Background: Adult-onset Still’s disease (AOSD) is a systemic autoinflammatory condition of unknown aetiology. AOSD is presumed to have a polygenic basis but there is genetic and clinical overlap with monogenic autoinflammatory disorders[1]. Genetic studies thus far have been limited and although a role for NLRP3 inflammasome in disease pathogenesis has been postulated, further research is needed to elucidate the precise aetiology. Objectives: Here, we conducted detailed genetic and inflammatory biomarker analysis of a large AOSD patient cohort to investigate the underlying pathology and identify novel targets for potential treatment. Methods: In this study we included 103 AOSD patients. We performed whole exome sequencing on 60 AOSD cases (at a mean combined read depth of approximately 100×), to identify rare germline variants and somatic mutations from virtual gene panels. Differential gene expression profiles between AOSD cases (n=27) and healthy controls (n=10) were investigated using Bulk RNAseq performed on whole blood. We also conducted cytokine profiling on the full cohort, and measured NLRP3 inflammasome activation using a custom assay and Type I Interferon (IFN) score using a novel method. The genetic landscape and biomarker datasets were compared to test whether there was a cumulative effect of rare genetic variants on the inflammatory disease signature. Results: We found a strong genetic association between AOSD (Figure 1), and the presence of rare germline variants (n=106, CADD>15, MAF<1%) and somatic mutations (n=26) within genes associated with monogenic autoinflammatory disorders and clonal haematopoiesis of indeterminate potential. Transcriptome analysis revealed 2834 differentially expressed genes, with significant enrichment in pathways associated with neutrophil degranulation and the innate immune system. Profiling of inflammatory markers (Figure 2) showed significantly elevated ASC/NLRP3 speck levels in AOSD compared to healthy controls (p=0.0001), and AOSD patients with resistant, arthritis predominant disease (p<0.0001). AOSD patients also showed significant elevation in several cytokines: IL-18 (p<0.0001), IL-6 (p<0.0001), IL-23 (p<0.0001), IL-12p70 (p=0.0005), IFNγ (p=0.0002) and IFN-α2 (p=0.0009). Likewise, the Type I IFN score was also significantly higher in AOSD compared to healthy controls (p=0.0015) and other autoinflammatory diseases. Conclusion: Our study demonstrated considerable genetic complexity within AOSD and the potential utility of the ASC/NLRP3 speck assay for disease stratification and a potential treatment target. Many enriched variants may not, by themselves, be sufficient to cause disease but could contribute to a polygenic model for AOSD. REFERENCES: [1] Giacomelli, R., Ruscitti, P. & Shoenfeld, Y. A comprehensive review on adult onset Still’s disease. J Autoimmun 93, 24-36, doi:10.1016/j.jaut.2018.07.018 (2018). Acknowledgements: Funding from European Union’s Horizon 2020 research and innovation programme (ImmunAID). Prof Sinisa Savic is supported by a senior fellowship from Kennedy Trust. Disclosure of Interests: Leon Chang: None declared, Joanne Topping: None declared, Fatima Nadat: None declared, James Poulter: None declared, Christopher Watson: None declared, Clive Carter: None declared, Linda Pournara: None declared, Jan Zernicke: None declared, Rebecca Ross: None declared, Catherine Cargo: None declared, Francesco Del Galdo: None declared, Jürgen Rech Speaker honoraria from BMS, Novartis and SOBI, Consulting fees from BMS, Novartis and SOBI, Research grant form Novartis, and Sobi, Bruno Fautrel Honoraria from AbbVie, Amgen, Biogen, BMS, Celltrion, Fresenius Kabi, Galapagos, Lilly, Medac, MSD, NORDIC Pharma, Novartis, Pfizer, Roche, Sandoz, SOBI, UCB, Viatris, Research grants from AbbVie, Lilly, MSD, Novartis, Pfizer, Eugen Feist Received speaker’s honorarium from Pfizer, Abbvie, Lilly, Galapagos, BMS, Sandoz, Novartis, Sobi, Roche, Janssen, Sanofi, UCB, Consultant of: Abbvie, Lilly, Galapagos, Sanofi, Novartis, Grant/research support from: Pfizer, BMS, Roche, Lilly; Novartis, Galapagos, Michael McDermott: None declared, Sinisa Savic Speaker’s honorarium Takeda, Novartis, SOBI, BioCryst; Research grants: SOBI, Novartis, CSL Behring, Received honorarium for participating on advisory boards from Novartis, Takeda, BioCryst, KalVista, Celldex Therapeutics, CSL Behring, SOBI, Pharming.Figure 1Rare germline variant analysis. A) Bar chart illustrating the number of rare germline variants identified in each AOSD case. B) Rare variant distribution within each gene panel category (CHIP-associated, Autoinflammation and Type I Interferonopathies). C) Bar charts illustrating the most frequently mutated genes within the cohort. Bar segments with a checkered pattern denote variants significantly enriched within the AOSD cohort. D) Bar charts indicating rare variants identified in multiple AOSD cases. Checkered bars indicate significantly enriched variants. Figure 2ASC/NLRP3 specks levels in AOSD and disease control cohorts. A) ASC/NLRP3 specks in healthy controls (HC, n= 30) were compared to AOSD (n=103), SIJA (n=12), Schnitzler (n=10), CAPS (n=11) and FMF (n=31) cohorts. B) Interferon (IFN) activity score. Boxplots comparing IFN scores between healthy controls and AOSD, SIJA, Schnitzler, CAPS and FMF cases. Statistical significance is denoted by * =p<0.05; ** =p<0.01; *** =p<0.001; **** =p<0.0001.

