The Societal Impact of Inclisiran in England: Evidence From a Population Health Approach.

OBJECTIVES: As first-in-class cholesterol-lowering small interfering ribonucleic acid, inclisiran provides effective reductions in low-density lipoprotein-cholesterol to achieve better cardiovascular (CV) health. We estimate the health and socioeconomic effects of introducing inclisiran according to a population health agreement in England. METHODS: Building on the inclisiran cost-effectiveness model, a Markov model simulates health gains in terms of avoided CV events and CV deaths because of add-on inclisiran treatment for patients aged 50 years and older with pre-existing atherosclerotic CV disease. These are translated into socioeconomic effects, defined as societal impact. To that end, we quantify avoided productivity losses in terms of paid and unpaid work productivity and monetize them according to gross value added. Furthermore, we calculate value chain effects for paid work activities, drawing on value-added multipliers based on input-output tables. The derived value-invest ratio compares avoided productivity losses against the increased healthcare costs. RESULTS: Our results show that 138 647 CV events could be avoided over a period of 10 years. The resulting societal impact amounts to £8.17 billion, whereas additional healthcare costs are estimated at £7.94 billion. This translates into a value-invest ratio of 1.03. CONCLUSIONS: Our estimates demonstrate the potential health and socioeconomic value of inclisiran. Thereby, we highlight the importance to treat CVD and illustrate the impact that a large-scale intervention can have on population health and the economy.

Retrospective analysis of real-world treatment patterns and clinical outcomes in patients with advanced non-small cell lung cancer starting first-line systemic therapy in the United Kingdom.

BACKGROUND: The treatment landscape for advanced non-small cell lung cancer (aNSCLC) has evolved rapidly since immuno-oncology (IO) therapies were introduced. This study used recent data to assess real-world treatment patterns and clinical outcomes in aNSCLC in the United Kingdom. METHODS: Electronic prescribing records of treatment-naive patients starting first-line (1 L) treatment for aNSCLC between June 2016 and March 2018 (follow-up until December 2018) in the United Kingdom were assessed retrospectively. Patient characteristics and treatment patterns were analyzed descriptively. Outcomes assessed included overall survival (OS), time to treatment discontinuation, time to next treatment, and real-world tumor response. RESULTS: In all, 1003 patients were evaluated (median age, 68 years [range, 28-93 years]; 53.9% male). Use of 1 L IO monotherapy (0-25.9%) and targeted therapy (11.8-15.9%) increased during the study period, but chemotherapy remained the most common 1 L treatment at all time points (88.2-58.2%). Median OS was 9.5 months (95% CI, 8.8-10.7 months) for all patients, 8.1 months (95% CI, 7.4-8.9 months) with chemotherapy, 14.0 months (95% CI, 10.7-20.6 months) with IO monotherapy, and 20.2 months (95% CI, 16.0-30.5 months) with targeted therapy. In the 28.6% of patients who received second-line treatment, IO monotherapy was the most common drug class (used in 51.6%). CONCLUSIONS: Although use of 1 L IO monotherapy for aNSCLC increased in the United Kingdom during the study period, most patients received 1 L chemotherapy. An OS benefit for first-line IO monotherapy vs chemotherapy was observed but was numerically smaller than that reported in clinical trials. Targeted therapy was associated with the longest OS, highlighting the need for improved treatment options for tumors lacking targetable mutations.

Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome.

It was recently reported that the recBC mutants of Escherichia coli, deficient for DNA double-strand break (DSB) repair, have a decreased copy number of their terminus region. We previously showed that this deficit resulted from DNA loss after post-replicative breakage of one of the two sister-chromosome termini at cell division. A viable cell and a dead cell devoid of terminus region were thus produced and, intriguingly, the reaction was transmitted to the following generations. Using genome marker frequency profiling and observation by microscopy of specific DNA loci within the terminus, we reveal here the origin of this phenomenon. We observed that terminus DNA loss was reduced in a recA mutant by the double-strand DNA degradation activity of RecBCD. The terminus-less cell produced at the first cell division was less prone to divide than the one produced at the next generation. DNA loss was not heritable if the chromosome was linearized in the terminus and occurred at chromosome termini that were unable to segregate after replication. We propose that in a recB mutant replication fork breakage results in the persistence of a linear DNA tail attached to a circular chromosome. Segregation of the linear and circular parts of this "σ-replicating chromosome" causes terminus DNA breakage during cell division. One daughter cell inherits a truncated linear chromosome and is not viable. The other inherits a circular chromosome attached to a linear tail ending in the chromosome terminus. Replication extends this tail, while degradation of its extremity results in terminus DNA loss. Repeated generation and segregation of new σ-replicating chromosomes explains the heritability of post-replicative breakage. Our results allow us to determine that in E. coli at each generation, 18% of cells are subject to replication fork breakage at dispersed, potentially random, chromosomal locations.

Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme.

Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type) or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant.

Mutations Affecting Potassium Import Restore the Viability of the Escherichia coli DNA Polymerase III holD Mutant.

Mutants lacking the ψ (HolD) subunit of the Escherichia coli DNA Polymerase III holoenzyme (Pol III HE) have poor viability, but a residual growth allows the isolation of spontaneous suppressor mutations that restore ΔholD mutant viability. Here we describe the isolation and characterization of two suppressor mutations in the trkA and trkE genes, involved in the main E. coli potassium import system. Viability of ΔholD trk mutants is abolished on media with low or high K+ concentrations, where alternative K+ import systems are activated, and is restored on low K+ concentrations by the inactivation of the alternative Kdp system. These findings show that the ΔholD mutant is rescued by a decrease in K+ import. The effect of trk inactivation is additive with the previously identified ΔholD suppressor mutation lexAind that blocks the SOS response indicating an SOS-independent mechanism of suppression. Accordingly, although lagging-strand synthesis is still perturbed in holD trkA mutants, the trkA mutation allows HolD-less Pol III HE to resist increased levels of the SOS-induced bypass polymerase DinB. trk inactivation is also partially additive with an ssb gene duplication, proposed to stabilize HolD-less Pol III HE by a modification of the single-stranded DNA binding protein (SSB) binding mode. We propose that lowering the intracellular K+ concentration stabilizes HolD-less Pol III HE on DNA by increasing electrostatic interactions between Pol III HE subunits, or between Pol III and DNA, directly or through a modification of the SSB binding mode; these three modes of action are not exclusive and could be additive. To our knowledge, the holD mutant provides the first example of an essential protein-DNA interaction that strongly depends on K+ import in vivo.

Familial hypercholesterolaemia in UK primary care: a Clinical Practice Research Datalink study of an under-recognised condition.

BACKGROUND: Studies utilising genotyping methods report the prevalence of familial hypercholesterolaemia to be as high as one in 137 of the adult population. AIM: To estimate the prevalence of familial hypercholesterolaemia measured by clinically coded diagnosis, associated treatments, and lipid measurements observed in UK primary care. DESIGN AND SETTING: This was a retrospective analysis using the Clinical Practice Research Datalink (CPRD) GOLD database. METHOD: Patients aged ≥18 years and actively registered on the index date (30 June 2018) formed the study cohort. Point prevalence of familial hypercholesterolaemia for 2018 was estimated overall and for each nation of the UK. Patients with familial hypercholesterolaemia were stratified into primary and secondary prevention groups, defined as those with/without a prior diagnosis of atherosclerotic cardiovascular disease. Prevalence estimates and extrapolations were replicated for these subgroups. Baseline demographic, lipid, and clinical characteristics for the prevalent cohort were presented. RESULTS: In total, 4048 patients with familial hypercholesterolaemia formed the study cohort. The estimated familial hypercholesterolaemia prevalence for the UK was 16.4 per 10 000 (95% confidence interval [CI] = 16.0 to 16.9). Of these, 2646 (65.4%) patients with familial hypercholesterolaemia had a recent prescription for lipid-lowering therapy. Mean lipid levels were lower for those treated with lipid-lowering therapy compared with those untreated: 5.34 mmol/L (SD 1.50) versus 6.25 mmol/L (SD 1.55) for total cholesterol and 3.15 mmol/L (SD 1.34) versus 3.96 mmol/L (SD 1.36) for low-level density lipoprotein cholesterol. CONCLUSION: The estimated prevalence of familial hypercholesterolaemia was one in 608 of the population, less than expected from other studies, which may indicate that familial hypercholesterolaemia is under-recognised in UK primary care. Over one-third of diagnosed patients were undertreated and many did not achieve target goals, placing them at risk of cardiovascular events.

