Effect of HIV infection and antiretroviral therapy initiation on genome-wide DNA methylation patterns.

BACKGROUND: Previous epigenome-wide association studies have shown that HIV infection can disrupt the host DNA methylation landscape. However, it remains unclear how antiretroviral therapy (ART) affects the HIV-induced epigenetic modifications. METHODS: 184 individuals with HIV from the NEAT001/ANRS143 clinical trial (with pre-ART and post-ART samples [96 weeks of follow-up]) and 44 age-and-sex matched individuals without HIV were included. We compared genome-wide DNA methylation profiles in whole blood between groups adjusting for age, sex, batch effects, and DNA methylation-based estimates of leucocyte composition. FINDINGS: We identified 430 differentially methylated positions (DMPs) between HIV+ pre-ART individuals and HIV-uninfected controls. In participants with HIV, ART initiation modified the DNA methylation levels at 845 CpG positions and restored 49.3% of the changes found between HIV+ pre-ART and HIV-uninfected individuals. We only found 15 DMPs when comparing DNA methylation profiles between HIV+ post-ART individuals and participants without HIV. The Gene Ontology enrichment analysis of DMPs associated with untreated HIV infection revealed an enrichment in biological processes regulating the immune system and antiviral responses. In participants with untreated HIV infection, DNA methylation levels at top HIV-related DMPs were associated with CD4/CD8 ratios and viral loads. Changes in DNA methylation levels after ART initiation were weakly correlated with changes in CD4+ cell counts and the CD4/CD8 ratio. INTERPRETATION: Control of HIV viraemia after 96 weeks of ART initiation partly restores the host DNA methylation changes that occurred before antiretroviral treatment of HIV infection. FUNDING: NEAT-ID Foundation and Instituto de Salud Carlos III, co-funded by European Union.

Inhaled ciclesonide for outpatient treatment of COVID-19 in adults at risk of adverse outcomes: a randomised controlled trial (COVERAGE).

OBJECTIVES: To assess the efficacy of inhaled ciclesonide in reducing the risk of adverse outcomes in COVID-19 outpatients at risk of developing severe illness. METHODS: COVERAGE is an open-label, randomized controlled trial. Outpatients with documented COVID-19, risk factors for aggravation, symptoms for ≤7 days, and absence of criteria for hospitalization are randomly allocated to either a control arm or one of several experimental arms, including inhaled ciclesonide. The primary efficacy endpoint is COVID-19 worsening (hospitalization, oxygen therapy at home, or death) by Day 14. Other endpoints are adverse events, maximal follow-up score on the WHO Ordinal Scale for Clinical Improvement, sustained alleviation of symptoms, cure, and RT-PCR and blood parameter evolution at Day 7. The trial's Safety Monitoring Board reviewed the first interim analysis of the ciclesonide arm and recommended halting it for futility. The results of this analysis are reported here. RESULTS: The analysis involved 217 participants (control 107, ciclesonide 110), including 111 women and 106 men. Their median age was 63 years (interquartile range 59-68), and 157 of 217 (72.4%) had at least one comorbidity. The median time since first symptom was 4 days (interquartile range 3-5). During the 28-day follow-up, 2 participants died (control 2/107 [1.9%], ciclesonide 0), 4 received oxygen therapy at home and were not hospitalized (control 2/107 [1.9%], ciclesonide 2/110 [1.8%]), and 24 were hospitalized (control 10/107 [9.3%], ciclesonide 14/110 [12.7%]). In intent-to-treat analysis of observed data, 26 participants reached the composite primary endpoint by Day 14, including 12 of 106 (11.3%, 95% CI: 6.0%-18.9%) in the control arm and 14 of 106 (13.2%; 95% CI: 7.4-21.2%) in the ciclesonide arm. Secondary outcomes were similar for both arms. DISCUSSION: Our findings are consistent with the European Medicines Agency's COVID-19 task force statement that there is currently insufficient evidence that inhaled corticosteroids are beneficial for patients with COVID-19.

Molecular profiling of advanced soft-tissue sarcomas: the MULTISARC randomized trial.