The precise link between inflammation and pathogenesis of myelodysplastic syndrome (MDS) is yet to be fully established. We developed a novel method to measure ASC/NLRP3 protein specks which are specific for the NLRP3 inflammasome only. We combined this with cytokine profiling to characterise various inflammatory markers in a large cohort of patients with lower risk MDS in comparison to healthy controls and patients with defined autoinflammatory disorders (AIDs). The ASC/NLRP3 specks were significantly elevated in MDS patients compared to healthy controls (p < 0.001) and these levels were comparable to those found in patients with AIDs. The distribution of protein specks positive only for ASC was different to ASC/NLRP3 ones suggesting that other ASC-containing inflammasome complexes might be important in the pathogenesis of MDS. Patients with MDS-SLD had the lowest levels of interleukin (IL)-1β, tumour necrosis factor (TNF), IL-23, IL-33, interferon (IFN) γ and IFN-α2, compared to other diagnostic categories. We also found that inflammatory cytokine TNF was positively associated with MDS progression to a more aggressive form of disease and IL-6 and IL-1β with time to first red blood cell transfusion. Our study shows that there is value in analysing inflammatory biomarkers in MDS, but their diagnostic and prognostic utility is yet to be fully validated.

Background: Cystic fibrosis (CF) is associated with increased resting energy expenditure. However, the introduction of elexacaftor/tezacaftor/ivacaftor (ETI) has resulted in a paradigm shift in nutritional status for many people with CF, with increase body mass index and reduction in the need for nutritional support. While these changes are likely to reflect improved clinical status and an associated downregulation of energy expenditure, they may also reflect drug-induced alterations in metabolic perturbations within CF cells. We hypothesise that some of these changes relate to normalisation of mitochondrial respiration in CF. Methods: Using wild-type (WT) and F508del/F508del CFTR human bronchial epithelial cell lines (HBE cell lines) and baby hamster kidney (BHK) cells we examined the impact of ETI on cellular metabolism. We monitored mitochondrial respiration, using Seahorse extracellular flux assays and monitored mitochondrial reactive oxygen species (mROS) and intracellular calcium levels by flow cytometry. Results: Increased mitochondrial respiration was found in HBE cell lines and BHK cells expressing CFTR F508del/F508del when assessing basal, maximal, spare respiratory capacities and ATP production, as well as increased mitochondrial ROS generated via forward electron transport. ETI significantly decreased basal, maximal, spare respiratory capacity and ATP production to WT levels or below. Calcium blocker, BAPTA-AM normalised mitochondrial respiration, suggesting a calcium-mediated mechanism. ETI decreased intracellular calcium levels in CF cells to the same extent as BAPTA-AM, highlighting the importance of calcium and chloride in mitochondrial respiration in CF. Conclusions: CF cell lines exhibit increased mitochondrial respiration, which can be downregulated by ETI therapy through mechanisms involving calcium.

OBJECTIVE:Adult-onset Still disease (AOSD) is a systemic autoinflammatory disorder (AID) of unknown etiology. Genetic studies have been limited. Here, we conducted detailed genetic and inflammatory biomarker analysis of a large cohort with AOSD to investigate the underlying pathology and identify novel targets for potential treatment. METHODS:We investigated AOSD cases (n = 60) for rare germline and somatic variants using whole exome sequencing with virtual gene panels. Transcriptome profiles were investigated by bulk RNA sequencing whole blood. Cytokine profiling was performed on an extended patient cohort (n = 106) alongside measurements of NLRP3 inflammasome activation using a custom assay and type I interferon (IFN) score using a novel method. RESULTS:We observed higher than expected frequencies of rare germline variants associated with monogenic AIDs in AOSD cases (AOSD 38.4% vs healthy controls [HCs] 20.4%) and earlier onset of putative somatic variants associated with clonal hematopoiesis of indeterminate potential. Transcriptome profiling revealed a positive correlation between Still Activity Score and gene expression associated with the innate immune system. ASC/NLRP3 specks levels and type I IFN scores were significantly elevated in AOSD cases compared with HCs (P = 0.0001 and 0.0015, respectively), in addition to several cytokines: interleukin (IL)-6 (P < 0.0001), IL-10 (P < 0.0075), IL-12p70 (P = 0.0005), IL-18 (P < 0.0001), IL-23 (P < 0.0001), IFN-α2 (P = 0.0009), and IFNγ (P = 0.0002). CONCLUSION:Our study shows considerable genetic complexity within AOSD and demonstrates the potential utility of the ASC/NLRP3 specks assay for disease stratification and targeted treatment. The enriched genetic variants identified may not by themselves be sufficient to cause disease, but may contribute to a polygenic model for AOSD.

