Clinical and histological features of immune-mediated necrotising myopathy: a multi-centre South Australian cohort study

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Clinical and histological features of immune-mediated necrotising myopathy: a multi-centre South Australian cohort study Jessica Day , Sophia Otto , Kathy Cash , Vidya Limaye PII: DOI: Reference:

S0960-8966(20)30029-8 https://doi.org/10.1016/j.nmd.2020.02.003 NMD 3797

To appear in:

Neuromuscular Disorders

Received date: Revised date: Accepted date:

20 October 2019 23 December 2019 5 February 2020

Please cite this article as: Jessica Day , Sophia Otto , Kathy Cash , Vidya Limaye , Clinical and histological features of immune-mediated necrotising myopathy: a multi-centre South Australian cohort study, Neuromuscular Disorders (2020), doi: https://doi.org/10.1016/j.nmd.2020.02.003

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Highlights 

Immune-mediated necrotising myopathy is clinically and histologically heterogeneous

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Many patients present with severe weakness and few recover full power by one year

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Complement deposition on myofibres closely correlates with clinical severity

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Aboriginal and Torres Strait islander patients present with a severe phenotype

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Running Title: Characterising immune-mediated necrotising myopathy Clinical and histological features of immune-mediated necrotising myopathy: a multicentre South Australian cohort study. Jessica Day1. 2., Sophia Otto2. 3., Kathy Cash3., Vidya Limaye2. 4. 1. Experimental Therapeutics Laboratory, Hanson Institute, School of Pharmacy and Medical Science, University of South Australia, Adelaide, SA, Australia. 2. Royal Adelaide Hospital, Adelaide, SA, Australia. 3. SA Pathology, Adelaide, SA, Australia. 4. Discipline of Medicine, University of Adelaide, Adelaide, SA, Australia. Author Contributions: JD – hypothesis initiation and revision, research design, conducting experiments, acquiring data, analysing data, writing manuscript, acquiring funding VL – SAMD custodian, hypothesis initiation and revision, research design, acquiring data, manuscript revision, acquiring funding KC – hypothesis revision, experimental design, conducting experiments, laboratory supervision of JD, manuscript revision SO – hypothesis revision, acquiring data, manuscript revision, providing materials and laboratory space Corresponding author details: Dr Jessica Day [email protected]

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Abstract Immune-mediated necrotising myopathy (IMNM) is a recently described entity. We describe a cohort of South Australian IMNM patients in order to define the spectrum of disease, characterise features that distinguish IMNM from other idiopathic inflammatory myopathy (IIM) subtypes and identify factors associated with clinically severe disease. Subjects were identified from the South Australian Myositis Database (SAMD), a histologically defined registry. Consecutive muscle sections from patients with IMNM (n = 62), other forms of IIM (n = 60) and histologically normal muscle (n = 17) were stained using immunohistochemistry and graded. Clinical information was collected from the SAMD and through retrospective chart review. IMNM patients displayed clinical and histological heterogeneity. While most (67%) were profoundly weak at presentation, 24% exhibited mild-moderate weakness and 9% had normal power. Histological myonecrosis ranged from minor to florid. The amount of myofibre complement deposition was closely associated with clinical severity. Patients of Aboriginal and Torres Strait Islander heritage and those with anti-SRP autoantibodies present with a severe phenotype. Despite intense immunotherapy, few IMNM patients recovered full power at one year follow up. The identification of clinical, serological and histological features which are associated with severe forms of the disease may have diagnostic and therapeutic utility. Key Words: Myositis; Idiopathic Inflammatory Myopathy; Immune-mediated necrotising myopathy; Immune-mediated necrotizing myopathy; Necrotising autoimmune myopathy; necrotizing autoimmune myopathy; Indigenous health.

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1. Introduction The idiopathic inflammatory myopathies (IIMs) are a group of systemic autoimmune diseases characterised primarily by muscle inflammation but also potentially accompanied by a range of extra-muscular manifestations. Dermatomyositis (DM), polymyositis (PM) and inclusion body myositis (IBM) constitute well-characterised subtypes of IIM, with the entity of non-specific idiopathic inflammatory myopathy (NSIIM) being more recently described[1]. Whilst these IIM subtypes are distinguished on clinical, serological and histological grounds, they are unified by the presence of a typically prominent intramuscular lymphocytic infiltrate. However, over the past 15 years, descriptions of a distinct form of proximal myopathy characterised by prominent necrosis, regeneration, myophagocytosis and minimal lymphocytic infiltrate that responds to immunotherapy have emerged[2]. This entity has been termed immune-mediated necrotising myopathy (IMNM; also known as necrotising autoimmune myopathy, NAM) and has been conceptualised within the spectrum of IIM. There is a growing body of literature that seeks to define and characterise this IIM subtype with respect to aetiopathogenesis, environmental and genetic risk factors, autoantibody profiles, clinico-histological features, prognosis and response to treatment[36]. Complicating these endeavours is the heterogeneity of IMNM, the recognition that myonecrosis is a non-specific feature that may result from immune- and non-immune factors, and the fact that IMNM is not necessarily accompanied by detectable autoantibodies or other features of autoimmunity. These uncertainties have implications for the systematic study of these conditions and may hinder accurate diagnosis and treatment. Comprehending what the diagnosis of IMNM means in a local context is important to enable meaningful comparisons with international cohorts.

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Herein we systematically describe the clinical and histological features of a consecutive cohort of patients diagnosed with IMNM in order to better understand the spectrum of disease, characterise the features that distinguish it from other forms of IIM, identify distinct phenotypes within South Australia and contribute further to the important Australian research[7-11] accumulating on this emerging condition. We additionally characterise the features that distinguish IMNM from other forms of IIM and identify factors associated with clinically severe forms of IMNM. 2. Methods 2.1 Subjects Muscle tissue, serological and clinical data were obtained from the South Australian Myositis Database (SAMD), a histologically-defined registry of patients with PM, DM, IBM, NSIIM and ‘necrotising myopathy’. The histological criteria for recruitment to the SAMD has previously been described for PM, DM and IBM[12]. ‘Necrotising myopathy’ is defined as the presence of myofibre necrosis and an absence of features consistent with other forms of myopathy. Of these patients, only those with a clinical diagnosis of IMNM or NAM made by a rheumatologist with myositis expertise were included in the current study. Cases of NSIIM have evidence of muscle inflammation (lymphocytic infiltration or MHC-I or MHC-II upregulation) but insufficient histological or clinical features to allow sub-classification. Some cases were excluded due to missing clinical information, alternative diagnosis, failure to fulfil published classification criteria [1, 13] or technical difficulties (Supplementary Figure 1). Consecutive cases of IMNM diagnosed between 2001-2016 were compared to a select subset of patients with PM, DM, IBM and NSIIM diagnosed between 2013-2016. Histologically normal control muscle constituted muscle specimens obtained from subjects