BACKGROUND: Soft-tissue sarcomas (STS) represent a heterogeneous group of rare tumors including more than 70 different histological subtypes. High throughput molecular analysis (next generation sequencing exome [NGS]) is a unique opportunity to identify driver mutations that can change the usual one-size-fits-all treatment paradigm to a patient-driven therapeutic strategy. The primary objective of the MULTISARC trial is to assess whether NGS can be conducted for a large proportion of metastatic STS participants within a reasonable time, and, secondarily to determine whether a NGS-guided therapeutic strategy improves participant's outcome. METHODS: This is a randomized, multicentre, phase II/III trial inspired by the design of umbrella and biomarker-driven trials. The setting plans up to 17 investigational centres across France and the recruitment of 960 participants. Participants aged at least 18 years, with unresectable locally advanced and/or metastatic STS confirmed by the French sarcoma pathological reference network, are randomized according to 1:1 allocation ratio between the experimental arm "NGS" and the standard "No NGS". NGS will be considered feasible if (i) NGS results are available and interpretable, and (ii) a report of exome sequencing including a clinical recommendation from a multidisciplinary tumor board is provided to investigators within 7 weeks from reception of the samples on the biopathological platform. A feasibility rate of more than 70% is expected (null hypothesis: 70% versus alternative hypothesis: 80%). In terms of care, participants randomized in "No NGS" arm and who fail treatment will be able to switch to the NGS arm at the request of the investigator. DISCUSSION: The MULTISARC trial is a prospective study designed to provide high-level evidence to support the implementation of NGS in routine clinical practice for advanced STS participants, on a large scale. TRIAL REGISTRATION: clinicaltrial.gov NCT03784014 .

Epigenetic age acceleration changes 2 years after antiretroviral therapy initiation in adults with HIV: a substudy of the NEAT001/ANRS143 randomised trial.

BACKGROUND: DNA methylation-based estimators of biological age are reliable biomarkers of the ageing process. We aimed to investigate a range of epigenetic ageing biomarkers in a substudy of the NEAT001/ANRS143 clinical trial, which compared ritonavir-boosted darunavir with either raltegravir or tenofovir disoproxil fumarate and emtricitabine in antiretroviral therapy (ART)-naive adults. METHODS: We analysed frozen whole blood samples from 168 ART-naive participants with HIV from the NEAT001/ANRS143 trial, before ART initiation and after 2 years of ART (84 participants on ritonavir-boosted darunavir with raltegravir and 84 participants on ritonavir-boosted darunavir with tenofovir disoproxil fumarate and emtricitabine). We also included 44 participants without HIV with a similar age and sex distribution. We analysed DNA methylation. Epigenetic age estimators (Horvath's clock, Hannum's clock, GrimAge, and PhenoAge) and estimated leucocyte compositions were generated using Horvath's New Online Methylation Age Calculator and Houseman's method. We calculated epigenetic age acceleration measures for each estimator of epigenetic age. The NEAT001/ANRS143 trial is registered with ClinicalTrials.gov, NCT01066962. FINDINGS: Compared with the HIV-uninfected group, ART-naive participants with HIV showed higher epigenetic age acceleration (EAA) according to all EAA estimators (mean 2·5 years, 95% CI 1·89-3·22 for Horvath-EAA; 1·4 years, 0·74-1·99 for Hannum-EAA; 2·8 years, 1·97-3·68 for GrimAge-EAA; and 7·3 years, 6·40-8·13 for PhenoAge-EAA), with all differences being statistically significant except for Hannum-EAA (Horvath-EAA p=0·0008; Hannum-EAA p=0·059; GrimAge-EAA p=0·0021; and PhenoAge-EAA p<0·0001). Epigenetic ageing was more pronounced in participants who had CD4 counts less than 200 cells per µL (significant for PhenoAge and Hannum's clock, p=0·0015 and p=0·034, respectively) or viral loads over 100 000 copies per mL at baseline (significant for PhenoAge, p=0·017). After 2 years of ART, epigenetic age acceleration was reduced, although PhenoAge and GrimAge remained significantly higher in participants with HIV compared with participants without HIV (mean difference 3·69 years, 95% CI 1·77-5·61; p=0·0002 and 2·2 years, 0·47-3·99; p=0·013, respectively). There were no significant differences in the ART effect on epigenetic ageing between treatment regimens. At baseline, participants with HIV showed dysregulation of DNA methylation-based estimated leucocyte subsets towards more differentiated T-cell phenotypes and proinflammatory leucocytes, which was also partly restored with ART. INTERPRETATION: ART initiation partly reversed epigenetic ageing associated with untreated HIV infection. Further studies are needed to understand the long-term dynamics and clinical relevance of epigenetic ageing biomarkers in people with HIV. FUNDING: NEAT-ID Foundation.

