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Ruxolitinib in Patients With Corticosteroid-Refractory or Corticosteroid-Dependent Chronic Graft-Versus-Host Disease: 3-Year Final Analysis of the Phase III REACH3 Study.
In REACH3 (ClinicalTrials.gov identifier: NCT03112603), ruxolitinib was investigated versus best available therapy (BAT) for 3 years in patients with steroid-refractory/dependent chronic graft-versus-host-disease (SR/D-cGVHD). Patients received ruxolitinib (10 mg twice daily) or BAT for 24 weeks; thereafter (weeks 24-156), patients continued randomized treatment, entered long-term survival follow-up, or crossed over from BAT to ruxolitinib. In 329 randomly assigned patients (ruxolitinib: 165; BAT: 164), the median failure-free survival (FFS) was 38.4 months for ruxolitinib versus 5.7 months for BAT (hazard ratio, 0.36 [95% CI, 0.27 to 0.49]). Median duration of response (DOR) was not reached for ruxolitinib versus 6.4 months for BAT. Ruxolitinib-treated patients had a higher probability of FFS (ruxolitinib: 56.5%; BAT: 18.2%) and maintaining a response (ruxolitinib: 59.6%; BAT: 26.7%) at 36 months. Median overall survival was not reached. Nonrelapse mortality and malignancy relapse/recurrence events were low. In 70 patients who crossed over to ruxolitinib, the overall response rate (50.0%) at week 24 and best overall response (81.4%) during the crossover period were consistent with the primary analysis of randomly assigned patients. No new safety signals were observed. Ruxolitinib provided longer FFS and DOR than BAT, demonstrating sustained efficacy and manageable safety over 3 years of follow-up in patients with SR/D-cGVHD.
Validation of the "Patient-Acceptable Symptom State" Question as Outcome Measure in AChR Myasthenia Gravis: A Multicentre, Prospective Study.
INTRODUCTION: Patient Acceptable Symptom State (PASS) is emerging as a valuable subjective measure of the overall myasthenia gravis (MG)-related burden. This study aimed at identifying PASS-positive thresholds for the most used clinical scales, investigating whether PASS and MGFA post-intervention status capture different aspects of the disease outcome, and identifying clinical variables associated with PASS=YES response. METHODS: Adult AChR-MG patients were prospectively enrolled at two Italian Centres (Rome: index cohort; Florence: validation cohort). PASS thresholds for MG-ADL, QMG, and MG-QOL15r were defined in the index cohort by ROC analysis and validated in the validation cohort; predictors of favorable PASS were identified by multivariable analysis. RESULTS: This study included 173 patients (44% females, median age at onset: 53 years). PASS=YES patients had significantly lower median MG-ADL, QMG, and MG-QOL15r scores, with the following thresholds for PASS=YES: MG ADL ≤ 2, QMG ≤ 8 and MG-QOL15r ≤ 6. The MG-ADL (OR = 0.46, 95% CI = 0.36-0.60, p
Engineering antisense oligonucleotides for targeted mRNA degradation through lysosomal trafficking.
Antisense oligonucleotides (ASOs) can modulate gene expression at the mRNA level, providing the ability to tackle conventionally undruggable targets and usher in an era of personalized medicine. A key mode of action for ASOs relies upon RNase H-engagement in the nucleus, however, most mature mRNA is present in the cytoplasm. This disconnect limits the efficacy and biomedical applications of ASOs. In this paper, we have established a new mechanism of action for achieving potent and targeted mRNA knockdown by leveraging a lysosomal degradation pathway. To achieve this, we employ autophagosome-tethering compound (ATTEC) technology that utilises bifunctional small molecules for lysosomal trafficking. In this manner, to induce degradation of target mRNA located in the cytoplasm, we conjugated an ATTEC warhead, ispinesib, to RNase H-inactive ASOs. These fully 2'-O-methylated RNase H-inactive ASOs have higher chemical stability and tighter mRNA binding than conventional 'gapmer' sequences, but cannot be recognised by RNase H. Using our lysosomal trafficking antisense oligonucleotide (LyTON) technology, we show significant lysosome-dependent knockdown of multiple molecular targets in various cell lines, via transfection and gymnotic uptake. The LyTON modification is also able to boost the knockdown efficacy of RNase H-active 'gapmer' ASOs. Engineered to degrade mRNA independent of RNase H recognition, LyTONs will enable gene silencing using oligonucleotide chemistries with higher chemical stability, tighter mRNA binding affinity, and improved cell delivery profiles. This will enable us to target a wider range of disease-relevant mRNA, potentially leading to the development of new therapies.