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with myalgia, weakness or unexplained creatine phosphokinase (CK) elevations, but which lacked gross myopathic features, inflammatory infiltrate or MHC I expression. Detailed clinical and serological data were collected prospectively in a Microsoft Access database approved by the Research Ethics Committees of South Australian teaching hospitals. This included information regarding the presence of weakness, dysphagia, extramuscular manifestations and statin exposure. Patients are routinely asked about symptoms of dysphagia and referred for a speech pathology assessment if they have consistent symptoms. Missing information was collected retrospectively, where possible. Details regarding symptom duration, synchronous cancer (cancer diagnosis within 3 years[14, 15] of IIM), immunotherapy, death and strength at one year post-diagnosis were collected retrospectively. Cumulative corticosteroid exposure at biopsy was recorded as the prednisolone-equivalent dose. Patients who attended the Royal Adelaide Hospital had disease activity and damage assessments recorded using the Core Set Measures recommended by the International Myositis Assessment and Clinics Studies Group (IMACS). These included the following: 1) Manual Muscle Testing 8 (MMT8), 2) Patient and physician global assessments by visual analogue scale (VAS; PtG and DrG respectively), 3) Health Assessment Questionnaire (HAQ) Disability Index, 4) the muscle activity component of the Myositis Disease Activity Assessment Tool by VAS (MDAAT-muscle), 5) CK and 6) assessment of muscle damage by VAS (Myositis Damage Index; MDI). Baseline clinical assessments were the first assessments recorded following diagnosis. If these assessments occurred more than 3 months post biopsy, they were excluded from correlation statistics. ‘Peak CK’ was the highest CK level within 3 months of biopsy.

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Most patients were tested for the following myositis specific antibodies (MSAs) and myositis associated antibodies (MAAs) using the Euroline Myositis Profile 3 Immunoblot: anti-signalrecognition particle (SRP), anti-histidyl-tRNA synthetase (Jo1), anti-threonyl-tRNA synthetase (PL7), anti-alanyl-tRNA synthetase (PL12), anti-glycyl-tRNA synthetase (EJ), antiisoleucyl-tRNA synthetase (OJ), anti-Mi2, anti-Ro52, anti-Ku, anti-PMSCl75 and anti-U1RNP. Nineteen IMNM patients were tested for anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) antibodies using a commercial enzyme-linked immunoassay (ELISA, PathWest, Perth, Australia). Clinical, serological and histological subgroups of IMNM were defined and compared. Mildmoderate presentation was defined as normal power or MRC ≥ 4/5 (or MMT ≥ 7/10) in the weakest muscle without dysphagia; severe presentation was defined as MRC ≤ 4-/5 (or MMT ≤ 6/10) in any muscle. The serological IMNM subgroups were defined as: seronegative IMNM, anti-SRP+ IMNM and anti-HMGCR+ IIM, consistent with recently published classification criteria[3]. Only patients who underwent both anti-HMGCR ELISA and Euroline myositis immunoblot testing were included in this analysis. We also analysed IMNM patients according to the presence of certain clinical or demographic characteristics, namely: 1) Aboriginal or Torres Strait Islander (ATSI) heritage and 2) exposure to statin medications. 2.2 Muscle biopsy. Muscle samples were obtained via surgical biopsy or needle biopsy with a University College Hospital (UCH) needle, frozen in isopentane cooled using liquid nitrogen[16] and stored at 190°C until sectioning. Consecutive 9µm-thick cryostat sections of diseased and normal muscle were placed on coated slides. Haematoxylin and eosin (H&E) staining was performed on the first and last section of each series to visualise infiltrating cell types and myofibre 7

morphology and to confirm that the histopathology was unchanged in the consecutive series of sections collected for immunostaining. Sections were air dried for 30 minutes then stored at -80°C until use (1 – 68 days).

2.3 Immunohistochemistry Immunohistochemistry was performed using an autostainer (Dako Autostainer Plus). Slides were washed in buffer (TA-999-TT, Thermo Scientific) then blocked for 5 minutes with 2% H2O2 in methanol to reduce non-specific background staining due to endogenous peroxidase. The sections were washed again with buffer then incubated for 5 minutes with a commercial protein blocking agent (TA-060-PBQ, Thermo Scientific). After a further wash with buffer, slides were incubated with primary antibody for 30 minutes. Primary antibodies used were anti-MHC I (HLA-ABC) antibody at 1/2000 (M0736, Dako), anti-CD45 antibody at 1/400 (M0701, Dako), anti-CD68 antibody at 1/8000 (M0814, Dako), anti-CD8 antibody at 1/75 (M7103, Dako), anti-C5b-9 (membrane attack complex; MAC) antibody at 1/100 (M0777, Dako), anti-microtubule-associated protein 1A/1B-light chain 3 (LC3) antibody at 1/50 (AM1800a, Abgent), anti-neonatal myosin heavy chain (MHCn) at 1/100 (NCL-MHCn). Primary antibodies were diluted in commercial antibody diluent (TA-125-ADQ, Thermo Scientific). After washing, slides were incubated in commercial primary antibody enhancer (TL-060-PB, Thermo Scientific) for 10 minutes prior to washing and incubation with a commercial detection system comprising a universal secondary antibody formulation with anti-Mouse IgG and anti-Rabbit IgG specificity, conjugated to horseradish peroxidase polymer (TL-060-PH, Thermo Scientific) for 15 minutes. After washing in buffer, slides were

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incubated with peroxidase-compatible liquid substrate chromagen system (Dako K3468) for 10 minutes, followed by haematoxylin counterstaining. Positive controls were included with every staining procedure to ensure consistency across immunohistochemistry tests. In addition, a negative control was performed by omitting a primary antibody in every staining procedure to evaluate for non-specific binding of the universal secondary antibody polymer. Muscle samples from all participants were stained for MHC I, CD45, CD68, MAC, LC3 and MHCn. Eighty-one samples also underwent staining for CD8. 2.4 Quantification of immunohistochemical staining Sections stained for MAC, MHC I, LC3 and MHCn were analysed and graded in a semiquantitative manner by a muscle pathologist (SO) and a trained investigator (JD) using traditional microscopy evaluation according to a pre-determined grading scale (MAC, MHC I and LC3 grade range: 0 – 3+; MHCn grade range: 0 – 4+; Supplementary Table 1). Both investigators graded the slides twice on separate occasions. The degree of peri-fascicular accentuation of MHC I staining was noted for each case (none, mild-moderate, marked). H&E stained sections were graded for necrosis (grade range: 0 – 4+). The entire biopsy section was graded. The mean values from each grading session are reported. For quantification of CD45+ leucocytes, CD8+ lymphocytes and CD68+ macrophages, JD performed manual cell counts of 10 randomly selected high power fields (HPFs, magnification x 400) and calculated the average number of cells per section. Grading of each histological parameter was completed for the entire cohort before proceeding to the next. At the time of grading, the evaluating investigators (JD, SO) were blinded to the clinical details and the grades assigned to other immunoproteins. 9

