepinephrine and dopamine re-uptake inhibitor, has been evaluated for the treatment of obesity. Scrutiny of the weight loss data from a phase II study (GlaxoSmithKline study OBS20001) showed a wide variation in weight loss response following GW320659 treatment and the possibility that the study population might include subgroups with enhanced weight loss response. Methods Pharmacogenetic analysis was performed in 191 subjects prospectively ascertained from a Phase II dose ranging study to evaluate the influence of genotype on weight loss efficacy and safety of GW320659 in obese subjects. Results Common genetic polymorphisms in the drug target (norepinephrine transporter protein 1, SLC6A2) and mechanism pathway (NMDA receptor channel NR1 subunit, GRIN1) were associated with increased weight loss following GW320659 treatment in a proportion (36%) of the study population. In the patient subgroup selected for these genotypes, GW320659 (15 mg/day) produced a significant difference in mean weight loss of 7.84 kg (SD 5.23, n=14), compared to 2.53 kg (SD 5.17, n=24) in the subgroup that did not possess the genotypes (P=0.006). This subgroup also showed a highly significant weight loss response for GW320659 compared to placebo (+0.31 kg, SD 3.32, n=16) with the same genotypes (P<0.0001). In addition, there was no difference in placebo response between either subgroup. Conclusions Polymorphisms in SLC6A2 and GRIN1 could be used to maximize effective obesity pharmacotherapy by norepinephrine/dopamine transporter inhibitors by identifying patients that may be predisposed to particularly good treatment weight loss response.

Several lines of evidence support a role for CCL2 (monocyte chemotactic protein-1) and its receptor CCR2 in the development of atherosclerosis. The aim of the present study was to determine the association of the CCR2 Val64Ile polymorphism with the development of coronary artery disease in the WOSCOPS study sample set. A total of 443 cases and 1003 controls from the West of Scotland Coronary Prevention Study (WOSCOPS) were genotyped for the Val64Ile polymorphism in the CCR2 gene. Genotype frequencies were compared between cases and controls. The CCR2 Val64Ile polymorphism was found not to be associated with coronary events in this study population (odds ratio 1.15, 95% CI 0.82-1.61, p = 0.41). This case-control study does not support an association of the CCR2 Val64Ile polymorphism with coronary artery disease in the WOSCOPS sample set and does not confirm a possible protective role for CCR2 Val64Ile in the development of coronary artery disease.

Summary Functional genomics approaches can overcome current limitations that hamper oncology drug development such as lack of robust target identification and clinical efficacy. Here we performed genome-scale CRISPR-Cas9 screens in 204 human cancer cell lines from 12 cancer-types and developed a data-driven framework to prioritise cancer therapeutic candidates. We integrated gene cell fitness effects with genomic biomarkers and target tractability for drug development to systematically prioritise new oncology targets in defined tissues and genotypes. Furthermore, we took one of our most promising dependencies, Werner syndrome RecQ helicase, and verified it as a candidate target for tumours with microsatellite instability. Our analysis provides a comprehensive resource of cancer dependencies, a framework to prioritise oncology targets, and nominates specific new candidates. The principles described in this study can transform the initial stages of the drug development process contributing to a new, diverse and more effective portfolio of oncology targets.

Abstract The homozygous oimloim mouse, a model of moderate‐to‐severe human osteogenesis imperfecta, contains a G‐nucleotide deletion in the Cola‐2 gene (the murine proα2(I) collagen gene) that results in accumulation of α1(I) homotrimer collagen. Although these mice have a distinctive phenotype that includes multiple fractures and deformities, genotyping is necessary to distinguish them from their wildtype (+/+) and heterozygote ( oim/+ ) littermates. In this study, the dye primer and dye terminator chemistry methods, in combination with automated direct DNA sequencing, were compared for accuracy and ease in genotyping. A total of 82 mice from 14 litters were bred and genotyped, this resulted in 18 +/+, 35 oim /+, and 29 oim/oim mice. The dye primer and dye terminator chemistry methods worked equally well for identification of the deletion mutation and thus the genotype of all of the mice. However, the dye terminator method was found to be superior on the basis of the reduced amount of sample handling and reduced quantity of reagent required.