The emergence of Fanconi anaemia type S: a phenotypic spectrum of biallelic BRCA1 mutations.
BRCA1 is involved in the Fanconi anaemia (FA) pathway, which coordinates repair of DNA interstrand cross-links. FA is a rare genetic disorder characterised by bone marrow failure, cancer predisposition and congenital abnormalities, caused by biallelic mutations affecting proteins in the FA pathway. Germline monoallelic pathogenic BRCA1 mutations are known to be associated with hereditary breast/ovarian cancer, however biallelic mutations of BRCA1 were long predicted to be incompatible with embryonic viability, hence BRCA1 was not considered to be a canonical FA gene. Despite this, several patients with biallelic pathogenic BRCA1 mutations and FA-like phenotypes have been identified - defining a new FA type (FA-S) and designating BRCA1 as an FA gene. This report presents a scoping review of the cases of biallelic BRCA1 mutations identified to date, discusses the functional effects of the mutations identified, and proposes a phenotypic spectrum of BRCA1 mutations based upon available clinical and genetic data. We report that this FA-S cohort phenotype includes short stature, microcephaly, facial dysmorphisms, hypo/hyperpigmented lesions, intellectual disability, chromosomal sensitivity to crosslinking agents and predisposition to breast/ovarian cancer and/or childhood cancers, with some patients exhibiting sensitivity to chemotherapy. Unlike most other types of FA, FA-S patients lack bone marrow failure.
Clinical significance of preleukemic somatic GATA1 mutations in children with Down syndrome.
Children with Down syndrome (DS) have a high risk of GATA1-associated myeloid leukemia (ML-DS) before age 4. Somatic N-terminal GATA1 mutations (GATA1s) are necessary, but not sufficient, for ML-DS, but their significance at birth for individual babies and whether mutations occur after birth is unclear. To address these questions, we performed a prospective study of DS newborns using next-generation sequencing-based GATA1 mutation analysis with detailed hematologic and clinical evaluation and follow-up for the window of ML-DS risk. Of 450 DS neonates, 113 (25%) had GATA1s mutations of which 20/113 (17.7%) were multiple and 59 (52%) were clinically silent. Variant allele frequency (VAF) varied from 0.3-89%. VAF positively correlated (p<0.0001) with % blasts, leukocytes, dyserythro- and dysmegakaryopoiesis scores and clinical disease and negatively with hemoglobin, although only 4/113 were anemic. GATA1s mutations were detected from 28 weeks(w) gestation; the highest frequency (45%) was at 34-35w while mutation frequency in early fetal samples (<20w) was only <4% (2/57). GATA1s clones (VAF, % blasts) fell rapidly post-natally becoming undetectable by 6 months (6m) except in neonates who developed ML-DS. 7/110 surviving neonates (6.4%) developed ML-DS at a median age of 17.5m. GATA1s clone size at birth was the only predictor of subsequent ML-DS. No neonates lacking GATA1s mutations acquired mutations after birth or developed ML-DS. Taken together, the fetal environment is essential for GATA1s mutation selection and expansion of GATA1s clones. Rates of leukemic transformation of GATA1s clones detected at birth are low but clones that persist beyond 6 months transformed.