2.5 Statistics Statistical analysis was performed using STATA version 14.0. Values were expressed as the median and the 25% to 75% interquartile range (IQR). Shapiro-Wilk W tests and visualisation of scatter plots were performed to test for normality of data. Two group comparisons were performed using the Mann-Whitney U test as the data were non-parametric. When three or more groups were compared, a Kruskal-Wallis H test was conducted to identify whether a statistically significant difference existed, followed by a post hoc Dunn’s test. A Bonferroni correction was applied for multiple comparisons. Fisher’s exact test was used to analyse categorical data. Spearman correlations were performed to analyse associations between radiological grades and continuous or ordinal parameters. The number of cases analysed is indicated if a full data set was not available. P values < 0.05 were considered significant. 3. Results 3.1 Inter-rater reliability of grading Inter-rater reliability was acceptable (κ 0.66 – 0.75) for all histological parameters (Supplementary Table 2). 3.2 Clinical features of South Australian IMNM patients Of the 773 patients in the South Australian Myositis Database (1993 – 2016), 20% were recorded as having a ‘necrotising myopathy’ histologically (Supplementary Figure 1). The remaining patients had histological diagnoses of DM (10%), PM (31%), IBM (23%), NSIIM (10%) and ‘Other (e.g. sarcoidosis, vasculitis; 10%). Consecutive cases of IMNM diagnosed between 2001-2016 are analysed herein (Supplementary Figure 1) and compared to a subset of DM, PM, IBM and NSIIM patients diagnosed between 2013-2016.

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3.2.1 Statin exposure and diabetes are common amongst South Australian IMNM patients The clinical characteristics of participants at presentation are shown in Table 1. Details regarding autoantibody profiles are provided in Supplementary Table 3. Forty-four IMNM patients underwent Euroline immunoblot testing and nineteen underwent anti-HMGCR testing. Anti-HMGCR antibody was the most common antibody detected (n = 9) in IMNM patients whilst other autoantibodies were uncommon: anti-SRP (n = 5), anti-PL7 (n = 4), anti-Ro (n = 3), anti-Mi2 (n = 2) and anti-PMSCL75 (n = 1). The anti-PL7+ patients lacked clinical features of anti-synthetase syndrome and exhibited widespread necrosis that was not limited to a perifascicular distribution. One patient exhibited mild perifascicular accentuation of myonecrosis but was anti-Jo1 antibody negative on the Extractable Nuclear Antigen (ENA) panel. This patient did not undergo Euroline immunoblot testing, meaning that presence of a non-Jo-1 anti-synthetase antibody is not excluded, though notably the patient had no extra-muscular manifestations of antisynthetase syndrome, making this unlikely. For one anti-Mi2+ IMNM patient, the autoantibody was borderline positive and disappeared on repeat testing, suggesting a false positive. The other anti-Mi2+ IMNM patient also featured pANCA and myeloperoxidase positivity, prominent histological necrosis and lacked rash or histopathology suggestive of DM or vasculitis. Exclusion of this case from the analysis did not affect the pattern of our results. One PM patient was antiHMGCR+, but the predominant histological feature was inflammation with evidence of primary myofibre invasion by lymphocytes. Compared with DM and PM, patients with IMNM were less likely to be MAA-positive, to exhibit extra-muscular features or have non-IIM autoimmune disease, but were more likely to have been prescribed statins (all p < 0.05). The most common extramuscular manifestations amongst IMNM patients were inflammatory arthritis (n = 6) and Raynaud’s 11

phenomenon (n = 3). One anti-HMGCR+ IMNM patient also exhibited the Shawl sign and a seronegative IMNM patient with severe weakness had concomitant pulmonary fibrosis. Cardiac involvement was not reported in any IMNM patient. Creatine kinase was elevated in IMNM patients compared with DM, IBM and NSIIM (all p < 0.05). Diabetes was common among IMNM patients, however most diabetic patients (82%) were on statin therapy.

3.2.2 Heterogeneous clinical presentations were observed Most IMNM patients had a high serum CK (> 1000 in 87%), profound weakness and a subacute presentation, although heterogeneity was observed. For instance, 16% (9/58) of IMNM patients presented with symptoms lasting > 1 year and CK was normal in 10% (6/62). Of the 55 IMNM patients with adequate clinical information to determine strength at presentation, five (9%) had normal power, 13 (24%) had mild-moderate weakness (MRC ≤ 4/5 or MMT ≤ 6/10I n the weakest muscle without dysphagia) and thirty-seven (67%) had a severe presentation.

3.2.3 A high proportion of IMNM patients require intense immunomodulatory therapy Data relating to treatment and death was available for eighty-seven patients in the year post biopsy. A high proportion of IMNM patients were treated with intravenous methylprednisolone (IVMP) whereas non-IMNM IIM patients rarely received this therapy (Table 1). When administered, most IMNM patients received IVMP within a week of the diagnostic muscle biopsy (81%, 17/21). Intravenous immunoglobulin (IVIG) was also commonly employed in IMNM patients (61%). Nine patients commenced rituximab within 12 months of diagnosis and IMNM was over-represented in this group (IMNM = 7, DM = 2). Three IMNM patients who initially presented with mild weakness and minor necrosis 12

deteriorated over several months despite oral disease-modifying agents (DMARDs), eventually requiring admission for IVMP and rituximab or IVIG. The remaining ‘mildmoderate presenters’ with follow up information available (n = 10) received a weaning course of prednisolone plus oral DMARDs and/or IVIG with improvement or stabilisation in muscle strength. 3.2.4 Prognosis is poor in IMNM Deaths within a year occurred in nine IMNM patients and one DM patient. Three patients (DM = 1, IMNM = 2) died of cancer or complications related to chemotherapy, while the remaining 7 IMNM patients died as a consequence of profound muscle weakness, infectious complications of immunosuppression and immobility, pulmonary embolism or marked functional deterioration in the setting of complex comorbidities resulting in a decision to provide supportive care only. All IMNM patients who died within a year had a severe presentation. Many surviving ‘severe presenters’ had persistent profound weakness at one year despite intense immunotherapy (Figure 1). Few IMNM patients had normal power at one year follow-up, regardless of the severity of the disease at initial presentation (Figure 1).