Abstract Recently, a new rapid system for fetal fibronectin (fFN) analysis (TLI IQ ) has been developed with accuracy equal to the gold standard, ELISA (enzyme‐linked immunosorbent assay) testing. The objective of this quality improvement project was to confirm that a reduction in intervention and a reduced length of stay did occur, as expected, after the testing of fFN was introduced at a tertiary referral hospital (National Women's Hospital) for women in threatened preterm labour. There was a reduction in investigation and treatment of women after fFN was introduced. The average length of stay reduced from 3.8 to 2.9 days after testing was introduced. For the women who were fFN negative, the average length of stay was 1.7 days.

We report on a myotonic dystrophy (DM) family exhibiting instability of normal sized (CTG)n alleles in the DM kinase gene on the non-DM chromosome. At least two mutational events involving normal DM alleles must have occurred in this family; one was characterised as a 34-35 (CTG)n repeat mutation. These findings represent a dissociation between (CTG)n repeat instability and myotonic dystrophy. Furthermore, this family highlights genetic counselling issues relating to the pathogenicity of alleles at the upper end of the normal size range and the risk of further expansion into the disease range.

Upon gene therapy (GT) for adenosine deaminase (ADA) deficient-SCID and Wiskott-Aldrich Syndrome (WAS), gene-corrected hematopoietic stem/progenitor cells (HSPC) generated a stable genetically engineered hematopoietic system where each vector-marked cell is univocally barcoded by a vector integration site (IS). To study the dynamics of human hematopoietic system, we collected by LAM-PCR+Illumina sequencing 28.539.414 sequence reads corresponding to 89.373 IS tagging clones belonging to 13 different cell types purified from the bone marrow and the peripheral blood of 4 WAS patients up to 48 months after GT. We firstly identified identical IS shared among CD34+ progenitors, and mature Myeloid/Lymphoid cells as markers of the real-time clonal output of vector-marked HSPC clones in vivo. We unraveled the nature and timing of short, intermediate and long term HSPC output showing that CD34+ clones active at 3-6 months after GT are not detectable at later follow up. Distinct waves of HSPC diversity were observed during the first 6-9 months after GT reaching a homeostatic equilibrium only by 12 months. By unsupervised clustering of IS similarities among lineages we unveiled diverse HSPC output towards lymphoid, myeloid and megakaryo-erythroid cells showing hints of a NK cells origin distinct from T and B cells. We exploited IS similarities to infere and test hematopoietic hierarchies by combining conditional probability distributions and static/dynamic graphical models of dependencies. We also estimated by mark-recapture approaches that just few thousands clones (1185-2884) are responsible for the long-term maintenance of the whole genetically engineered hematopoietic system. The ongoing analyses on IS collected from 7 distinct CD34+ subtypes isolated from GT patients will further increase HSPC tracking resolution. To evaluate the preservation of activity by transplanted HSPC we exploited data derived from the IS-based tracking of 4.845 clones in ADA-SCID patients performed for up to 6 years after GT. We showed that identical IS are consistently detected at multiple lineages level even several years after GT. Strikingly, by semi-quantitative PCRs on specific vector-genome junctions we tracked a fluctuating but consistent output of marked HSPC over a period of 5 years without the manifestation of clonal quiescence phases. Since gamma-retroviral vector used in this GT trial actively transduce only replicating cells, this provide the first evidence that HSPC, awaken from dormancy in vitro, can still retain in vivo long-term activity. Overall our work constitute the first molecular tracking of hematopoietic system in humans. The information gathered are crucial for the design of therapeutic approaches for a broad spectrum of hematological diseases and tumors.