Correlation between blood telomere length and CD4+ CD8+ T-cell subsets changes 96 weeks after initiation of antiretroviral therapy in HIV-1-positive individuals.

In 31 participants who started first-line antiretroviral therapy in the NEAT 001/ANRS 143 clinical trial, we found after 96 weeks a statistically significant increase in blood telomere length (TL) of 0.04 (T/S Ratio) (p = 0.03). This increase was positively correlated with both the change in the percentage of CD4+ T-cells and with the decrease of CD38+ molecules on Central Memory CD8+ and negatively correlated with the change in the percentage of CD4+ Effector Memory cells. Increase in TL could be an expression of immune reconstitution and the associated decrease in immune activation. We acknowledge for the low statistical power due to the small sample size and the potential for false positive results due to multiple testing. Hence, further studies are needed to confirm these observations.

Body composition and adipokines changes after initial treatment with darunavir-ritonavir plus either raltegravir or tenofovir disoproxil fumarate-emtricitabine: A substudy of the NEAT001/ANRS143 randomised trial.

BACKGROUND: Comparison of changes in body composition, adipokines and inflammatory markers after initial therapy with a nucleos(t)ide reverse transcriptase inhibitor (N(t)RTI)- sparing or containing regimen are scarce. DESIGN: Randomised Clinical Trial. METHODS: This is the body composition substudy of NEAT 001/ANRS 143, a randomised trial comparing darunavir/ritonavir (DRV/r) plus either raltegravir (RAL) or tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) in 805 ART naïve HIV-infected adults. The primary endpoint was percentage change in limb fat at week 96. Secondary endpoints were associations among these changes and metabolic markers (IL-6, insulin, leptin, adiponectin, FGF-23). RESULTS: 126 subjects (61 DRV/r + RAL and 65 DRV/r + TDF/FTC) were included. The rate of change in BMI between groups for RAL versus TDF/FTC at week 96 was 1.5% per 48-week period (p = 0.015). The rate of change in limb fat mass, trunk fat mass, total body fat and total lean mass was for RAL versus TDF/FTC at week 96 was 2.5% (p = 0.38), 7.3% ((p = 0.021), 4.9% (p = 0.061) and 1.3% (p = 0.12) respectively. Baseline insulin and leptin levels were correlated with baseline limb fat and trunk fat mass [r = 0.31 (p = 0.0043)/r = 0.28 (p = 0.0011) for limb fat, and r = 0.63 (p<0.0001)/r = 0.50(p<0.0001) for trunk fat]. After adjustment, a 10% faster increase in leptin between baseline and week 48 was associated with a more rapid increase in limb fat at week 48 (0.5% per 48 weeks, p<0.001), total body fat mass (0.6% per 48 weeks, p<0.001), and trunk fat mass (0.3% per 48 weeks, p = 0.0026). CONCLUSIONS: After week 96 a N(t)RTI sparing regimen of DRV/r + RAL produced a numerically greater percentage increase in body composition variables with only change in trunk fat mass and BMI being significant.

Patient Self-Reported Adherence to Ritonavir-Boosted Darunavir Combined With Either Raltegravir or Tenofovir Disoproxil Fumarate/Emtricitabine in the NEAT001/ANRS143 Trial.