3.3 Histological features of South Australian IMNM patients 3.3.1 Histological heterogeneity was observed amongst IMNM patients While median necrosis grades were high in IMNM patients (Figure 2A), heterogeneity was observed. Twenty patients (32%) had mild necrosis ( 10% fibres) and in many cases this consisted of a few scattered necrotic muscle cells. Conversely in 9 patients (15%), myonecrosis was widespread (> 50% fibres). Necrosis was also frequently present in nonIMNM patients (Figure 2A). However, muscle from patients with IMNM featured high ‘necrosis: leucocyte’ ratios, consistent with the notion that this condition is typically defined 13

as featuring more necrosis relative to inflammation (Figure 2B). Indeed, this was the only histological index that statistically differentiated IMNM from all other IIM subtypes. The overall numbers of intramuscular inflammatory cells in IMNM patients did not differ from DM, PM or NSIIM patients (Supplementary Figure 2). Myoregeneration and myofibre complement deposition was notable in IMNM patients although these features were also present in other IIM subtypes (Figure 2C, 2E, 2F). While MHC-I staining was less marked than for other IIM subtypes (Figure 2D), a considerable minority (24%; 15/62) of IMNM patients exhibited widespread (≥ grade 2) staining on histologically normal myofibres. Sarcolemmal MHC-I positivity was observed in anti-SRP+, anti-HMGCR+ and seronegative individuals. Perifascicular accentuation of MHC-I staining occurred in two anti-SRP+ patients and five seronegative IMNM patients (e.g., Supplementary Figure 3). Capillary MAC staining was marked (grade 3) in 29% (18/62) of IMNM patients and median grades were equivalent to that observed in DM (2 [1.5-3] versus 2 [1.5 – 2.5], p = 0.22). Of note, dermatomyositis patients had higher corticosteroid exposure at biopsy (Table 1, p < 0.05), which may have influenced the histology. 3.3.2 Complement deposition was observed on necrotic, non-necrotic and regenerating myofibres in IMNM. Sarcoplasmic MAC staining localised to necrotic fibres (Figure 3A-B), and these processes correlated (rs 0.71, p < 0.001). However, absent MAC staining on apparently necrotic cells was also observed in some IMNM patients (e.g., Figure 3C-D), indicating that complementinduced cytolysis may not be the sole mechanism of muscle cell breakdown. Like others[17, 18], we observed granular sarcolemmal MAC deposition on non-necrotic and regenerating myofibres in IMNM patients (Figure 3E-H), suggesting that it may be an early or initiating

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factor in the muscle cell death process and additionally identifying regenerating myocytes as a potential target of the cytolytic attack. 3.3.3 LC3 (Autophagy Marker) Fibres positive for LC3, a marker of autophagy, were frequently observed in muscle obtained from IMNM patients. Indeed LC3 grades in IMNM did not significantly differ from IBM patients. In IMNM, the abnormal LC3 staining localised to necrotic and regenerating fibres. A fine, diffuse sarcoplasmic staining pattern was seen in regenerating fibres, while a coarse granularity observed in necrotic fibres (Supplementary Figure 4). In IBM, LC3 staining was coarse and/or localised to cells featuring rimmed vacuoles. 3.3.4 Histological associations with clinical disease severity Patients with severe presentation had significantly higher levels of both sarcolemmal and sarcoplasmic MAC deposition (Figure 4), and these histological features were negatively associated with strength (Table 2). Most patients with severe presentations had moderatesevere necrosis, however marked myonecrosis (Grade 3) was present in 2 patients with normal power. In one such patient the necrosis was focal, as sections from another area of the same muscle revealed normal histology. Both patients also had evidence of profound myoregeneration (Grade 3), which could explain their normal power. Both the degree of myonecrosis and sarcoplasmic MAC deposition were associated with the rapidity of presentation (Table 2). All nine patients who died within 12 months had evidence of moderate-severe myofibre MAC deposition and myonecrosis on biopsy. Myofibre regeneration correlated negatively with muscle damage as determined by first MDI (Table 2). Creatine kinase levels correlated positively with myonecrosis and sarcoplasmic MAC deposition and negatively with strength and symptom duration in IMNM (Table 2). The 15

number of intramuscular macrophages, leucocytes and LC3 positive fibres were higher in severely affected patients, although the differences were not as profound as for complement deposition (Figure 4). Associations between clinical severity at presentation or clinical disease activity measures and other histological features such as MHC-I positivity, capillary MAC deposition or the number of cytotoxic T cells in IMNM patients were not observed (data not shown).

3.4 Clinical associations: ethnicity, statins and autoantibodies 3.4.1 Aboriginal and Torres Strait Islander patients present with a severe form of IMNM Sixteen percent (9/55) of IMNM patients were of ATSI heritage compared to 2% (1/59) of non-IMNM IIM patients (p = 0.006). By comparison, 2016 census data indicate that ATSI people represent 2% of the South Australian population[19], indicating an overrepresentation of this ethnicity within our IMNM cohort. ATSI IMNM patients were younger than non-ATSI IMNM patients and were markedly affected by the disease (Table 3). They were more likely to have dysphagia, severe weakness, distal in additional to proximal weakness, marked muscle cell MAC deposition and tended towards higher serum CK levels and more histological necrosis. Treatment details were available for five ATSI IMNM patients, and all required IVMP and ≥ 2 other immunomodulatory agents (IVIG, rituximab, methotrexate and/or cyclophosphamide). Despite intensive treatment, three died within a year of diagnosis.