Risk of major adverse cardiovascular events associated with elevated low-density lipoprotein cholesterol in a population with atherosclerotic cardiovascular disease with and without type 2 diabetes: a UK database analysis using the Clinical Practice Research Datalink.

OBJECTIVES: To estimate the 12-month probabilities of major adverse cardiovascular events (MACE) and non-cardiovascular death in patients with atherosclerotic cardiovascular disease (ASCVD) and elevated low-density lipoprotein cholesterol (LDL-C). DESIGN: A retrospective database analysis. SETTING: UK primary care. PARTICIPANTS: Patients were selected from the Clinical Practice Research Datalink (Aurum) linked to Hospital Episode Statistics inpatient and Office of National Statistics mortality datasets. Patients with an ASCVD diagnosis between 01 January 2010 and 31 May 2018 and LDL-C ≥2.6 mmol/L were selected. PRIMARY OUTCOMES: Primary outcomes were 12-month risk of (1) MACE (composite of revascularisation, unstable angina, non-fatal myocardial infarction, non-fatal stroke and cardiovascular death) and (2) non-cardiovascular mortality. Kaplan-Meier survival analysis estimated the probability of each outcome. A Cox proportional hazards model explored covariates associated with MACE. RESULTS: Of 102 245 study patients, 16 501 (16.1%) had a diagnosis of type 2 diabetes (T2DM). 65.5% of those with and 49.9% of those without T2DM had a lipid-lowering therapy (LLT) 6 months prior to index diagnosis. Twelve-month probability of MACE was 7.9% for non-T2DM and 11.8% for T2DM. LDL-C was significantly associated with risk of MACE (HR=1.19 (95% CI 1.16 to 1.22) per 1 mmol/L). History of acute coronary syndrome, other coronary heart disease, stroke and T2DM significantly increased the risk of MACE. Ezetimibe (0.88 (95% CI 0.79 to 0.99)) and low-intensity statins (0.88 (95% CI 0.79 to 0.97)) were associated with reduced 12-month MACE risk.and low-intensity statins 0.88 (95% CI 0.79 to 0.97) CONCLUSION: We confirmed the association between elevated LDL-C and MACE. Many patients with ASCVD and elevated LDL-C were untreated with LLT. With the increasing demands on general practitioners, initiatives aimed at improving identification and treatment of at-risk patients within primary care should be considered.

Comparative efficacy of non-statin lipid-lowering therapies in patients with hypercholesterolemia at increased cardiovascular risk: a network meta-analysis.

OBJECTIVE: Network meta-analysis was used to derive estimates of the relative efficacy of inclisiran, evolocumab, alirocumab, bempedoic acid, and ezetimibe in patients with hypercholesterolemia and/or at increased cardiovascular risk due to elevated low-density lipoprotein cholesterol taking maximum tolerated dose statins. METHODS: Clinical trials published through February 2021 comparing percent change from baseline in low-density lipoprotein cholesterol were identified via a systematic review. Bayesian network meta-analyses were performed for patients with atherosclerotic cardiovascular disease and/or high cardiovascular risk on maximally tolerated statins in the base case, which included 23 trials. RESULTS: Results from the base-case analyses demonstrated that inclisiran, evolocumab, and alirocumab provide superior efficacy over placebo, bempedoic acid, and ezetimibe in terms of reduction in low-density lipoprotein cholesterol. Inclisiran was also comparable to alirocumab (mean difference: 0.78% [95% CrI: -8.35, 9.88]) and evolocumab (8.16%, [95% CrI: -1.82, 18.49]). Findings of a scenario which also included trials conducted in patients with heterozygous familial hypercholesterolemia were consistent with the base case. There was evidence of statistical heterogeneity across the included trials, roughly equivalent to variation of 5-10% change in low-density lipoprotein cholesterol, suggesting that any differences between treatments that were greater than 5-10% are generalizable. CONCLUSIONS: This study provides insight regarding the comparative efficacy of drugs for which no head-to-head trials exist and suggests that inclisiran, alirocumab, and evolocumab are expected to provide similar clinically meaningful improvements in low-density lipoprotein cholesterol in patients with hypercholesterolemia on maximally tolerated statins who are at increased cardiovascular risk.