In 2006, a linear immunological continuum of autoinflammatory and autoimmune disorders ranging from monogenic diseases of innate immunity at one end to monogenic diseases of adaptive immunity at the other end was proposed to classify these conditions. Deep immunophenotyping has now revealed a cell-based nosology of these disorders.

Rheumatoid arthritis (RA) is a relatively common systemic autoimmune disease with an estimated prevalence of approximately 1% worldwide. Patients present predominantly with symmetrical small joint inflammatory arthritis, which involves dysregulated immune responses, leading to bone and cartilage deformities due to extensive erosive damage. The introduction of biological based therapies for the management of this life-altering condition, over the past three decades, has led to marked improvements in patients’ quality of life. A wide range of both innate and adaptive immune cells are involved in the pathogenesis of RA, with a complex interplay of cytokines, T-cells, B-cells, and dendritic cells. Some of these cells have been successfully targeted in the treatment of RA by the use of biologics-based therapies. For example, rituximab therapy blocks B cell activation and abatacept effectively blocks T cell activation in patients with RA. Despite these advances, there remain some patients who are resistant to all current therapeutic options, which has encouraged further research into understanding the primary signal transduction pathways that mediate the disease. In this review we discuss the roles of the main signalling pathways, including metabolic reprogramming that have been implicated in RA disease progression, in order to develop a conceptual framework for more precise deployment of existing therapies, and to provide a rationale for producing molecular inhibitors of these pathways. Improved knowledge of the many intracellular signalling pathways in RA will complement current precision medicine strategies, particularly for the patients with difficult-to-treat RA, and especially in those with multidrug resistance disease.

Inflammation is a key driver in the pathogenesis of cystic fibrosis (CF). We assessed the effectiveness of elexacaftor/tezacaftor/ivacaftor (ETI) therapy on downregulating systemic and immune cell-derived inflammatory cytokines. We also monitored the impact of ETI therapy on clinical outcome. Adults with CF, heterozygous for F508del (n = 19), were assessed at baseline, one month and three months following ETI therapy, and clinical outcomes were measured, including sweat chloride, lung function, weight, neutrophil count and C-reactive protein (CRP). Cytokine quantifications were measured in serum and following stimulation of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) and adenosine triphosphate and analysed using LEGEND plex™ Human Inflammation Panel 1 by flow cytometry (n = 19). ASC specks were measured in serum and caspase-1 activity and mRNA levels determined from stimulated PBMCs were determined. Patients remained stable over the study period. ETI therapy resulted in decreased sweat chloride concentrations (p < 0.0001), CRP (p = 0.0112) and neutrophil count (p = 0.0216) and increased percent predicted forced expiratory volume (ppFEV1) (p = 0.0399) from baseline to three months, alongside a trend increase in weight. Three months of ETI significantly decreased IL-18 (p< 0.0011, p < 0.0001), IL-1β (p<0.0013, p = 0.0476), IL-6 (p = 0.0109, p = 0.0216) and TNF (p = 0.0028, p = 0.0033) levels in CF serum and following PBMCs stimulation respectively. The corresponding mRNA levels were also found to be reduced in stimulated PBMCs, as well as reduced ASC specks and caspase-1 levels, indicative of NLRP3-mediated production of pro-inflammatory cytokines, IL-1β and IL-18. While ETI therapy is highly effective at reducing sweat chloride and improving lung function, it also displays potent anti-inflammatory properties, which are likely to contribute to improved long-term clinical outcomes.

Background Producing transparent interpretable algorithms summarizing clinical trial outcomes to accurately predict individual patient’s responses would be a significant advance. We hypothesized that software designed to analyze biomedical data, based on evolutionary computation (EC), could produce summary algorithmic biomarkers from a clinical trial, predictive of individual responses to therapy. Methods and Findings A previously published randomized double-blind placebo controlled clinical trial was analyzed. Patients with active rheumatoid arthritis on a stable dose of methotrexate and naive to anti-tumor necrosis factor biologic therapy, were randomized to receive infliximab or placebo. The primary endpoint was synovial disease activity assessed by magnetic resonance imaging. Secondary endpoints included the Disease Activity Score 28 (DAS28). Baseline peripheral blood gene expression variable data were available for 59 patients, plus the treatment variable, infliximab or placebo, yielding a total of 52,379 baseline variables. The binary dependent variable for analysis was DAS28 response, defined by a decrease in DAS28 score of ≥1.2, at 14 weeks. At 14 weeks, 20 of the 30 patients receiving infliximab had responded, and ten of the 29 patients receiving placebo had responded. The software derived