3.4.2 Clinical heterogeneity was observed amongst anti-HMGCR positive IMNM patients. Statistical comparison of seronegative (n = 8), anti-HMGCR+ (n = 9) and anti-SRP+ (n = 5) IMNM subgroups was limited by small numbers. Descriptive analysis of their clinical and 16

histological features is provided in Table 5. All anti-SRP+ patients presented with rapid or subacute onset severe proximal weakness associated with florid histopathology and high serum CK (range 6800 – 11358). Three (60%) had dysphagia. All received IVMP within two weeks of muscle biopsy followed by ≥ 2 immunomodulatory agents, however neuromuscular outcomes at one year were poor. Seronegative IMNM patients were also profoundly weak at presentation, however neuromuscular outcomes at one year varied with 3 (38%) significantly improving on immunotherapy (Table 4). Deaths in this group related to complications from cancer (n = 2) and muscle weakness (n = 1). Clinical heterogeneity was observed amongst anti-HMGCR+ IMNM patients. For instance, six antiHMGCR+ IMNM patients had profound weakness at presentation, two had mild proximal lower limb weakness and one had myalgia but normal strength. Symptom duration also varied markedly in anti-HMGCR+ IMNM with five presenting subacutely (1-3 months) and the remainder exhibiting a chronic (1-2 years, n = 2) or very chronic (> 4 years, n = 2) disease course. Two anti-HMGCR+ patients (22%) deteriorated despite immunotherapy, while power stabilised or improved in the remainder. Extra-muscular manifestations were infrequent in all IMNM patients, regardless of autoantibody status. Statin use was common in both seronegative and anti-HMGCR+ patients but not in anti-SRP+ patients. 3.4.3 Statin-exposed IMNM patients feature less intramuscular inflammation than statinunexposed patients Statin-exposed IMNM patients were older (66 [59-72] versus 57 [45-64] years, p = 0.01), were less likely to exhibit Raynaud’s phenomenon (2% versus 27%, p = 0.02) and trended to less non-IIM autoimmune disease than statin-unexposed IMNM (5% versus 27%, p = 0.06). They featured less leucocyte (p = 0.02) and macrophage (p = 0.03) infiltration but similar necrosis grades and serum CK levels compared with statin un-exposed IMNM patients. Of 17

note, 44% (4/9) of statin-unexposed IMNM patients were anti-SRP+ versus 3% (1/31) of statin-exposed IMNM, which might explain the high levels of inflammation seen in the former. A relative paucity of leucocytes in the statin-exposed group was observed regardless of HMGCR antibody status (Supplementary Figure 5), indicating that histological inflammation is not a useful discriminator between statin-associated seronegative IMNM and statin-induced anti-HMGCR+ IMNM.

4. Discussion Immune-mediated necrotising myopathy is a relatively recently described entity. To our knowledge, this is the largest Australian study to systematically describe the histological spectrum of this disease in a consecutive cohort and determine associations with clinical disease severity. Consistent with previous research, we found that many IMNM patients feature a subacute trajectory, high serum CK levels, profound muscle weakness, few extramuscular manifestations and evidence of myonecrosis, complement deposition and regeneration histologically. Most patients have persistent weakness at one year, despite immunotherapy. Whilst previous descriptions of IMNM have underscored a paucity of inflammation and absent MHC-I expression[1], our data confirm more recent reports that these features can occur[3, 6, 20-22] and their presence should not preclude this diagnosis. Rather, it is the degree of myonecrosis relative to inflammation that appears to differentiate this condition from other IIM subtypes. Like previous investigators, we have observed perifascicular accentuation of MHC-I[21] and capillary complement deposition[23] to be a feature in some patients. Overall, most findings reported in this study are consistent with

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literature emerging from other institutions, indicating that the South Australian IMNM cohort is comparable with international cohorts.

Nonetheless, some of our observations are unique to a South Australian population. For instance, patients of ATSI heritage were over-represented within the IMNM cohort, developed IMNM more commonly than other IIM subtypes and presented with a severe phenotype. This is in keeping with observations of an over-representation of ATSI patients among cases of statin-associated necrotising myopathy[10], with studies demonstrating an increased prevalence of autoimmune and inflammatory rheumatological conditions generally in this population[24] and with evidence that autoimmune disease in ATSI patients is associated with a more severe disease trajectory and increased complications leading to death[25]. Moreover, the true burden of these conditions within this population is likely to be underestimated as ATSI patients are less likely to access specialist healthcare services for rheumatic conditions in general[26]. Whether the differences in disease severity we observed are attributable to socioeconomic factors, or whether there is something inherently different to the disease pathophysiology in these patients is unclear and warrants further study. Ethnic variation in the presentation of IMNM has been observed elsewhere. For instance, African American patients tend towards more weakness and higher serum CK levels compared with their Caucasian counterparts[27, 28], Pacific Islander patients are over-represented in New Zealand studies of IMNM[29] and statin exposure appears less common in Asian patients with anti-HMGCR+ IMNM compared to European cohorts[22]. Genomic and epidemiological studies that seek to elucidate the reasons underpinning the ethnic discrepancies observed within our cohort are required.

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This study lends support to the wealth of evidence implicating complement-associated cytolysis as the major cause of myotoxicity in necrotising myopathies[18, 30-33]. Myonecrosis may occur via antibody-dependent activation of MAC in IMNM, as sarcolemmal expression of SRP and HMGCR antigen, binding of their corresponding autoantibodies and deposition of MAC as has been previously demonstrated[18]. The mechanism leading to surface expression of intracellular proteins such as SRP and HMGCR in IMNM patients are unclear, however antibody-induced surface redistribution of antigens has been shown to involve the cytoskeletal proteins actin, myosin and tubulin in cultured glomerular epithelial cells[33, 34]. The observation of sarcolemmal MAC deposition in seronegative IMNM may suggest the presence of as-yet undetected MSAs, or that an antibody-independent mechanism might also be involved, as has been demonstrated in DM[35]. Sarcolemmal MAC deposition is likely to be an early or initiating factor in the process of myonecrosis, which may explain why these histological appearances were often uncoupled in the present study. Of course, complement deposition may not be the sole mechanism of myotoxicity in these heterogeneous conditions, as suggested by our findings of absent MAC expression in some cases despite H&E appearances suggestive of muscle cell breakdown. This could also indicate that our MAC detection system lacks sensitivity, or that these abnormal cells are undergoing a process other than necrosis. Regardless, this study confirms complement deposition on myofibres to be a marker of disease severity in IMNM, which likely reflects its role as a mediator of myonecrosis. Whether complement activation inhibitors, such as eculizumab, would be of therapeutic benefit in these diseases warrants consideration.

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Abnormal LC3 staining in regenerating and necrotic fibres of IMNM patients was observed in this study and has been reported previously[36, 37]. The LC3 protein is a marker of autophagy, a highly regulated process in which cells engulf, degrade and recycle cellular contents through the formation of unique double-membraned vesicles (autophagosomes) which then fuse with lysosomes. LC3 is critically involved in autophagosome biogenesis, and high cellular levels of this protein may either indicate increased autophagy activity[38] or an accumulation of autophagosomes due to autophagic impairment, as in IBM[39]. Importantly, aggregation of LC3 has been shown to occur in an autophagy-independent manner[40] and thus the accumulation of LC3 observed in necrotic cells may be nonspecific. Nonetheless, the observation LC3 is expressed in some regenerating myofibres in IMNM patients is of interest, particularly in light of evidence that autophagy may play a beneficial role in activation of satellite cells upon muscle injury[41]. Critically, immature myofibres are also implicated in the perpetuation of autoimmune myopathies[42] and whether these histological features reflect a pathological or reparative process in IMNM requires further experimental studies.

