Autoantibody to GNAS in Early Detection of Hepatocellular Carcinoma: A Large-Scale Sample Study Combined with Verification in Serial Sera from HCC Patients

The aim of this study was to explore the value of autoantibody to GNAS in the early detection of hepatocellular carcinoma (HCC). In a large-scale sample set of 912 participants (228 cases in each of HCC, liver cirrhosis (LC), chronic hepatitis B (CHB), and normal controls (NCs) groups), autoantibody to GNAS was detected with a positive result in 47.8% of HCC patients, which was significantly higher than that in patients with LC (35.1%), CHB (19.7%), and NCs (19.7%). Further analysis showed that the frequency of autoantibody to GNAS started increasing in compensated cirrhosis patients (37.0%) with a jump in decompensated cirrhosis patients (53.2%) and reached a peak in early HCC patients (62.4%). The increasing autoantibody response to GNAS in patients at different stages was closely associated with the progression of chronic liver lesions. The result from 44 human serial sera demonstrated that 5 of 11 (45.5%) HCC patients had elevated autoantibody to GNAS before and/or at diagnosis of HCC. Moreover, 46.1% and 62.4% of high positive rates in alpha-fetoprotein (AFP) negative and early-stage HCC patients can supplement AFP in early detection of HCC. These findings suggest that autoantibody to GNAS could be used as a potential biomarker for the early detection of HCC.

Paradoxical sex-specific patterns of autoantibody response to SARS-CoV-2 infection

Background Pronounced sex differences in the susceptibility and response to SARS-CoV-2 infection remain poorly understood. Emerging evidence has highlighted the potential importance of autoimmune activation in modulating the acute response and recovery trajectories following SARS-CoV-2 exposure. Given that immune-inflammatory activity can be sex-biased in the setting of severe COVID-19 illness, the aim of the study was to examine sex-specific autoimmune reactivity to SARS-CoV-2 in the absence of extreme clinical disease. Methods In this study, we assessed autoantibody (AAB) reactivity to 91 autoantigens previously linked to a range of classic autoimmune diseases in a cohort of 177 participants (65% women, 35% men, mean age of 35) with confirmed evidence of prior SARS-CoV-2 infection based on presence of antibody to the nucleocapsid protein of SARS-CoV-2. Data were compared to 53 pre-pandemic healthy controls (49% women, 51% men). For each participant, socio-demographic data, serological analyses, SARS-CoV-2 infection status and COVID-19 related symptoms were collected by an electronic survey of questions. The symptoms burden score was constructed based on the total number of reported symptoms (N = 21) experienced within 6 months prior to the blood draw, wherein a greater number of symptoms corresponded to a higher score and assigned as more severe burden. Results In multivariable analyses, we observed sex-specific patterns of autoreactivity associated with the presence or absence (as well as timing and clustering of symptoms) associated with prior COVID-19 illness. Whereas the overall AAB response was more prominent in women following asymptomatic infection, the breadth and extent of AAB reactivity was more prominent in men following at least mildly symptomatic infection. Notably, the observed reactivity included distinct antigens with molecular homology with SARS-CoV-2. Conclusion Our results reveal that prior SARS-CoV-2 infection, even in the absence of severe clinical disease, can lead to a broad AAB response that exhibits sex-specific patterns of prevalence and antigen selectivity. Further understanding of the nature of triggered AAB activation among men and women exposed to SARS-CoV-2 will be essential for developing effective interventions against immune-mediated sequelae of COVID-19.