an algorithm, with 4 gene expression variables plus treatment assignment and 12 mathematical operations, that correctly predicted responders versus non-responders for all 59 patients with available gene expression data, giving 100% accuracy, 100% sensitivity and 100% specificity. We present the algorithm to provide transparency and to enable verification. Excluding the 4 gene expression variables, we then derived similarly predictive algorithms with 4 other gene expression variables. We hypothesized that the software could derive algorithms as predictors of treatment response to anti-tumor necrosis factor biologic therapy using just these 8 gene expression variables using previously published independent datasets from 6 rheumatoid arthritis studies. In each validation analysis the accuracy of the predictors we derived surpassed those previously reported by the original study authors. Conclusions and Relevance Software based on EC summarized the outcome of a clinical trial, with transparent biomarker algorithms correctly predicted the clinical outcome for all 59 RA patients. The biomarker variables were validated in 6 independent RA cohorts. This approach simplifies and expedites the development of algorithmic biomarkers accurately predicting individual treatment response, thereby enabling the deployment of precision medicine, and, in the future, providing a basis for dynamic labeling of prescription drugs. Original Trial Registration used for analysis: ClinicalTrials.gov registration: [NCT01313520][1]### Competing Interest StatementCompeting interests: In accordance with the journal?s policy we report that the authors of this manuscript have the following competing interests: - PL is the Chief Executive Officer, a founder and employee of Liquid Biosciences. - PL is a Director of Ignite Biomedical. - KH is an unpaid advisor to Liquid Biosciences. - PL and KH own stock in Liquid Biosciences. - MMcD, DH and VS have no competing interests. - There are no additional declarations from the authors relevant to this research relating to employment, consultancy, products in development, patents, or revenues from marketed products to declare. - Ignite Biomedical is developing a predictive test based on the biomarkers reported in the manuscript.### Clinical TrialN/A because all the data used were de-identified and publicly available in prior publications.### Funding StatementYes### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Not applicable because all the data used were de-identified and publicly available, neither ethics committee approval nor informed consent were required.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesThe authors confirm that the data underlying the findings are fully available without restriction from the Gene Expression Omnibus archive: GEO accession GSE58795, GSE5392, GSE12051, GSE15258, GSE33377, GSE78068 and GSE20690. Other relevant data are in the paper and Supporting Information files. We have also made the pivotal discovery algorithm in the manuscript available in different formats and also the provided the data for the 4 gene expression variables it contains available in the Supporting Information Files to facilitate validation. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01313520&atom=%2Fmedrxiv%2Fearly%2F2024%2F01%2F30%2F2024.01.29.24301910.atom

Objectives: Malnutrition is a hallmark of disease in cystic fibrosis (CF). The introduction of elexacaftor/tezacaftor/ivacaftor (ETI) has been associated with increased body mass index (BMI). Preliminary data from a UK cohort study (Igloo-CF), suggests that while BMI increases with ETI therapy, energy intake decreases. We hypothesised that ETI could normalise cellular metabolism in CF, and herein, we assess bioenergetics in two different drug exposed cell models each expressing wild-type (WT) or DF508/DF508 CFTR. Methods: Human bronchial epithelial cells (HBECs), NuLi-1 WT and CuFi-1 DF508/DF508 and baby hamster kidney cells (BKHs) BHK WT and BHK DF508/DF508 were cultured under basal conditions in the presence of ETI [E (3 µM), T (5/10 µM) and I (2.5/5 µM)] for 48 h. Extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) were measured using XFe96 Extracellular Flux Analyzer. L-lactate secretion and intracellular succinate levels were measured using colorimetric assay kits. Results: Increased mitochondrial metabolism was found in HBECs and BHK cells expressing the CFTR DF508/DF508 mutation when assessing maximal (p = 0.006) and spare respiratory capacities (p = 0.001). There were significant increases in cellular succinate levels (p <0.0001) and a trend towards elevated lactate. ETI significantly decreased glycolysis (p < 0.0001), maximal (p = 0.0002) and spare respiratory capacity (p = 0.0001) and intracellular succinate (p < 0.0001) to HC levels. BAPTA-AM and thapsigargin also normalised mitochondrial metabolism and succinate levels in CF cells, suggesting a calcium mediated mechanism. Conclusion: CF cell lines exhibit increased mitochondrial and glycolytic metabolism which can be downregulated by ETI therapy. These changes may impact the energy requirements of patients with CF and provide a partial potential explanation for the paradoxically increased BMI despite falling dietary energy input. We thank Anil Mehta for critical comments.