We have shown IMNM to be a heterogeneous condition, both clinically and histologically. Although IMNM is frequently a severe and disabling disease, mild presentations occur and the histological feature of myonecrosis can be minor. Histological heterogeneity among IMNM has been noted elsewhere[3] and hinders academic efforts to classify and study the disease. Importantly, such variability also generates uncertainty regarding diagnosis and appropriate management amongst clinicians. As such, documenting the histological spectrum of IMNM and elucidating the associations between histology and disease severity in IMNM could inform clinical practice and were key aims of the current study. Using 21

immunohistochemical staining procedures routinely available in most diagnostic neuromuscular laboratories, we determined that the degree of sarcolemmal and sarcoplasmic complement deposition correlates closely with the clinical presentation and more so than the degree of inflammation, myofibre regeneration, MHC-I expression or autophagic protein accumulation. We additionally showed a correlation between serum CK and the degree of myofibre necrosis, sarcoplasmic MAC deposition and muscle strength, confirming this to be a useful biomarker of disease activity in IMNM. Our observation that IMNM patients with clinically mild presentations and low-grade histological changes can subsequently deteriorate underscores the need for vigilance and close clinical monitoring.

Our study has a number of limitations. The observational design means that we can identify associations but not causality. Despite the multi-centre nature of our study, the rarity of these conditions and our application of inclusion criteria meant our sample size was small, particularly when we performed subgroup analyses. This limits the robustness of our statistical analysis, although many of our findings are consistent with previous reports. Not all patients were treatment naïve at biopsy, which would have affected the histological parameters. Not all IMNM patients underwent anti-HMGCR testing as this assay has only become available in recent years. Data regarding immunotherapy, strength and mortality outcomes in the first year post diagnosis were collected retrospectively and relied on physician documentation in the clinical notes. Our cases were selected from a histologically defined registry and confirmed to have clinical IMNM by referring to the rheumatological diagnosis. Cases of ‘necrotising myopathy’ that received an alternative clinical diagnosis (e.g. toxic myopathy, rhabdomyolysis, anti-synthetase syndrome) were excluded. While this conceivably introduces bias it makes the finding of marked histological heterogeneity even 22

more noteworthy. Not all patients had Euroline immunoblots performed, meaning that some classified as IMNM histologically might have had undetected anti-synthetase antibodies. This would represent a misclassification. However, only one such patient exhibited perifasicular accentuation of necrosis and this patient was negative for anti-Jo-1 antibodies on ENA. The strengths of our study include the multi-centre setting, the number of patients analysed and the blinded, systematic assessment of histological features. To our knowledge, this is the largest study systematically examining the histological and clinical features in a consecutive cohort of Australian IMNM patients.

5. Conclusions Immune-mediated necrotising myopathy is a heterogeneous condition. Most patients have persistent weakness at one year despite immunotherapy. Severity at presentation is closely associated with the degree of necrosis and complement deposition on muscle cells. Aboriginal and Torres Strait Islander peoples appear to present with a more severe form of IMNM, and the mechanisms underlying this should be explored in future research.

23

Acknowledgements: Professor Peter Blumbergs (SA Pathology, Royal Adelaide Hospital) for hypothesis revision. Ms Bernice Gutschmidt (SA Pathology) for technical assistance with experimental process. Funding Sources Jessica Day is personally supported by an Australian National Health and Medical Research Council post-graduate scholarship [1114970], a Royal Adelaide Hospital Dawes top-up scholarship and a Victorian ARA top-up scholarship. The research was funded by a Myositis Association of Australia research grant. There are no conflicts of interest to declare. Ethics This study was approved by the Central Adelaide Local Health Network ethics committee, Adelaide, Australia.

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27

Figure Captions

Figure 1. Neuromuscular status at one year in IMNM patients according to their disease severity at presentation. Most IMNM patients had a persistent neuromuscular deficit at one year, regardless of disease severity at presentation. Severe presentations defined as MMT ≤ 6/10 or MRC ≤4-/5 in any muscle at presentation; Mild-moderate presentations defined as MMT ≥ 7/10 or MRC ≥ 4/5 in the weakest muscle at presentation.

28

Figure 2. Histological features of South Australian IMNM patients. Only the ‘necrosis: leucocyte’ ratio was significantly higher in IMNM versus all other IIM subtypes (B). * p < 0.05;

29

** p < 0.001. See supplementary figures for: LC3 grades, MAC capillary grades, CD45+ cell count, CD68+ cell count. IMNM, immune-mediated necrotising myopathy; MAC, membrane attack complex; MHC-I, major histocompatibility complex I; MHCn, neonatal myositis heavy chain.

30

Figure 3. Patterns of MAC staining in IMNM. A. H&E stain of muscle from a 47 year old woman with profound proximal, paraspinal, bulbar and respiratory muscle weakness and anti-PL-7 positivity demonstrating widespread

31

necrosis. B. MAC staining revealed widespread sarcolemmal and sarcoplasmic positivity. Magnification x 100. C. H&E from a 63 year old man with anti-SRP+ IMNM demonstrating two cells exhibiting features of necrosis (pallor, swelling, hyalinisation) and one profoundly necrotic cell. D. Only the profoundly necrotic cell exhibited positive MAC staining. Magnification x 400. E. H&E of muscle obtained from a 66 year man with IMNM demonstrating non-necrotic myofibres that stained positively for MAC (F.) Magnification x 600 G. Neonatal myosin heavy chain (MHCn) staining of muscle obtained from a 52 year old man with seronegative IMNM demonstrating numerous regenerating myofibres that also exhibit delicate sarcolemmal MAC expression (H.) Magnification x 400. H&E, haematoxylin and eosin; IMNM, immune-mediated necrotising myopathy; MAC, membrane attack complex; SRP, signal recognition particle.