BACKGROUND: The NEAT001/ANRS143 trial demonstrated noninferiority of ritonavir-boosted darunavir combined with either raltegravir (RAL + DRV/r) or tenofovir disoproxil fumarate/emtricitabine (TDF/FTC + DRV/r) in HIV-positive, antiretroviral-naive adults. In post hoc analyses, however, RAL + DRV/r showed inferiority in patients with baseline CD4 <200/mm and HIV-1 RNA ≥100,000 copies per milliliter. This preplanned ancillary study was conducted to assess whether differences in adherence might explain efficacy results. SETTING: Phase III, open-label, randomized, multicenter study in 15 European countries (ClinicalTrials.gov, NCT01066962). METHODS: Seven hundred seventy-four participants self-reported adherence (modified AIDS Clinical Trials Group questionnaire) over 96 weeks [383 RAL + DRV/r (twice daily; 5 pills/day), 391 TDF/FTC + DRV/r (once daily; 4 pills/day)]. Primary endpoint was ≥95% versus <95% adherence to prescribed doses recorded (1) over the last 4 days or (2) on the visual analogue scale over the last 30 days. RESULTS: Characteristics, except age, were similar between arms; 9% had CD4 <200 cells/mm and HIV-1 RNA ≥100,000 copies per milliliter. Adherence ≥95% in the last 4 days (P = 0.029) or at the visual analogue scale (P = 0.0072) was higher with TDF/FTC + DRV/r than with RAL + DRV/r. Adherence ≥95% over the last 4 days was associated with lower probability of virological failure (P = 0.015). Adherence in patients with baseline CD4 <200 cells/mm and HIV-1 RNA ≥100,000 copies per milliliter was similar to the rest of the population, and not significantly associated with efficacy measures, with no significant differences between arms. CONCLUSION: Adherence was high and slightly better in the TDF/FTC + DRV/r than in the RAL + DRV/r arm. No convincing evidence was found that higher failure rate in the RAL + DRV/r arm in the subgroup with worse baseline viroimmunological status is caused by adherence differences.

Blood Telomere Length Changes After Ritonavir-Boosted Darunavir Combined With Raltegravir or Tenofovir-Emtricitabine in Antiretroviral-Naive Adults Infected With HIV-1.

Background: Tenofovir is a potent inhibitor of human telomerase. The clinical relevance of this inhibition is unknown. Methods: NEAT001/ANRS143 is a randomized trial that showed noninferiority over 96 weeks of ritonavir-boosted darunavir plus raltegravir versus tenofovir disoproxil fumarate/emtricitabine in 805 antiretroviral antiretrovrial-naive HIV-infected adults. We compared changes in whole-blood telomere length measured with quantitative polymerase chain reaction in 201 randomly selected participants (104 raltegravir and 97 tenofovir disoproxil fumarate/emtricitabine). We performed multivariable estimative and predictive linear regression. Results: At week 96, participants receiving tenofovir disoproxil fumarate/emtricitabine had a statistically significant higher gain in telomere length than participants receiving raltegravir. Difference in mean telomere length change between groups (tenofovir disoproxil fumarate/emtricitabine minus raltegravir) from baseline to week 96 adjusted by baseline telomere length was 0.031 (P = .009). This difference was not significantly confounded by age, gender, known duration of HIV infection, CD4 (baseline/nadir), CD8 cells, CD4/CD8 ratio, HIV viral load (baseline/week 96), tobacco and alcohol consumption, statins, or hepatitis C. Conclusion: Antiretroviral-naive HIV-infected adults receiving ritonavir-boosted darunavir and tenofovir disoproxil fumarate/emtricitabine had a significant higher gain in blood telomere length than those receiving ritonavir-boosted darunavir and raltegravir, suggesting a better initial recovery from HIV-associated immunosenescence.

Bone mineral density and inflammatory and bone biomarkers after darunavir-ritonavir combined with either raltegravir or tenofovir-emtricitabine in antiretroviral-naive adults with HIV-1: a substudy of the NEAT001/ANRS143 randomised trial.