H3K27me3 conditions chemotolerance in triple-negative breast cancer.

The persistence of cancer cells resistant to therapy remains a major clinical challenge. In triple-negative breast cancer, resistance to chemotherapy results in the highest recurrence risk among breast cancer subtypes. The drug-tolerant state seems largely defined by nongenetic features, but the underlying mechanisms are poorly understood. Here, by monitoring epigenomes, transcriptomes and lineages with single-cell resolution, we show that the repressive histone mark H3K27me3 (trimethylation of histone H3 at lysine 27) regulates cell fate at the onset of chemotherapy. We report that a persister expression program is primed with both H3K4me3 (trimethylation of histone H3 at lysine 4) and H3K27me3 in unchallenged cells, with H3K27me3 being the lock to its transcriptional activation. We further demonstrate that depleting H3K27me3 enhances the potential of cancer cells to tolerate chemotherapy. Conversely, preventing H3K27me3 demethylation simultaneously to chemotherapy inhibits the transition to a drug-tolerant state, and delays tumor recurrence in vivo. Our results highlight how chromatin landscapes shape the potential of cancer cells to respond to initial therapy.

Modeling the impact of patient treatment preference on health outcomes in relapsing-remitting multiple sclerosis.

Aims: Model how moving from current disease-modifying drug (DMD) prescribing patterns for relapsing-remitting multiple sclerosis (RRMS) observed in the United Kingdom (UK) to prescribing patterns based on patient preferences would impact health outcomes over time.Materials and methods: A cohort-based Markov model was used to measure the effect of DMDs on long-term health outcomes for individuals with RRMS. Data from a discrete choice experiment were used to estimate the market shares of DMDs based on patient preferences (i.e. preference shares). These preference shares and real-world UK market shares were used to calculate the effect of prescribing behavior on relapses, disability progression, and quality-adjusted life-years (QALYs). The incremental benefit of patient-centered prescribing over current practices for the UK RRMS population was then estimated; scenario and sensitivity analyses were also conducted.Results: Compared to current prescribing practices, when UK patients with RRMS were treated following patient preferences, health outcomes were improved. This population was expected to experience 501,690 relapses and gain 1,003,263 discounted QALYs over 50 years under patient-centered prescribing practices compared to 538,417 relapses and 958,792 discounted QALYs under current practices (-6.8% and +4.6%, respectively). Additionally, less disability progression was observed when prescribed treatment was based on patient preferences. In a scenario analysis where only oral treatments were considered, the results were similar, although the magnitude of benefit was smaller. Number of relapses was most sensitive to how the annualized relapse rate was modeled; disability progression was most sensitive to mortality rate assumptions.Limitations: Treatment efficacy estimates applied to various models in this study were based on data derived from clinical trials, rather than real-world data; the impact of patient-centered prescribing on treatment adherence and/or switching was not modeled.Conclusions: The population of UK RRMS patients may experience overall health gains if patient preferences are better incorporated into prescribing practices.

CD44 regulates epigenetic plasticity by mediating iron endocytosis.

CD44 is a transmembrane glycoprotein linked to various biological processes reliant on epigenetic plasticity, which include development, inflammation, immune responses, wound healing and cancer progression. Although it is often referred to as a cell surface marker, the functional regulatory roles of CD44 remain elusive. Here we report the discovery that CD44 mediates the endocytosis of iron-bound hyaluronates in tumorigenic cell lines, primary cancer cells and tumours. This glycan-mediated iron endocytosis mechanism is enhanced during epithelial-mesenchymal transitions, in which iron operates as a metal catalyst to demethylate repressive histone marks that govern the expression of mesenchymal genes. CD44 itself is transcriptionally regulated by nuclear iron through a positive feedback loop, which is in contrast to the negative regulation of the transferrin receptor by excess iron. Finally, we show that epigenetic plasticity can be altered by interfering with iron homeostasis using small molecules. This study reveals an alternative iron-uptake mechanism that prevails in the mesenchymal state of cells, which illuminates a central role of iron as a rate-limiting regulator of epigenetic plasticity.