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
The immune-epithelial-stromal interactions underpinning intestinal damage in celiac disease (CD) are incompletely understood. To address this, we performed single-cell transcriptomics (RNA sequencing; 86,442 immune, parenchymal and epithelial cells; 35 participants) and spatial transcriptomics (20 participants) on CD intestinal biopsy samples. Here we show that in CD, epithelial populations shifted toward a progenitor state, with interferon-driven transcriptional responses, and perturbation of secretory and enteroendocrine populations. Mucosal T cells showed numeric and functional changes in regulatory and follicular helper-like CD4+ T cells, intraepithelial lymphocytes, CD8+ and γδ T cell subsets, with skewed T cell antigen receptor repertoires. Mucosal changes remained detectable despite treatment, representing a persistent immune-epithelial 'scar'. Spatial transcriptomics defined transcriptional niches beyond those captured in conventional histological scores, including CD-specific lymphoid aggregates containing T cell-B cell interactions. Receptor-ligand spatial analyses integrated with disease susceptibility gene expression defined networks of altered chemokine and morphogen signaling, and provide potential therapeutic targets for CD prevention and treatment.
Iron deficiency causes aspartate-sensitive dysfunction in CD8+ T cells.
Iron is an irreplaceable co-factor for metabolism. Iron deficiency affects >1 billion people and decreased iron availability impairs immunity. Nevertheless, how iron deprivation impacts immune cell function remains poorly characterised. We interrogate how physiologically low iron availability affects CD8+ T cell metabolism and function, using multi-omic and metabolic labelling approaches. Iron limitation does not substantially alter initial post-activation increases in cell size and CD25 upregulation. However, low iron profoundly stalls proliferation (without influencing cell viability), alters histone methylation status, gene expression, and disrupts mitochondrial membrane potential. Glucose and glutamine metabolism in the TCA cycle is limited and partially reverses to a reductive trajectory. Previous studies identified mitochondria-derived aspartate as crucial for proliferation of transformed cells. Despite aberrant TCA cycling, aspartate is increased in stalled iron deficient CD8+ T cells but is not utilised for nucleotide synthesis, likely due to trapping within depolarised mitochondria. Exogenous aspartate markedly rescues expansion and some functions of severely iron-deficient CD8+ T cells. Overall, iron scarcity creates a mitochondrial-located metabolic bottleneck, which is bypassed by supplying inhibited biochemical processes with aspartate. These findings reveal molecular consequences of iron deficiency for CD8+ T cell function, providing mechanistic insight into the basis for immune impairment during iron deficiency.
Iron and the immune system.
Iron is a cofactor for hundreds of enzymes and biochemical processes that support cellular metabolism across the kingdoms of life. Because of this, the host and pathogen compete for iron as a vital resource. Moreover, research has shown that iron acquisition and iron trafficking have substantial effects on the immune system. This is especially important because iron-related disorders - both deficiency and overload - are common worldwide. In this Review, we describe how immune cells acquire and use iron, which branches of the immune system are most affected by iron and how changes in iron availability can affect infectious diseases, autoinflammatory disorders and antitumour immunity. We also discuss key unanswered questions and potential therapeutic opportunities to manipulate immunity by controlling iron trafficking.
Continuous Indexing of Fibrosis (CIF): improving the assessment and classification of MPN patients.
The grading of fibrosis in myeloproliferative neoplasms (MPN) is an important component of disease classification, prognostication and monitoring. However, current fibrosis grading systems are only semi-quantitative and fail to fully capture sample heterogeneity. To improve the quantitation of reticulin fibrosis, we developed a machine learning approach using bone marrow trephine (BMT) samples (n = 107) from patients diagnosed with MPN or a reactive marrow. The resulting Continuous Indexing of Fibrosis (CIF) enhances the detection and monitoring of fibrosis within BMTs, and aids MPN subtyping. When combined with megakaryocyte feature analysis, CIF discriminates between the frequently challenging differential diagnosis of essential thrombocythemia (ET) and pre-fibrotic myelofibrosis with high predictive accuracy [area under the curve = 0.94]. CIF also shows promise in the identification of MPN patients at risk of disease progression; analysis of samples from 35 patients diagnosed with ET and enrolled in the Primary Thrombocythemia-1 trial identified features predictive of post-ET myelofibrosis (area under the curve = 0.77). In addition to these clinical applications, automated analysis of fibrosis has clear potential to further refine disease classification boundaries and inform future studies of the micro-environmental factors driving disease initiation and progression in MPN and other stem cell disorders.