BACKGROUND: Osteopenia, osteoporosis, and low bone mineral density are frequent in patients with HIV. We assessed the 96 week loss of bone mineral density associated with a nucleoside or nucleotide reverse transcriptase inhibitor (NtRTI)-sparing regimen. METHODS: Antiretroviral-naive adults with HIV were enrolled in 78 clinical sites in 15 European countries into a randomised (1:1), open-label, non-inferiority trial (NEAT001/ANRS143) assessing the efficacy and safety of darunavir (800 mg once per day) and ritonavir (100 mg once per day) plus either raltegravir (400 mg twice per day; NtRTI-sparing regimen) or tenofovir (245 mg once per day) and emtricitabine (200 mg once per day; standard regimen). For this bone-health substudy, 20 of the original sites in six countries participated, and any patient enrolled at one of these sites who met the following criteria was eligible: plasma viral loads greater than 1000 HIV RNA copies per mL and CD4 cell counts of fewer than 500 cells per µL, except in those with symptomatic HIV infection. Exclusion criteria included treatment for malignant disease, testing positive for hepatitis B virus surface antigen, pregnancy, creatinine clearance less than 60 mL per min, treatment for osteoporosis, systemic steroids, or oestrogen-replacement therapy. The two primary endpoints were the mean percentage changes in lumbar spine and total hip bone mineral density at week 48, assessed by dual energy x-ray absorptiometry (DXA) scans. We did the analysis with an intention-to-treat-exposed approach with antiretroviral modifications ignored. The parent trial is registered with ClinicalTrials.gov, number NCT01066962, and is closed to new participants. FINDINGS: Between Aug 2, 2010, and April 18, 2011, we recruited 146 patients to the substudy, 70 assigned to the NtRTI-sparing regimen and 76 to the standard regimen. DXA data were available for 129, 121 and 107 patients at baseline, 48 and 96 weeks respectively. At week 48, the mean percentage loss in bone mineral density in the lumbar spine was greater in the standard group than in the NtRTI-sparing group (mean percentage change -2.49% vs -1.00%, mean percentage difference -1.49, 95% CI -2.94 to -0.04; p=0.046). Total hip bone mineral density loss was similarly greater at week 48 in the standard group than in the NtRTI-sparing group (mean percentage change -3.30% vs -0.73%; mean percentage difference -2.57, 95% CI -3.75 to -1.35; p<0.0001). Seven new fractures occurred during the trial (two in the NtRTI-sparing group and five in the standard group). INTERPRETATION: A raltegravir-based regimen was associated with significantly less loss of bone mineral density than a standard regimen containing tenofovir disoproxil fumarate, and might be a treatment option for patients at high risk of osteopenia or osteoporosis who are not suitable for NtRTIs such as abacavir or tenofovir alafenamide. FUNDING: The European Union Sixth Framework Programme, Inserm-ANRS, Ministerio de Sanidad y Asuntos Sociales de España, Gilead Sciences, Janssen Pharmaceuticals, and Merck Laboratories.

Ritonavir-boosted darunavir combined with raltegravir or tenofovir-emtricitabine in antiretroviral-naive adults infected with HIV-1: 96 week results from the NEAT001/ANRS143 randomised non-inferiority trial.

BACKGROUND: Standard first-line antiretroviral therapy for HIV-1 infection includes two nucleoside or nucleotide reverse transcriptase inhibitors (NtRTIs), but these drugs have limitations. We assessed the 96 week efficacy and safety of an NtRTI-sparing regimen. METHODS: Between August, 2010, and September, 2011, we enrolled treatment-naive adults into this randomised, open-label, non-inferiority trial in treatment-naive adults in 15 European countries. The composite primary outcome was change to randomised treatment before week 32 because of insufficient virological response, no virological response by week 32, HIV-1 RNA concentration 50 copies per mL or higher at any time after week 32; death from any cause; any new or recurrent AIDS event; or any serious non-AIDS event. Patients were randomised in a 1:1 ratio to receive oral treatment with 400 mg raltegravir twice daily plus 800 mg darunavir and 100 mg ritonavir once daily (NtRTI-sparing regimen) or tenofovir-emtricitabine in a 245 mg and 200 mg fixed-dose combination once daily, plus 800 mg darunavir and 100 mg ritonavir once daily (standard regimen). This trial was registered with ClinicalTrials.gov, number NCT01066962. FINDINGS: Of 805 patients enrolled, 401 received the NtRTI-sparing regimen and 404 the standard regimen, with median follow-up of 123 weeks (IQR 112-133). Treatment failure was seen in 77 (19%) in the NtRTI-sparing group and 61 (15%) in the standard group. Kaplan-Meier estimated proportions of treatment failure by week 96 were 17·8% and 13·8%, respectively (difference 4·0%, 95% CI -0·8 to 8·8). The frequency of serious or treatment-modifying adverse events were similar (10·2 vs 8·3 per 100 person-years and 3·9 vs 4·2 per 100 person-years, respectively). INTERPRETATION: Our NtRTI-sparing regimen was non-inferior to standard treatment and represents a treatment option for patients with CD4 cell counts higher than 200 cells per µL. FUNDING: European Union Sixth Framework Programme, Inserm-ANRS, Gilead Sciences, Janssen Pharmaceuticals, Merck Laboratories.