Unraveling the effect of proliferative stress in vivo in hematopoietic stem cell gene therapy mouse study

The hematopoietic system of patients enrolled in hematopoietic stem cells (HSC) gene therapy (GT) treatments is fully reconstituted upon autologous transplantation of engineered stem cells. HSCs highly proliferate up to full restoration of homeostasis and compete for niche homing and engraftment. The impact of the proliferation stress in HSC on genetic instability remains an open question that cured patients advocate for characterizing long-term safety and efficacy. The accumulation of somatic mutations has been widely used as a sensor of proliferative stress. Vector integration site (IS) can be used as a molecular tool for clonal identity, inherited by all HSC progeny, to uncover lineage dynamics in vivo at single-cell level. Here we characterized at single-clone granularity the proliferative stress of HSCs and their progeny over time by measuring the accumulation of mutations from the DNA of each IS. To test the feasibility of the approach, we set-up an experimental framework that combines tumor-prone Cdkn2a-/- and wild type (WT) mouse models of HSC-GT and molecular analyses on different hematopoietic cell lineages after transplantation of HSCs transduced with genotoxic LV (LV.SF.LTR) or GT-like non-genotoxic LV (SIN.LV.PGK). The Cdkn2a-/- mouse model provided the experimental conditions to detect the accumulation of somatic mutations, since the absence of p16INK4A and p19ARF enhances the proliferative potential of cells that have acquired oncogenic mutations. As expected, mice transplanted with Cdkn2a-/- Lin- cells marked with LV.SF.LTR (N=24) developed tumors significantly earlier compared to mock (N=20, p<0.0001), while mice treated with SIN. LV.PGK (N=23) did not. On the other side, mice that received WT Lin- cells treated with LV.SF.LTR (N=25) or SIN.LV.PGK (N=24) vector have not developed tumors. Given this scenario, we expect that Cdkn2a-/- Lin- cells transduced with LV.SF.LTR are associated with higher mutation rates compared to the SIN.LV.PGK group and wild type control mice. The composition of peripheral blood, lymphoid (B and T) and myeloid compartments was assessed by FACS on samples collected every 4 weeks and IS identification. More than 200,000 IS have been recovered. To identify the presence of somatic mutations, the genomic portions of sequencing reads flanking each different IS were analyzed with VarScan2. The accumulation rates of mutations have been evaluated by our new Mutation Index (MI) which normalizes the number of mutations by clones and coverage. Considering that a large portion of IS has been discarded since not covered by a minimum number of 5 unique reads (genomes), the remaining number of IS contained >90% of reads in each group. The MI increased over time in both LV.SF.LTR groups, with higher values for the Cdkn2a-/-. On the other hand, treatment with SIN.LV.PGK resulted in lower MI in both groups compared to LV.SF.LTR groups, reflecting the higher clonal composition of the cells treated with the SIN.LV.PGK and the phenomenon of insertional mutagenesis in the LV.SF.LTR. Moreover, the higher MI values of the SIN.LV.PGK Cdkn2a-/- group compared with the WT group proved the induction of DNA fragility. Our results showed that the analysis of the accumulation of somatic mutations at single clone unraveled HSC proliferation stress in vivo, combining for the first time the analysis of acquired mutations with IS. We are now applying our model to different clinical trials, and studying HSCs sub- clonal trees by symmetric divisions, previously indistinguishable by IS only. Our study will open the doors to in vivo long-term non-invasive studies of HSC stability in patients

HIV-1 mediated insertional activation of STAT5B promotes the formation of a viral reservoir in T regulatory cells

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Normalization of clonal diversity in gene therapy studies using shape constrained splines

Viral vectors are used to insert genetic material into semirandom genomic positions of hematopoietic stem cells which, after reinfusion into patients, regenerate the entire hematopoietic system. Hematopoietic cells originating from genetically modified stem cells will harbor insertions in specific genomic positions called integration sites, which represent unique genetic marks of clonal identity. Therefore, the analysis of vector integration sites present in the genomic DNA of circulating cells allows to determine the number of clones in the blood ecosystem. Shannon diversity index is adopted to evaluate the heterogeneity of the transduced population of gene corrected cells. However, this measure can be affected by several technical variables such as the DNA amount used and the sequencing depth of the library analyzed and therefore the comparison across samples may be affected by these confounding factors. We developed an advanced spline-regression approach that leverages on confounding effects to provide a normalized entropy index. Our proposed method was first validated and compared with two state of the art approaches in a specifically designed in vitro assay. Subsequently our approach allowed to observe the expected impact of vector genotoxicity on entropy level decay in an in vivo model of hematopoietic stem cell gene therapy based on tumor prone mice

Characteristics of Recurrent Ischemic Stroke after Embolic Stroke of Undetermined Source: Secondary Analysis of a Randomized Clinical Trial