Paraneoplastic neurological syndromes: a practical approach to diagnosis and management

Paraneoplastic neurological syndromes (PNS) are the immune-mediated effects of a remote cancer and are characterised by an autoantibody response against antigens expressed by the tumour. Classically, well-characterised ‘onconeuronal’ antibodies target intracellular antigens and hence cannot access their antigens across intact cell membranes. The pathogenic mediators are likely to be neuronal-specific T cells. There is a variable response to immunotherapies and the clinical syndrome helps to direct the search for a specific set of tumours. By contrast, many newly emerging autoantibodies with oncological associations target cell surface epitopes and can exert direct pathogenic effects on both the central and peripheral nervous systems. Patients with these cell-surface directed autoantibodies often clearly respond to immunotherapies. Overall, the clinical, serological and oncological features in an individual patient helps determine the clinical relevance of the syndrome and hence guide its management. We summarise current knowledge and a practical approach to the investigation, diagnosis, treatment and outcomes of patients with suspected PNS.

Abstract P472: Edil3 Autoantibodies Do Not Distinguish Kawasaki Disease (KD) From Febrile Controls, But Are Acutely Lower In Children With Cardiac Involvement.

Kawasaki Disease (KD) is a childhood vasculitis, marked by prolonged fevers and coronary artery inflammation/aneurysms. Epidemiologic and demographic data support that a single preceding infectious agent may lead to KD, however, the cause remains unknown. The leading theory on pathogenesis is a post-infectious self-limiting autoimmune response, but this is still controversial. A recent study of individuals with KD demonstrated an association with autoantibody response to EDIL3 (also known as DEL-1). EDIL3 is a structural glycoprotein expressed by both macrophages and vascular endothelium. This presents an attractive potential target for investigation, as EDIL3 is known to modulate inflammation via leukocyte binding and infiltration through vessel walls. Additionally, EDIL3 is highly expressed in aortic and coronary artery tissue samples, showing some of the highest non-neural expression. Genetic variants of Del-1 have been associated with risk of intracranial aneurysms. Due to this burgeoning literature, we interrogated our cohort of plasma samples from febrile children, including Kawasaki disease, to confirm specificity of targeting to EDIL3. Plasma samples from febrile children and children with KD, including timecourse samples, were interrogated with ELISA assays using full-length recombinant EDIL3 (LSBio). In contrary to the published report, EDIL3 binding does not appear to be specific to KD when compared to febrile controls inclusive of a variety of disease etiologies (viral, bacterial, etc.). On serial samples (pre-IVIG, post-IVIG and convalescent) from the two different patient cohorts, antibodies binding EDIL3 are elevated in post-IVIG samples. This further supports general autoantibody reactivity against EDIL3 as IVIG is a pooled blood product from thousands of individuals typically. Intriguingly, in comparison of samples from children with or without any coronary artery Z scores above 2.5, EDIL3 autoantibody responses were actually lower in those with dilated coronary arteries. Current efforts are focused on identify specific human autoantibodies against EDIL3. Implications of these findings will be explored.

Serum Anti-PDLIM1 Autoantibody as Diagnostic Marker in Ovarian Cancer

BackgroundSerum autoantibodies (AAbs) against tumor-associated antigens (TAAs) could be useful biomarkers for cancer detection. This study aims to evaluate the diagnostic value of autoantibody against PDLIM1 for improving the detection of ovarian cancer (OC).MethodsImmunohistochemistry (IHC) test in tissue array containing 280 OC tissues, 20 adjacent tissues, and 8 normal ovarian tissues was performed to analyze the expression of PDLIM1 in tissues. Enzyme-linked immunosorbent assay (ELISA) was employed to measure the autoantibody to PDLIM1 in 545 sera samples from 182 patients with OC, 181 patients with ovarian benign diseases, and 182 healthy controls.ResultsThe results of IHC indicated that 84.3% (236/280) OC tissues were positively stained with PDLIM1, while no positive staining was found in adjacent or normal ovarian tissues. The frequency of anti-PDLIM1 autoantibody was significantly higher in OC patients than that in healthy and ovarian benign controls in both training (n=122) and validation (n=423) sets. The area under the curves (AUCs) of anti-PDLIM1 autoantibody for discriminating OC from healthy controls were 0.765 in training set and 0.740 in validation set, and the AUC of anti-PDLIM1 autoantibody for discriminating OC from ovarian benign controls was 0.757 in validation set. Overall, it was able to distinguish 35.7% of OC, 40.6% of patients with early-stage, and 39.5% of patients with late-stage. When combined with CA125, the AUC increased to 0.846, and 79.2% of OC were detected, which is statistically higher than CA125 (61.7%) or anti-PDLIM1(35.7%) alone (p<0.001). Also, anti-PDLIM1 autoantibody could identify 15% (18/120) of patients that were negative with CA125 (CA125 <35 U/ml).ConclusionsThe anti-PDLIM1 autoantibody response in OC patients was positively correlated with PDLIM1 high expression in OC tissues, suggesting that the autoantibody against PDLIM1 might have the potential to be a novel serological biomarker of OC, serving as a complementary measure of CA125, which could improve the power of OC detection.