Figure 4. IMNM patients with severe presentations had high levels of myonecrosis, LC3 positive fibres and complement deposition on myofibres (A). Intramuscular inflammation was also more pronounced in severely affected IMNM patients (B.). No significant differences were observed between these groups of patients with regards to numbers of infiltrating CD8+ T cells, MHC-I grades or MAC capillary grades (data not shown). Severe presentations defined as MMT ≤ 6/10 or MRC ≤4-/5 in any muscle at presentation; Mild32

moderate presentations defined as MMT ≥ 7/10 or MRC ≥ 4/5 in the weakest muscle at presentation. IMNM, immune-mediated necrotising myopathy; MAC, membrane attack complex; MMT8, manual muscle testing 8; MRC, medical research council scale for muscle strength.

Table 1. Clinical characteristics of participants at baseline and immunotherapy administered in the first year following diagnosis. Clinical and Demographic features of study subjects IMNM

DM

PM

IBM

NSIIM

62

18

13

14

15

64 (57 – 69)

55 (47 – 70)

60 (45 – 66)

68 (64 – 79)

59 (47 – 69)

32 (51%)

12 (67%)

7 (54%)

7 (50%)

10 (67%)

49 (15 – 151)

104 (88 – 274)

136 (122 – 244)

n = 58

n=8

n=9

n=9

n=5

9/58 (16%)

1/8 (13%)

2/9 (22%)

9 /9 (100%)

1/5 (20%)

ATSI

9/55 (16%)

1 (6%)

0 (0%)

0 (0%)

0 (0%)

Caucasian

43/55 (78%)

16 (89%)

12 (92%)

14 (100%)

13/14 (93%)

Asian

3/55 (5%)

1 (6%)

1 (8%)

0 (0%)

1/14 (7%)

MSA positivity b

18/44 (41%)

5/16 (31%)

3 (23%)

2/12 (17%)

2/13 (15%)

MAA positivity

4/44 (9%)

8/17 (47%)

6 (46%)

2/12 (17%)

3/13 (23%)

Prednisolone a (PNL, mg) Cumulative PNL (mg) a

0 (0 – 0)

20 (0 – 55)

0 (0 – 7)

0 (0 – 0)

0 (0 – 0)

N Age (years)

a

Female Symptom duration a (days) Symptoms > 1 year

1275 (1096 – 2718)

92 (75 – 130)

n = 48

n = 12

n=9

n = 13

n = 14

0 (0 – 0)

840 (150 – 2625)

0 (0 – 370)

0 (0 – 0)

105 (0 – 350)

n = 35

n = 12

n=8

n = 13

n=9

45/56 (80%)

1/16 (6%)

1 (8% )

7 (50%)

4/14 (29%)

Diabetes a

27/50 (54%)

4/15 (27%)

1 (8%)

2 (14%)

1/14 (7%)

Cancer d

9/58 (16%)

3/16 (19%)

2/12 (17%)

0 (0%)

1/14 (7%)

EM feature e

10/58 (17%)

17 (94%)

7 (54%)

5 (36%)

9/14 (64%)

Other AI disease

5/52 (10%)

9 (50%)

7 (54%)

5 (36%)

6/13 (46%)

Statin exposure

Peak CK (IU/L)

c

4086

546

2237

470

400

(1467 – 10799)

(248 – 1942)

(897 – 3193)

(217 – 709)

(73 – 839)

33

Dysphagia a

Myalgia MMT8

f

a

16/50 (32%)

5/16 (31%)

2 (15%)

9 (69%)

4/14 (29%)

32/54 (59%)

7/16 (44%)

8/13 (62%)

5/14 (36%)

7/13 (54%)

76 (64 – 80)

70 (62 – 80)

74 (64 – 80)

64 (54 – 74)

80 (78 – 80)

n = 23

n=7

n=7

n=8

n=5

Immunotherapy in the first year post biopsy N

49

11

9

10

8

IVMP

21 (43%)

1 (9%)

0 (0%)

0 (0%)

0 (0%)

IVIG

30 (61%)

3 (27%)

1 (11%)

4 (40%)

1 (13%)

MTX

17 (35%)

10 (91%)

6 (67%)

3 (30%)

1 (13%)

AZA

9 (18%)

2 (18%)

4 (44%)

2 (20%)

3 (38%)

HCQ

0 (0%)

2 (18%)

2 (22%)

0 (0%)

2 (25%)

RTX

7 (14%)

2 (18%)

0 (0%)

0 (0%)

0 (0%)

CYC

1 (2%)

0 (0%)

0 (0%)

0 (0%)

0 (0%)

MMF

3 (6%)

2 (18%)

1 (9%)

0 (0%)

1 (13%)

a

At muscle biopsy; b Includes patients who did not undergo anti-HMGCR testing; c At symptom onset;

d

Within three years of IIM diagnosis; e Extramuscular feature, i.e. presence of: shawl sign, Gottron’s

papules or sign, heliotrope rash, interstitial lung disease, mechanic’s hands, inflammatory arthritis or Raynaud’s phenomenon; f First assessment post biopsy; AI, autoimmune; ATSI, Aboriginal and Torres Strait Islander; AZA, azathioprine; CK, creatinine phosphokinase (IU/L); CYC, cyclophosphamide; DM, dermatomyositis; IBM, inclusion body myositis; HCQ, hydroxychloroquine; IIM, idiopathic inflammatory myopathy; IMNM, immune-mediated necrotising myopathy; IVIG, intravenous immunoglobulin; IVMP, intravenous methylprednisolone; MAA, myositis associated autoantibody; MMF, mycophenolate mofetil; MMT8, manual muscle test 8 (0 – 80); MSA, myositis specific autoantibodies; MTX, methotrexate; N, number; NSIIM, non-specific idiopathic inflammatory myopathy; PM, polymyositis; PNL, prednisolone; RTX, rituximab.

34

Table 2. Correlations between histological and clinical parameters for IMNM patients. There were correlations between the histological parameters of myonecrosis and myofibre complement deposition and measures of disease activity such as strength and creatine kinase. These histological features were also associated with the rapidity of presentation. The degree of myoregeneration was inversely related to the degree of muscle damage as determined by the Myositis Damage Index (MDI) visual analogue scale.

Clinical and histological correlations in IMNM patients MMT8 correlations Peak CK

-0.46* (n = 23a)

Necrosis grades

-0.75** (n = 14 )

MAC sarcolemma grades

-0.79** (n = 14b)

MAC sarcoplasm grades

-0.87** (n = 14 )

b

b

Peak CK correlations N

51

Necrosis grades

0.41*

MAC sarcolemma grades

0.15

MAC sarcoplasm grades

0.45** Symptom duration correlations

Peak CK

-0.59** (n = 50 c)

Necrosis grades

-0.46** (n = 58)

MAC sarcolemma grades

-0.24 (n = 58)

MAC sarcoplasm grades

-0.46** (n = 58) MDI correlations

a

N

11

MHCn grades (regeneration)

-0.83**

b

c

Peak CK within 3 months of initial MMT8; muscle biopsies within 3 months of initial MMT8; Peak CK within 3 months of presentation. * p < 0.05; ** p < 0.01. CK, creatine kinase; IMNM, immune-mediated necrotising myopathy; MAC, membrane attack complex; MDI, myositis damage index visual analogue scale; MHCn, neonatal myosin heavy chain; MMT8, manual muscle testing 8.