Abstract Hematopoietic stem cells (HSC) are endowed with the unique role of generating an adequate and efficient pool of blood cells throughout human life. Data derived from clonal tracking of HSC activity and hematopoietic dynamics directly in vivo in humans would be of paramount importance for the design of therapies for hematological disorders and cancers. Our gene therapy (GT) clinical trials for adenosine deaminase (ADA) deficient-SCID and Wiskott-Aldrich Syndrome (WAS) based on the infusion of genetically engineered HSC, constitute unique clinical settings where each vector-marked progenitors and its blood cell progeny is traceable being univocally barcoded by a vector integration site (IS). To study early dynamics of hematopoietic reconstitution in humans, we collected by LAM-PCR + Illumina-Miseq sequencing 14.807.407 sequence reads corresponding to 71.981 IS tagging clones belonging to 13 different cell types purified from the bone marrow and the peripheral blood of 4 WAS patients up to 36 months after GT. We firstly identified and quantified identical IS shared among CD34+ progenitors, and mature Myeloid/Lymphoid cells as marker of the real-time clonal output of individual vector-marked HSC clones in vivo. We unraveled the timing of short, intermediate and long term HSC output showing that CD34+ clones active at 3-6 months after GT are not detectable at later follow up. By unsupervised clustering of IS similarities among lineages we unveiled diverse input of HSPC clonal differentiation towards lymphoid, myeloid and megakaryo-erythroid cells and found that NK cells have a distinct relationship with HSPC as compared to T and B cells. We also profiled the level of HSPC output overtime showing that early reconstitution is markedly skewed towards myeloid production. Importantly, clonogenic progenitors generated in vitro from ex vivo purified CD34+ patients’ cells, showed a IS profile coherent with that of freshly purified BM and PB cell types from the same time-point. We also studied population clonal entropy through 7 different diversity indexes and uncovered that progenitor output occurs in distinct waves during the first 6-9 months after transplantation reaching a “homeostatic equilibrium” only by 12 months after GT. At steady state we estimated by mark-recapture mathematical approaches that 1900-7000 transduced HSC clones were stably contributing to the progenitors repertoire for up to 3 years after infusion of gene corrected CD34+ cells. To evaluate the long-term preservation of activity by transplanted HSC we exploited data derived from the IS-based tracking of 4.845 clones in ADA-SCID patients performed for up to 6 years after GT. We showed that identical IS are consistently detected at multiple lineages level even several years after GT. Strikingly, by semi-quantitative PCRs on specific vector-genome junctions we tracked a fluctuating but consistent output of marked HSC over a period of 5 years without the manifestation of clonal quiescence phases. Additionally, since the gamma-retroviral vector used in ADA-SCID HSC-GT trial is able to transduce only actively replicating cells, we provided the first evidence that in vitro activated HSC, “awaken” from dormancy, can still, once infused, retain in vivo long-term activity in humans. We exploited IS similarities among the lineages for both WAS and ADA-SCID datasets to reconstruct the hematopoietic hierarchy by combining conditional probability distributions and static/dynamic graphical models of dependencies. Notably, preliminary data unveiled a link between myeloid progenitors and mature lymphoid cells that supports the recently suggested model of hematopoiesis based on a delayed branching of myeloid and lymphoid lineages. Further mathematical models are being applied to specifically study population dynamics and single HSPC contribution to hematopoiesis including stochastic models of neutral clonal drift. More detailed analysis are also being performed on IS collected from 7 distinct CD34+ subtypes isolated from GT patients and FACS sorted according to the most recent markers of HSPC differentiation. Overall our work constitute the first molecular tracking of individual hematopoietic clones in humans providing an unprecedented detailed analysis of HSC activity and dynamics in vivo. The information gathered will be crucial for the design of therapeutic approaches for a broad spectrum of hematological diseases and tumors. Disclosures Neduva: GSK: Employment. Dow:GSK: Employment.