Serum Peptide Immunoglobulin G Autoantibody Response in Patients with Different Central Nervous System Inflammatory Demyelinating Disorders

Previous efforts to discover new surrogate markers for the central nervous system (CNS) inflammatory demyelinating disorders have shown inconsistent results; moreover, supporting evidence is scarce. The present study investigated the IgG autoantibody responses to various viral and autoantibodies-related peptides proposed to be related to CNS inflammatory demyelinating disorders using the peptide microarray method. We customized a peptide microarray containing more than 2440 immobilized peptides representing human and viral autoantigens. Using this, we tested the sera of patients with neuromyelitis optica spectrum disorders (NMOSD seropositive, n = 6; NMOSD seronegative, n = 5), multiple sclerosis (MS, n = 5), and myelin-oligodendrocyte glycoprotein antibody-associated disease (MOGAD, n = 6), as well as healthy controls (HC, n = 5) and compared various peptide immunoglobulin G (IgG) responses between the groups. Among the statistically significant peptides based on the pairwise comparisons of IgG responses in each disease group to HC, cytomegalovirus (CMV)-related peptides were most clearly distinguishable among the study groups. In particular, the most significant differences in IgG response were observed for HC vs. MS and HC vs. seronegative NMOSD (p = 0.064). Relatively higher IgG responses to CMV-related peptides were observed in patients with MS and NMOSD based on analysis of the customized peptide microarray.

Mapping Autoantibodies in Children With Acute Rheumatic Fever

BackgroundAcute rheumatic fever (ARF) is a serious sequela of Group A Streptococcus (GAS) infection associated with significant global mortality. Pathogenesis remains poorly understood, with the current prevailing hypothesis based on molecular mimicry and the notion that antibodies generated in response to GAS infection cross-react with cardiac proteins such as myosin. Contemporary investigations of the broader autoantibody response in ARF are needed to both inform pathogenesis models and identify new biomarkers for the disease.MethodsThis study has utilised a multi-platform approach to profile circulating autoantibodies in ARF. Sera from patients with ARF, matched healthy controls and patients with uncomplicated GAS pharyngitis were initially analysed for autoreactivity using high content protein arrays (Protoarray, 9000 autoantigens), and further explored using a second protein array platform (HuProt Array, 16,000 autoantigens) and 2-D gel electrophoresis of heart tissue combined with mass spectrometry. Selected autoantigens were orthogonally validated using conventional immunoassays with sera from an ARF case-control study (n=79 cases and n=89 matched healthy controls) and a related study of GAS pharyngitis (n=39) conducted in New Zealand.ResultsGlobal analysis of the protein array data showed an increase in total autoantigen reactivity in ARF patients compared with controls, as well as marked heterogeneity in the autoantibody profiles between ARF patients. Autoantigens previously implicated in ARF pathogenesis, such as myosin and collagens were detected, as were novel candidates. Disease pathway analysis revealed several autoantigens within pathways linked to arthritic and myocardial disease. Orthogonal validation of three novel autoantigens (PTPN2, DMD and ANXA6) showed significant elevation of serum antibodies in ARF (p < 0.05), and further highlighted heterogeneity with patients reactive to different combinations of the three antigens.ConclusionsThe broad yet heterogenous elevation of autoantibodies observed suggests epitope spreading, and an expansion of the autoantibody repertoire, likely plays a key role in ARF pathogenesis and disease progression. Multiple autoantigens may be needed as diagnostic biomarkers to capture this heterogeneity.