Background Since the development of the EULAR recommendations for the use of imaging in large vessel vasculitis (LVV) in 2017, new data has emerged in the field of imaging techniques and their application in the diagnosis and follow-up of patients with giant cell arteritis (GCA) and Takayasu arteritis (TAK). Objectives To summarize the evidence on different imaging techniques for diagnosis, monitoring, and outcome prediction in LVV in order to inform a EULAR task force updating the recommendations for imaging in LVV. Methods Systematic literature review (SLR) on studies published between 2017-2022 on ultrasound (US), magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET)-CT/MRI and fluorescein angiography in patients with LVV (PROSPERO registration CRD42022360545). Eligible study designs included randomized controlled trials and observational studies but excluded case-controlled studies. Two reviewers independently performed data extraction, synthesis, and risk of bias assessment. For studies on diagnosis, meta-analyses were performed using data from both the original and updated SLR whenever possible. Pooled sensitivities and specificities were obtained by fitting random effects models for all studies and for studies with low risk of bias separately. Meta-analyses were performed in R version 4.2.1. using the “lme4” package. The description of observations without inferences and the heterogeneity of reported data precluded any meta-analysis for outcome prediction or monitoring. Results A total of 4696 references were identified. Thirty-eight studies on GCA (n=32), TAK (n=2), and GCA and TAK (n=4) were included through the update, adding up to eighty-one studies from both SLRs. Pooled sensitivities and specificities for US, MRI and PET-CT using a clinical diagnosis of GCA as the reference standard are depicted in Table 1. No studies on the diagnostic value of imaging techniques were found for TAK. The US evaluation of patients with suspected GCA, including the assessment of both cranial and extracranial vessels, showed a higher pooled sensitivity (95%CI) (89% [73%-96%] vs 70% [59%-79%]) and similar specificity (95%CI) (91% [83%-95%] vs 91% [84%-94%]) compared to only including cranial vessels. Studies on outcome prediction (n=5) and monitoring (n=10) reported change of signs of vasculitis along with disease activity and proposed composite scores comprising several vessel territories for US, MRI and PET-CT. Conclusion US, MRI and PET-CT revealed a good performance for the diagnosis of GCA. Assessing both cranial and extracranial vessels with US leads to a higher pooled sensitivity with a similar pooled specificity compared to an assessment limited to cranial vessels. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests Philipp Bosch Speakers bureau: Janssen, Grant/research support from: Pfizer, Milena Bond: None declared, Christian Dejaco Consultant of: AbbVie, Eli Lilly, Galapagos, Janssen, Novartis, Pfizer, Roche, Sanofi, Grant/research support from: AbbVie, Cristina Ponte Consultant of: AbbVie, Vifor, Pfizer, AstraZeneca, Grant/research support from: AbbVie, Vifor, Pfizer, AstraZeneca, Sarah Mackie Speakers bureau: Roche/Chugai, Vifor and Pfizer, Consultant of: Roche/Chugai, Sanofi, AbbVie, AstraZeneca, Louise Falzon: None declared, Wolfgang A. Schmidt Speakers bureau: Chugai, Novartis,Roche, and Sanofi, Consultant of: Chugai, GSK, Novartis, Roche,and Sanofi, Sofia Ramiro Consultant of: AbbVie, Eli Lilly, Galapagos, MSD, Novartis, Pfizer, Sanofi, UCB, Grant/research support from: AbbVie, Eli Lilly, Galapagos, MSD, Novartis, Pfizer, Sanofi, UCB.Table 1Pooled sensitivities and specificities of diagnostic studies on GCA with clinical diagnosis as reference standardIndex testNumber of studiesPooled sensitivity (95%CI)Pooled specificity (95%CI)All studiesUS230.76 (0.66,0.83)0.91 (0.86,0.94)MRI80.82 (0.76,0.86)0.92 (0.84,0.97)PET-CT50.80 (0.70,0.87)0.91 (0.67,0.98)Low risk of bias studiesUS80.88 (0.83,0.92)0.96 (0.86,0.99)MRI30.81 (0.71,0.89)0.98 (0.89,1.00)PET-CT40.76 (0.67,0.83)0.95 (0.71,0.99)GCA, giant cell arteritis; MRI, magnetic resonance imaging; PET-CT, positron emission tomography – computed tomography; US, ultrasound

Drug resistance is one of the clinical challenges that limits the effectiveness of chemotherapy. Recent reports suggest that the unfolded protein response (UPR) and endoplasmic reticulum stress-adaptation signalling pathway, along with increased activation of its inositol-requiring enzyme 1α (IRE1α) arm, may be contributors to the pathogenesis of colorectal cancer (CRC). Here, we aimed to target the IRE1α/XBP1 pathway in order to sensitise CRC cells to the effects of chemotherapy. The CT26 colorectal cell line was treated with tunicamycin, and then was exposed to different concentrations of 5-fluorouracil (5-FU), either alone and/or in combination with the IRE1α inhibitor, 4µ8C. An MTT assay, flow cytometry and RT-PCR were performed to determine cell growth, apoptosis and IRE1α activity, respectively. In vivo BALB/c syngeneic colorectal mice received chemotherapeutic drugs. Treatment responses, tumour sizes and cytotoxicity were assessed via a range of pathological tests. 4µ8C was found to inhibit the growth of CRC, at a concentration of 10 µg/ml, without detectable cytotoxic effects and also significantly enhanced the cytotoxic potential of 5-FU, in CRC cells. In vivo experiments revealed that 4µ8C, at a concentration of 50 µM/kg prevented tumour growth without any cytotoxic or metastatic effects. Interestingly, the combination of 4µ8C with 5-FU remarkably enhanced drug responses, up to 40–60

V EXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a recently reported late-onset hematoinflammatory disorder occurring predominantly in older men, due to acquired mutations in the X-linked UBA1 gene. 1 An emerging category of hematoinflammatory disorders are broadly defined as diseases caused by somatic mutations restricted to the blood, but results in systemic inflammation with multiorgan involvement and are associated with abnormal and/or premalignant bone marrow (BM) changes. 2 The inflammatory manifestations are driven in part by activation of the NLRP3 inflammasome and release of proinflammatory cytokines such as interleukin (IL)-1 β , IL-18, and IL-6. 3 The hematological manifestations include vacuoles in erythroid and myeloid precursors, macrocytosis, and multilineage cytopenias. Most patients are diagnosed with clonal cytopenia of unknown significance, with 31%–50% of cases also fulfilling diagnostic criteria for myelodysplastic syndrome (MDS). 4,5 Typical inflammatory manifestations include fever, weight loss, skin lesions, lung disease, joint involvement, and inflammatory eye disease, while VEXAS can clinically mimic inflammatory syndromes such as Sweet’s syndrome (acute febrile neutrophilic dermatosis), relapsing poly-chondritis, and polyarteritis nodosa. 4,5 The best characterized and, to date, most common pathogenic variants lead to

Background The NLRP3 inflammasome is a critical multi-molecular platform involved in mediating innate immune responses, and could play a role in Adult onset Still’s disease (AOSD). Upon NLRP3 inflammasome activation, a large protein complex assembles, resulting in the re-localisation of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), secretion of IL-1β and IL-18, and ASC aggregation into a single inflammasome speck. ASC specks are considered as potential biomarkers for inflammatory disorders, with subsequent release into the bloodstream during pyroptosis. Somatic variants in NLRP3 have also been observed in phenocopies of autoinflammatory disorders and their role in AOSD has been postulated. Objectives To investigate the role of NLRP3-derived ASC protein specks as a potential disease activity biomarker and NLRP3 genetic variants in the pathogenesis of AOSD. Methods Sera from 103 AOSD patients [treatment naïve, on treatment, and patients who previously failed several bDMARDs and were involved in CONSIDER clinical trial (n=30)] were analysed. These were compared to Systemic Juvenile Idiopathic Arthritis (sJIA, n=14), Cryopyrin Associated Periodic Syndrome (CAPS, n=9), Schnitzler’s syndrome (n=10) and Familial Mediterranean Fever (FMF, n=27) patients. Extracellular NLRP3-derived ASC specks were identified using flow cytometry (double positivity for ASC-PE and NLRP3-APC), gating for events around 1μm in size, in sera. To determine if ASC/NLRP3 speck levels provide additional biological information, the correlation between the specks and known biomarkers (such as IL-18 and C-reactive protein (CRP)) were analysed in sera. This involved cytokine profiling, using 13-plex Inflammatory LEGENDplex assay and high-sensitivity CRP ELISAs. Deep whole exome sequencing (WES x100) (analysis restricted to autoinflammatory panel) was carried out on DNA from the 30 CONSIDER trial patients. Results In serological analyses, extracellular ASC/NLRP3 speck levels were increased in AOSD patients compared to 32 healthy control (HC) sera (p<0.01, Figure 1A). ASC/NLRP3 levels defined three subgroups of AOSD patients (low, moderate and high). High ASC/NLRP3 levels were present in all pre-treatment sera from CONSIDER trial patients (p< 0.001), compared to HC, suggesting their role may be dependent on the stage of the disease process. Interestingly, these patients were still responsive to canakinumab, showing significant reduction in extracellular ASC/NLRP3 levels in CONSIDER clinical trial cohort (baseline to week 12), compared to placebo (p<0.01) (Figure 1B) [1]. There was no correlation between ASC/NLRP3 specks and CRP levels (Figure 1C) or ASC/NLRP3 and total IL-18. No germline or somatic variants in NLRP3 were identified from patients in the CONSIDER cohort despite very high levels of ASC/NLRP3 specks being detected in their serum. Conclusion This study involved development of an assay that quantifies extracellular ASC specks as a biomarker of NLRP3 activation, to improve diagnoses and classification of SAIDs, particularly those with sporadic or unknown causes, such as AOSD. Increased levels of extracellular NLRP3-derived ASC specks were found in sera of AOSD compared to HC and autoinflammatory disease controls. Our findings also demonstrate heterogeneity within AOSD cohorts, with high-ASC speck levels in therapy-resistant CONSIDER trial patients suggesting the role of ASC specks may be dependent on the particular stage of the disease. Further analysis of rare somatic variants in genes associated with myelodysplastic syndrome, another condition