Importance: The concept of embolic stroke of undetermined source (ESUS) unifies a subgroup of cryptogenic strokes based on neuroimaging, a defined minimum set of diagnostic tests, and exclusion of certain causes. Despite an annual stroke recurrence rate of 5%, little is known about the etiology underlying recurrent stroke after ESUS. Objective: To identify the stroke subtype of recurrent ischemic strokes after ESUS, to explore the interaction with treatment assignment in each category, and to examine the consistency of cerebral location of qualifying ESUS and recurrent ischemic stroke. Design, Setting, and Participants: The NAVIGATE-ESUS trial was a randomized clinical trial conducted from December 23, 2014, to October 5, 2017. The trial compared the efficacy and safety of rivaroxaban and aspirin in patients with recent ESUS (n = 7213). Ischemic stroke was validated in 309 of the 7213 patients by adjudicators blinded to treatment assignment and classified by local investigators into the categories ESUS or non-ESUS (ie, cardioembolic, atherosclerotic, lacunar, other determined cause, or insufficient testing). Five patients with recurrent strokes that could not be defined as ischemic or hemorrhagic in absence of neuroimaging or autopsy were excluded. Data for this secondary post hoc analysis were analyzed from March to June 2019. Interventions: Patients were randomly assigned to receive rivaroxaban, 15 mg/d, or aspirin, 100 mg/d. Main Outcomes and Measures: Association of recurrent ESUS with stroke characteristics. Results: A total of 309 patients (205 men [66%]; mean [SD] age, 68 [10] years) had ischemic stroke identified during the median follow-up of 11 (interquartile range [IQR], 12) months (annualized rate, 4.6%). Diagnostic testing was insufficient for etiological classification in 39 patients (13%). Of 270 classifiable ischemic strokes, 156 (58%) were ESUS and 114 (42%) were non-ESUS (37 [32%] cardioembolic, 26 [23%] atherosclerotic, 35 [31%] lacunar, and 16 [14%] other determined cause). Atrial fibrillation was found in 27 patients (9%) with recurrent ischemic stroke and was associated with higher morbidity (median change in modified Rankin scale score 2 [IQR, 3] vs 0 (IQR, 1]) and mortality (15% vs 1%) than other causes. Risk of recurrence did not differ significantly by subtype between treatment groups. For both the qualifying and recurrent strokes, location of infarct was more often in the left (46% and 54%, respectively) than right hemisphere (40% and 37%, respectively) or brainstem or cerebellum (14% and 9%, respectively). Conclusions and Relevance: In this secondary analysis of randomized clinical trial data, most recurrent strokes after ESUS were embolic and of undetermined source. Recurrences associated with atrial fibrillation were a minority but were more often disabling and fatal. More extensive investigation to identify the embolic source is important toward an effective antithrombotic strategy. Trial Registration: ClinicalTrials.gov Identifier: NCT02313909

Effectiveness of thrombectomy in stroke according to baseline prognostic factors: inverse probability of treatment weighting analysis of a population-based registry

Background and Purpose In real-world practice, the benefit of mechanical thrombectomy (MT) is uncertain in stroke patients with very favorable or poor prognostic profiles at baseline. We studied the effectiveness of MT versus medical treatment stratifying by different baseline prognostic factors. Methods Retrospective analysis of 2,588 patients with an ischemic stroke due to large vessel occlusion nested in the population-based registry of stroke code activations in Catalonia from January 2017 to June 2019. The effect of MT on good functional outcome (modified Rankin Score ≤2) and survival at 3 months was studied using inverse probability of treatment weighting (IPTW) analysis in three pre-defined baseline prognostic groups: poor (if pre-stroke disability, age >85 years, National Institutes of Health Stroke Scale [NIHSS] >25, time from onset >6 hours, Alberta Stroke Program Early CT Score 3), good (if NIHSS <6 or distal occlusion, in the absence of poor prognostic factors), or reference (not meeting other groups’ criteria). Results Patients receiving MT (n=1,996, 77%) were younger, had less pre-stroke disability, and received systemic thrombolysis less frequently. These differences were balanced after the IPTW stratified by prognosis. MT was associated with good functional outcome in the reference (odds ratio [OR], 2.9; 95% confidence interval [CI], 2.0 to 4.4), and especially in the poor baseline prognostic stratum (OR, 3.9; 95% CI, 2.6 to 5.9), but not in the good prognostic stratum. MT was associated with survival only in the poor prognostic stratum (OR, 2.6; 95% CI, 2.0 to 3.3). Conclusions Despite their worse overall outcomes, the impact of thrombectomy over medical management was more substantial in patients with poorer baseline prognostic factors than patients with good prognostic factors

Rivaroxaban versus aspirin for secondary prevention of ischaemic stroke in patients with cancer: a subgroup analysis of the NAVIGATE ESUS randomized trial

Background and purpose: Cancer is a frequent finding in ischaemic stroke patients. The frequency of cancer amongst participants in the NAVIGATE ESUS randomized trial and the distribution of outcome events during treatment with aspirin and rivaroxaban were investigated. Methods: Trial participation required a recent embolic stroke of undetermined source. Patients’ history of cancer was recorded at the time of study entry. During a mean follow-up of 11 months, the effects of aspirin and rivaroxaban treatment on recurrent ischaemic stroke, major bleeding and all-cause mortality were compared between patients with cancer and patients without cancer. Results: Amongst 7213 randomized patients, 543 (7.5%) had cancer. Of all patients, 3609 were randomized to rivaroxaban [254 (7.0%) with cancer] and 3604 patients to aspirin [289 (8.0%) with cancer]. The annual rate of recurrent ischaemic stroke was 4.5% in non-cancer patients in the rivaroxaban arm and 4.6% in the aspirin arm [hazard ratio (HR) 0.98, 95% confidence interval (CI) 0.78–1.24]. In cancer patients, the rate of recurrent ischaemic stroke was 7.7% in the rivaroxaban arm and 5.4% in the aspirin arm (HR 1.43, 95% CI 0.71–2.87). Amongst cancer patients, the annual rate of major bleeds was non-significantly higher for rivaroxaban than aspirin (2.9% vs. 1.1%; HR 2.57, 95% CI 0.67–9.96; P for interaction 0.95). All-cause mortality was similar in both groups. Conclusions: Our exploratory analyses show that patients with embolic stroke of undetermined source and a history of cancer had similar rates of recurrent ischaemic strokes and all-cause mortality during aspirin and rivaroxaban treatments and that aspirin appeared safer than rivaroxaban in cancer patients regarding major bleeds. www.clinicaltrials.gov (NCT02313909). © 2020 European Academy of Neurolog