POS0395 ANTI-ACETYLATED PROTEIN ANTIBODIES IN RHEUMATOID ARTHRITIS (RA): CLUES FOR THE STARTING POINT OF AUTOANTIBODY RESPONSES IN RA

Background:Rheumatoid arthritis (RA) is characterized by autoantibodies such as rheumatoid factor (RF) and anti-modified protein autoantibodies (AMPAs) like anti-citrullinated protein antibodies (ACPA) and anti-carbamylated protein antibodies (anti-CarP). Recently, another AMPA: anti-acetylated protein antibodies (AAPA) have been found in RA patients [1]. The prevalence of AAPA antibodies and their isotypes have yet to be determined. Since isotype profiles reflect the breadth of an immune response, the prevalence of AAPA isotypes in arthritis patients with and without RA can help to understand the relevance of this autoantibody response in RA.Objectives:To describe the prevalence of AAPA isotypes in arthritis patients with and without RA.Methods:In 650 RA patients fulfilling the 1987 RA criteria and 555 non-RA arthritis patients from the Leiden Early Arthritis Cohort, baseline serum samples were screened by ELISA for IgG, IgM and IgA to an acetylated- and control peptide that was based upon the CCP-2 backbone. The cutoff for positivity was based on 80 controls (mean + 2SD). A sample was considered positive if it was above the cutoff and was 0.1 optical density higher on the acetylated peptide than on the control peptide.Results:AAPA IgG was found in 36% of RA patients versus 6.7% of non-RA arthritis patients (figure 1a). Within RA patients, AAPA IgG antibodies were mostly present in the ACPA-(CCP-2) positive group (64% in ACPA-positive, compared to 5% in ACPA-negative). Levels of AAPA IgG and IgA were higher in RA patients than in healthy controls and non-RA arthritis patients (figure 1b), however, surprisingly, no difference in levels was found for IgM.When isotype profiles in AAPA- positive arthritis patients were compared, patients with RA were more often positive for two or more isotypes then patients without RA, and thus displayed considerably more overlap in AAPA isotypes compared to non-RA patients (table 1). Intriguingly, IgM AAPA was the most prevalent isotype in non-RA patients, versus IgG in RA patients.Table 1.Anti-acetylated protein antibody (AAPA) isotype overlap in AAPA positive patients.AAPA isotypeRA patients (=310) n (%)Non-RA arthritis patients (n=106) n (%)IgG+IgM-IgA-115 (37.1)28 (5.1)IgG-IgM+IgA-52 (16.8)48 (8.7)IgG-IgM-IgA+14 (4.5)13 (2.3)IgG+IgM+IgA-24 (7.7)3 (0.5)IgG+IgM-IgA+37 (11.9)4 (0.7)IgG-IgM+IgA+9 (2.9)8 (1.4)IgG+IgM+IgA+59 (19.0)2 (0.4)AAPA: anti-acetylated protein antibodies, RA: rheumatoid arthritisConclusion:AAPA are detected in one third of RA patients, and mainly in the ACPA-positive subgroup. The predominance of IgM AAPA in non-RA arthritis patients and healthy controls suggests that healthy persons can develop AAPA IgM without the development of RA. These results also suggest that in healthy individuals, AAPA responses can occur, but do not mature past the IgM-stage, while in RA patients, the AAPA-response does mature and might form a “starting point” for development of other AMPA leading to the concurrent present of several AMPA in disease.References:[1]Juarez, M., et al., Identification of novel antiacetylated vimentin antibodies in patients with early inflammatory arthritis. Ann Rheum Dis, 2016. 75(6): p. 1099-107.Disclosure of Interests:None declared

AB0668 THE ONSET OF RHEUMATOID ARTHRITIS AFTER COVID-19 – COINCIDENCE OR CONNECTED?