Abstract Many gene fusions have been reported in tumours and for most their role remains unknown. As fusions can be used clinically for diagnostic and prognostic purposes, and are targets for treatment, it is crucial to assess their functional implications in cancer. To investigate the role of fusions in tumor cell fitness, we developed a systematic analysis utilising RNA-sequencing data from 1,011 human cancer cell lines to functionally link 8,354 gene fusion events with genomic data, sensitivity to &gt;350 anti-cancer drugs and CRISPR-Cas9 loss-of-fitness information. Established clinically-relevant fusions were readily identified. Overall, functional fusions were rare, including those involving cancer driver genes, suggesting that many fusions are dispensable for tumor cell fitness. Novel therapeutically actionable fusions involving RAF1, BRD4 and ROS1 were verified in new histologies. In addition, recurrent YAP1-MAML2 fusions were identified as activators of Hippo-pathway signaling in multiple cancer types, supporting therapeutic targeting of Hippo signalling. Our approach discriminates functional fusions, identifying new drivers of carcinogenesis and fusions that could have important clinical implications. Significance We identify fusions as new potential candidates for drug repurposing and drivers of carcinogenesis. These results support histology agnostic marker-driven precision cancer medicine. Most fusions are not functional with implications for interpreting cancer fusions reported from clinical sequencing studies.

Background: Liver function tests (LFTs) are frequently requested blood tests which may indicate liver disease. LFTs are commonly abnormal, the causes of which can be complex and frequently under investigated. This can lead to missed opportunities to diagnose and treat liver disease at an early stage. We developed an automated investigation algorithm, which would maximise early diagnosis of liver related diseases. Our aim was to determine whether this new pathway of care, Intelligent Liver Function testing (iLFT) increased diagnosis of liver disease and was cost-effective. Methods: By adjusting the current laboratory test order and communications systems we were able to produce an automated system to further analyse abnormal LFTs on initial testing samples. We integrated an automated investigation algorithm into the laboratory management system, based on minimal diagnostic criteria, liver fibrosis estimation, and reflex testing for causes of liver disease. This algorithm then generated a diagnosis and/or management plan. A stepped-wedged trial design was utilised to compare LFT outcomes in General Practices in the 6 months before and after introduction of the iLFT system. Diagnostic outcomes were collated and compared. Findings: Using iLFT, the diagnosis of liver disease was increased by 43%. It was cost-effective with a low initial incremental cost-effectiveness ratio (ICER) of £284 per correct diagnosis, and a saving to the NHS of £3,216 per patient lifetime. Interpretation: iLFT increases liver diagnosis, improves quality of care, and is highly cost-effective. This can be achieved with minor changes to working practices and exploitation of functionality existing within modern laboratory diagnostics systems. Funding: Chief Scientist Office, Scottish Government. Declaration of Interest: None declared. Ethical Approval: The study was approved by the East of Scotland Research Ethics committee.

An estimated 80% of women with threatened preterm labor will go on to deliver at term. Nevertheless, the sequelae of preterm delivery are so threatening that affected women routinely are assessed by ultrasound and receive steroids as well as acute tocolytic therapy in the hospital. Estimating levels of fetal fibronectin (fFN) clearly has some predictive value, notably a negative predictive value of 99.5% for delivery within 1 week. Until now, fFN has been estimated using an enzyme-linked immunosorbent assay (ELISA), an expensive and time-consuming procedure. A new optical system, the TLIIq photometer, has a turnaround time of only 20 minutes and appears to be as accurate as the ELISA. Studies done to date to evaluate the TLIIq have given mixed results. In a 3-month period before the TLIIq system became available, 11 women met criteria for fFN testing. Gestational ages ranged from 27 to 33 weeks and gestational ages at delivery from 30 to 40 weeks. Nine other women, tested in a second 3-month period using the TLIIq system, had initial gestational ages from 25 to 34 weeks and gestational ages at delivery of 30 to 41 techniques. The rate of ultrasound study fell from 100% to 78% in the later period. Steroids were administered to 82% and 56% of women, respectively, and tocolytic therapy to the same proportions. The average hospital stay declined from 3.8 to 2.5 days. Of 6 TLIIq-negative women, 4 had ultrasound studies, 2 received steroids, 2 received tocolytic therapy, and the time in the hospital averaged 1.7 days. These prospectively collected findings, although on a small scale, suggest that a rapid test for fFN has the potential to limit time in the hospital for women with threatened preterm labor and thereby to confer emotional and social benefit.