associated with elevated ASC/NLRP3 specks and systemic inflammation, is currently underway, since this may provide an alternative explanation for our findings. Reference [1]Kedor C, Listing J, Zernicke J, ….. Feist E. Canakinumab for Treatment of Adult-Onset Still’s Disease to Achieve Reduction of Arthritic Manifestation (CONSIDER): phase II, randomised, double-blind, placebo-controlled, multicentre, investigator-initiated trial. Ann Rheum Dis. 2020;79(8):1090-1097. Acknowledgements ImmunAID, University of Leeds (LIRMM), CONSIDER clinical trial team and SOBI. Disclosure of Interests Joanne Topping: None declared, James Poulter: None declared, Fatima Nadat: None declared, Clive Carter: None declared, Jan Zernicke Grant/research support from: GALAPAGOS Biopharma Germany GmbH. The CONSIDER clinical trial study was sponsored by Novartis., ImmunAID consortium Speakers bureau: ImmunAID is a European consortium of researchers whose members have been paid speakers for (but not limited to); AbbVie, Amgen, Biogen, BMS, Celltrion, Fresenius Kabi, Galapagos, Gilead, Janssen, Lilly, Medac, MSD, NORDIC Pharma, Novartis, Pfizer, Roche, Sandoz, Sanofi-Genzyme, SOBI, UCB, Viatris, Takeda, BioCryst, CSL Behring., Consultant of: ImmunAID is a European consortium of researchers whose members have been paid consultants for (but not limited to); AbbVie, Amgen, Biogen, BMS, Celltrion, Fresenius Kabi, Galapagos, Gilead, Janssen, Lilly, Medac, MSD, NORDIC Pharma, Novartis, Pfizer, Roche, Sandoz, Sanofi-Genzyme, SOBI, UCB, Viatris, Takeda, CSL Behring, BioCryst, KalVista., Grant/research support from: ImmunAID is a European consortium of researchers whose members have received financial grants from (but not limited to); AbbVie, CSL Behring, Lilly, MSD, Novartis and Pfizer., Jürgen Rech Speakers bureau: Sobi and Novartis., Paid instructor for: Sobi and Novartis., Consultant of: Sobi and Novartis., Grant/research support from: Sobi and Novartis., Bruno Fautrel Speakers bureau: AbbVie, Amgen, Biogen, BMS, Celltrion, Fresenius Kabi, Galapagos, Gilead, Janssen, Lilly, Medac, MSD, NORDIC Pharma, Novartis, Pfizer, Roche, Sandoz, Sanofi-Genzyme, SOBI, UCB, Viatris., Consultant of: AbbVie, Amgen, Biogen, BMS, Celltrion, Fresenius Kabi, Galapagos, Gilead, Janssen, Lilly, Medac, MSD, NORDIC Pharma, Novartis, Pfizer, Roche, Sandoz, Sanofi-Genzyme, SOBI, UCB, Viatris., Grant/research support from: AbbVie, Lilly, MSD and Pfizer., Eugen Feist Speakers bureau: Abbvie, Galapagos, Lilly, Novartis, Pfizer, Sanofi, Sobi., Consultant of: Consultant for Abbvie, Galapagos, Lilly, Novartis, Sanofi., Grant/research support from: Galapagos, Lilly, Novartis, Pfizer. The CONSIDER clinical trial study was sponsored by Novartis., Michael McDermott: None declared, Sinisa Savic Speakers bureau: Novartis, Sobi, Takeda, BioCryst, CSL Behring., Consultant of: Novartis, SOBI, Takeda, CSL Behring, BioCryst, KalVista., Grant/research support from: Novartis, CSL Behring.

Background Imaging recommendations for primary large vessel vasculitis (LVV) were developed in 2018. Several new studies have emerged since then, and an update of the original statements was required. Objectives To update the recommendations for the use of imaging in LVV. Methods A systematic literature review update was performed to retrieve new evidence on ultrasound (US), magnetic resonance imaging (MRI), computed tomography (CT) and [18F]-fluorodeoxyglucose positron emission tomography (FDG-PET) for diagnosis, monitoring and outcome prediction in LVV. The task force consisted of 24 physicians, health professionals and patients from 14 countries. Results Three overarching principles and eight recommendations were agreed (Table 1). Compared to the 2018 version, US is now recommended as first line imaging test in all patients with suspected GCA, and axillary arteries should be included in the standard examination. As an alternative to US, cranial and extracranial arteries can be examined by FDG-PET or MRI. For Takayasu arteritis, MRI is the preferred imaging modality; CT or FDG-PET are alternatives. Although imaging is not routinely recommended for follow-up, US, FDG-PET or MRI may be used for assessing vessel abnormalities in LVV patients with suspected relapse, particularly when laboratory markers of inflammation are unreliable. MRA, CTA or US may be used for long-term monitoring of structural damage, particularly at sites of preceding vascular inflammation. Conclusion The 2023 recommendations provide up-to-date guidance for the role of imaging in the diagnosis and assessment of patients with (suspected) LVV. References NIL. Acknowledgments Funding provided by EULAR (Project number: QoC13). We would like to thank Louise Falzon for her help with the literature search strategy. We also thank Lorna Neill, Luca Cimino and Fabrizio Gozzi for their help with the update of the PICO questions. Disclosure of Interests Christian Dejaco Consultant of: AbbVie, Novartis, Janssen, Sanofi, Speakers bureau: Abbvie, Eli Lilly, Janssen, Novartis, Pfizer, Roche, Galapagos and Sanofi, Grant/research support from: AbbVie, Sofia Ramiro Consultant of: AbbVie, Eli Lilly, Galapagos, MSD, Novartis, Pfizer, Sanofi, UCB, Speakers bureau: AbbVie, Eli Lilly, Galapagos, MSD, Novartis, Pfizer, Sanofi, UCB, Grant/research support