Background:COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been suggested to induce autoimmune phenomena. Multiple studies have reported the presence of autoantibodies in patients with COVID-19. Also the presence of anti-citrullinated protein antibodies (ACPA) and flaring of rheumatoid arthritis (RA) after COVID-19 has been described.[1, 2] Furthermore, in rheumatology clinics patients may present with polyarthritis compatible with RA shortly after SARS-CoV-2 infection. However, it is unclear how often ACPA occur after COVID-19 and whether preceding COVID-19 impacts on disease presentation of RA and phenotype of the ACPA response.Objectives:This study aims to determine the seroprevalence of ACPA after COVID-19 and to investigate the association between preceding COVID-19 infection and disease presentation of new-onset RA, including clinical phenotype and autoantibody response.Methods:To estimate the prevalence of ACPA after COVID-19 we measured ACPA IgG in samples from 61 patients visiting the specialized post-COVID outpatient clinic of the LUMC 5 weeks after hospitalization, using routine tests or in-house ELISA. Furthermore, we identified 5 patients presenting with polyarthritis compatible with RA after SARS-CoV-2 infection. To study the impact of COVID-19 on disease presentation, we examined clinical phenotype, autoantibody isotype positivity and ACPA IgG variable domain (V-domain) glycosylation of these patients and compared these features to regular RA patients. Autoantibody isotypes, including rheumatoid factor (RF) IgM/IgA, anti-CCP2 IgG/ IgM/IgA and anti-carbamylated protein antibodies (anti-CarP) IgG were measured using in-house ELISA’s. The percentage of V-domain glycosylation of purified ACPA IgG was measured with UHPLC.Results:None of the 61 post-COVID patients tested positive for ACPA 5 weeks after hospitalization, except two patients previously diagnosed with ACPA-positive RA. Thus, we could not observe an increase in ACPA-positivity shortly after COVID-19. Of the 5 patients who developed polyarthritis compatible with RA after SARS-CoV-2 infection, the average age was 63.6 years and 2/5 were female. 4/5 patients had been hospitalized due to severe COVID-19. On average, joint complaints started 6.6 weeks after infection, although two patients reported symptoms before infection. 4/5 patients fulfilled the ACR 2010 criteria for RA. Three patients (patient 1, 4, 5) were phenotypically very similar to regular new-onset RA patients. Patient 3 had a history of seronegative RA and had been in DMARD-free remission for 5 years. She flared 6 weeks after SARS-CoV-2 infection. Patient 2 had a remarkably different presentation. He was admitted with a suspected septic polyarthritis or pneumonia with reactive polyarthritis 6 weeks after COVID-19. ACPA level was low positive. The patient died unexpectedly after two days and autopsy revealed dilating myocarditis of unclear underlying cause. No causative pathogen could be identified.Previous studies have shown that RA-patients are most often either seronegative or triple-positive for RF, ACPA and anti-CarP antibodies. Autoantibody measurements on sera of the post-COVID polyarthritis patients revealed a similar pattern (Figure 1A) with two patients being completely seronegative, and three patients positive for a range of autoantibodies. In all post-COVID samples, the percentage of ACPA IgG V-domain glycosylation was significantly increased compared to total IgG (Figure 1B), similar as in regular RA.Conclusion:In conclusion, we found that the seroprevalence of ACPA is not increased post-COVID and that most patients presenting with polyarthritis after COVID-19 resemble regular RA patients, both regarding clinical phenotype and autoantibody characteristics. Although sample size and follow-up was limited, it appears that RA post-COVID may be coincidence rather than connected.References:[1]Vlachoyiannopoulos et al. Ann Rheum Dis, 2020.[2]Perrot et al. The Lancet Rheumatology, 2020.Disclosure of Interests:None declared.