High-throughput single-cell ChIP-seq identifies heterogeneity of chromatin states in breast cancer.

Modulation of chromatin structure via histone modification is a major epigenetic mechanism and regulator of gene expression. However, the contribution of chromatin features to tumor heterogeneity and evolution remains unknown. Here we describe a high-throughput droplet microfluidics platform to profile chromatin landscapes of thousands of cells at single-cell resolution. Using patient-derived xenograft models of acquired resistance to chemotherapy and targeted therapy in breast cancer, we found that a subset of cells within untreated drug-sensitive tumors share a common chromatin signature with resistant cells, undetectable using bulk approaches. These cells, and cells from the resistant tumors, have lost chromatin marks-H3K27me3, which is associated with stable transcriptional repression-for genes known to promote resistance to treatment. This single-cell chromatin immunoprecipitation followed by sequencing approach paves the way to study the role of chromatin heterogeneity, not just in cancer but in other diseases and healthy systems, notably during cellular differentiation and development.

Synthesis and properties of oligonucleotides containing a cholesterol thymidine monomer.

Highly selective base-pair recognition makes DNA a suitable building block for orderly self-assembled structures. For some applications in nanotechnology DNA complexes need to be fixed onto surfaces. To fulfil this requirement on lipid membranes we have synthesised a thymidine monomer modified with a cholesterol moiety. Solution studies show that the melting temperature (Tm) of the duplex, with adjacent cholesterols on each strand, is much higher than that of the unmodified duplex.

Modulation of density and orientation of amphiphilic DNA on phospholipid membranes. II. Vesicles.

In the present series of papers, we describe the results of a systematic study on the anchoring of cholesterol-tagged oligonucleotides to phospholipid bilayers followed by membrane-assisted hybridization of the complementary strand in solution. This paper compares the behavior of two cholesteryl modified oligonucleotides, differing in the architecture and hydrophobicity of the lipophilic moiety, in the self-aggregation, hybridization, and insertion in phospholipid vesicle membranes. We have focused our attention on a singly substituted derivative (SC-ON(1)) and a multicholesterol (MC-ON(1)) derivative, where the cholesteryl units are inserted at the desired positions along a noncoupling T-sequence. The self-aggregation properties in solution are also explicitly taken into account and evaluated as competitive with respect to the adsorption at fluid or solid interfaces and to hybridization with the complementary ON(2) sequence. By exploring a wide range of ON derivative concentrations, different peculiar scenarios emerge for different hydrophobicity of the amphiphilic DNA guest molecules on the vesicles, in terms of distribution and conformation of the single strand and consequent coupling properties with the complementary strand in solution.

Modulation of density and orientation of amphiphilic DNA anchored to phospholipid membranes. I. Supported lipid bilayers.

In the present series of papers, we describe the results of a systematic study on the anchoring of cholesterol-tagged oligonucleotides to phospholipids bilayers followed by membrane-assisted hybridization of the complementary strand in solution. This paper describes the anchoring of novel cholesterol-modified DNA-18mers in supported lipid bilayers (SLB) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine: we compared the behavior of two anchoring functionalities differing in the number of cholesterol units and in the length of a spacer group. Quartz Crystal Microbalance with impedance monitoring (QCM-Z) measurements showed that both oligonucleotides insert into the bilayer membrane through cholesterol anchoring; however, dramatic differences, in terms of surface organization and thickness, are found as the number of anchoring units increases. In the case of multiple cholesterol units, a peculiar three-regimes concentration dependence was revealed and correlated to the effective size of the adsorbing units. Interestingly, for high oligonucleotide concentration, the adsorption process was rationalized in terms of a compaction model of amphiphilic DNA molecules. QCM-Z temperature cycles of the SLB-anchored double strands provided clear evidence for reversible hybridization at the bilayer interface.