from: AbbVie, Eli Lilly, Galapagos, MSD, Novartis, Pfizer, Sanofi, UCB, Milena Bond Consultant of: AbbVie, Philipp Bosch Speakers bureau: Janssen, Grant/research support from: Pfizer, Cristina Ponte Consultant of: AbbVie, Sanofi, Novartis, Vifor, AstraZeneca, GlaxoSmithKline, and Roche, Speakers bureau: Vifor, AstraZeneca, GlaxoSmithKline, and Roche, Sarah Mackie Consultant of: Roche/Chugai, Sanofi, AbbVie, AstraZeneca, Sanofi, GSK, Sparrow, Speakers bureau: Roche/Chugai, Vifor, Pfizer and Novartis, Torsten Bley Consultant of: BioTel Research, Chugai, Guerbet, Novartis, Roche and Siemens Healthineers, Speakers bureau: BioTel Research, Chugai, Guerbet, Novartis, Roche, Sanofi and Siemens Healthineers, Daniel Blockmans Consultant of: Roche and GSK, Sara Brolin Grant/research support from: Novartis, Ertugrul Cagri Bolek: None declared, Rebecca Cassie: None declared, Maria C. Cid Consultant of: GSK, SCL-Vifor, AbbVie, AstraZeneca and Janssen, Grant/research support from: Kiniksa Pharmaceuticals, Juan Molina Collada Consultant of: Abbvie, Lilly, Janssen, Novartis, Pfizer, UCB, MSD, Speakers bureau: Abbvie, Lilly, Janssen, Novartis, Pfizer, UCB, MSD, Bhaskar Dasgupta Consultant of: Novartis, Abbvie,Roche,Chugai,Sanofi, Grant/research support from: Novartis, Abbvie,Roche,Chugai,Sanofi, Berit Dalsgaard NIelsen Consultant of: Roche and Novartis, Speakers bureau: Roche and Novartis, Eugenio de Miguel Consultant of: Novartis, AbbVie, Pfizer, Janssen, Lilly, Speakers bureau: Abbvie, Novartis, Pfizer, Roche, Janssen, Lilly, MSD, BMS, UCB, Grunental and Sanofi, Grant/research support from: Novartis, AbbVie, Pfizer, Janssen, Lilly, Haner Direskeneli Consultant of: Abbvie and Novartis, Grant/research support from: Pfizer, Amgene, Celltrion, UCB and Roche, Christina Duftner Consultant of: Abbvie, AOP Orphan, Astra-Zeneca, Bristol-Myers-Squibb, Eli-Lilly, Janssen, Galapagos, Merck-Sharp-Dohme, Novartis, Pfizer, Roche, Sandoz, UCB, Vifor, Speakers bureau: Abbvie, AOP Orphan, Astra-Zeneca, Bristol-Myers-Squibb, Eli-Lilly, Janssen, Galapagos, Merck-Sharp-Dohme, Novartis, Pfizer, Roche, Sandoz, UCB, Vifor, Grant/research support from: Eli-Lilly, Pfizer, UCB, ALOJZIJA HOCEVAR: None declared, Anna Moltó Consultant of: AbbVie, BMS, Biogen, Eli Lilly, Galapagos, Janssen, MSD, Novartis, and UCB, Grant/research support from: AbbVie, BMS, Biogen, Eli Lilly, Galapagos, Janssen, MSD, Novartis, and UCB, Valentin Schäfer: None declared, Luca Seitz Grant/research support from: iQone and Sandoz, Riemer Slart Grant/research support from: Siemens Healtineers and Pfizer, Wolfgang Schmidt Consultant of: Abbvie, Chugai, GlaxoSmithKline, Medac, Novartis, Roche, and Sanofi, Speakers bureau: Abbvie, Chugai, GlaxoSmithKline, Medac, Novartis, Roche, and Sanofi.Table 1Recommendations for the use of imaging in large vessel vasculitis in clinical practiceOverarching principlesA. In patients with suspected GCA, an early imaging test is recommended to support the clinical diagnosis of GCA, assuming high expertise and prompt availability of the imaging technique. Imaging should not delay initiation of treatment.B. Imaging examination should be done by a trained specialist using appropriate equipment, standardized operational procedures and settings.C. In patients in whom there is a high clinical suspicion of GCA and a positive imaging result, the diagnosis of GCA may be made without an additional test (biopsy or further imaging). In patients with a low clinical probability and a negative imaging result, the diagnosis of GCA can be considered unlikely. In all other situations (including the case of an inconclusive imaging result), additional efforts towards a diagnosis are necessary.Recommendations1. Ultrasound of temporal and axillary arteries should be considered as the first imaging modality to investigate mural inflammatory changes in patients with suspected GCA.2. High resolution MRI or FDG-PET can be used as alternatives to ultrasound for the assessment of cranial arteries in patients with suspected GCA.3. FDG-PET, alternatively MRI or CT, can be used for the detection of mural inflammation or luminal changes of extracranial arteries in patients with suspected GCA.4. In patients with suspected TAK, MRI to investigate mural inflammation or luminal changes should be used as the first imaging test to make a diagnosis of TAK.5. FDG-PET, CT or ultrasound may be used as alternative imaging modalities in patients with suspected TAK. Ultrasound is of limited value for assessment of the thoracic aorta.6. Conventional angiography is not recommended for the diagnosis of GCA or TAK as it has been superseded by the previously mentioned imaging modalities.7. In case of a suspected relapse of GCA or TAK, particularly when laboratory markers of disease activity are unreliable, ultrasound, FDG-PET or alternatively MRI may be considered for the assessment of vessel abnormalities. Imaging is not routinely recommended for patients in clinical and biochemical remission.8. In patients with GCA or TAK, MRA, CTA or ultrasound of extracranial vessels may be used for long-term monitoring of structural damage, particularly at sites of preceding vascular inflammation. The frequency of screening as well as the imaging method applied should be decided on an individual basis.