35

Table 3. Clinical and histological features of ATSI and Non-ATSI IMNM patients. Patients of ATSI heritage were younger and presented a more severe IMNM phenotype, both clinically and histologically.

Comparison of ATSI and Non-ATSI IMNM patients ATSI IMNM patients

Non-ATSI IMNM patients

N

9

46

Age (years) a

57 (52 – 59)

64 (59 – 69)

0.02

7 (78%)

22/46 (48%)

0.10

8 (89%)

31/41 (76%)

0.36

8/8 (100%)

27/47 (63%)

0.04

Distal weakness a

3 (33%)

0/41 (0%)

0.004

Peak CK (IU/L)

20000 (3726 – 47200)

3748 (1424 – 10170)

0.06

Dysphagia a

6 (67%)

9/39 (23%)

0.02

Extramuscular IIM a features

2 (22%)

8/43 (19%)

0.56

MSA positivity

anti-PL7, n = 1 anti-PL7 & anti-SRP, n = 1 anti-HMGCR, n = 1

15/36 (42%)

0.52

MAA positivity

1/6 (17%)

3/36 (8%)

0.47

Necrosis grades

2.5 (2 – 2.5)

1.5 (1 – 2.5)

0.06

MAC sarcolemmal grades

2 (1 – 3)

1 (0.5 – 2)

0.04

MAC sarcoplasmic grades

2 (1 – 2)

1 (0 – 1.5)

0.007

Female Statin Exposure

b

Severe weakness

c

p

3/6 (50%)

a

At muscle biopsy; b at symptom onset; c Severe IMNM defined as MMT8 ≤ 6/10 or MRC ≤3/5 in any muscle. ATSI, Aboriginal and Torres Strait Islander; CK, creatinine phosphokinase; IMNM, immune-mediated necrotising myopathy; MAA, myositis associated antibody; MAC, membrane attack complex; MSA, myositis specific antibody; N, number.

36

Table 4. Characteristics of IMNM patients according to autoantibody status. Anti-SRP+ IMNM patients presented with severe weakness, florid histopathology. Neuromuscular outcomes at one year were poor despite early and intense immunotherapy. Seronegative IMNM patients presented with severe weakness, but the neuromuscular outcomes varied. Clinical and histological heterogeneity was observed amongst antiHMGCR+ IMNM patients at presentation and at follow-up. Descriptive statistics presented only.

Characteristics of IMNM patients according to autoantibody status Only patients who underwent both Euroline immunoblot and anti-HMGCR ELISA presented (n = 22) Seronegative Anti-SRP+ Anti-HMGCR+ N

8

5

9

65 (53– 75)

63 (57 – 67)

62 (60 – 64)

3 (38%)

3 (60%)

7 (78%)

29 (10 – 154)

31 (21 – 81)

92 (56 – 721)

0 (0%)

0 (0%)

3 (33%)

7 (88%)

1 (20%)

8 (89%)

Severe Deficit at Presentation

8/8 (100%)

5/5 (100%)

6/9 (67%)

Peak CK

5953 (1646 – 53458)

8995 (7466 – 11358)

5697 (3734 – 11028)

1 (13%)

3/5 (60%)

4/9 (44%)

2 (25%)

0 (0%)

2 (22%)

2 (25%)

0 (0%)

1 (11%)

IVMP < 2 weeks of diagnosis

5 (63%)

5 (100%)

4 (44%)

Necrosis grades

2 (1.3 – 2.5)

2 (2 – 2.5)

1.5 (1.5 – 2)

MAC sarcoplasmic grades

1.3 (0.5 – 1.8)

2 (1.5 – 2)

1 (0.5 – 1)

CD8+ cell count/HPF

0.3 (0.2 – 10.2)

3.6 (2.1 – 6.3)

2 (0.8 – 5)

CD45+ cell count/HPF

3.5 (2.0 – 23.3)

31.7 (16.6 – 33.7)

7.4 (4 – 12.3)

CD68+ cell count/HPF

20.5 (0.2 – 44.8)

38.8 (32.2 – 43.2)

26.3 (22.2 – 32.7)

MHC-I grades

0.8 (0 – 2)

2 (1.5 – 2.5)

1 (0.5 – 1.5)

Age (years)

a

Female Symptom duration (days)

a

Symptoms > 2 years Statin Exposure

Dysphagia

b

a

Extramuscular IIM features Cancer

a

c

Neuromuscular outcome at one year Death

3 (38%)

1 (20%)

0 (0%)

Severe deficit

2 (25%)

3 (60%)

1 (11%)

Mild-Moderate deficit

2 (25%)

0 (0%)

6 (67%)

Normal power

1 (13%)

0 (0%)

2 (22%)

Unknown

0 (0%)

1 (20%)

0 (0%)

a

at presentation; b at symptom onset; c within 3 years of IIM diagnosis. CK, creatinine phosphokinase; HMGCR, anti-3-hydroxy-2-methylglutaryl-CoA reductase; HPF, high power field (magnification x 400); IIM, idiopathic inflammatory myopathy; IMNM, immune-mediated necrotising myopathy; IVMP, intravenous methylprednisolone; MAC, membrane attack complex; MHC-I, major histocompatibility complex I; N, number; SRP, signal recognition particle.

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Table 5. Characteristics of IMNM patients according to autoantibody status. Descriptive statistics presented only. Characteristics of IMNM patients according to autoantibody status Only patients who underwent both Euroline immunoblot and anti-HMGCR ELISA presented (n = 22) Seronegative Anti-SRP+ Anti-HMGCR+ N

8

5

9

65 (53– 75)

63 (57 – 67)

62 (60 – 64)

3 (38%)

3 (60%)

7 (78%)

29 (10 – 154)

31 (21 – 81)

92 (56 – 721)

0 (0%)

0 (0%)

3 (33%)

7 (88%)

1 (20%)

8 (89%)

Severe Deficit at Presentation

8/8 (100%)

5/5 (100%)

6/9 (67%)

Peak CK

5953 (1646 – 53458)

8995 (7466 – 11358)

5697 (3734 – 11028)

1 (13%)

3/5 (60%)

4/9 (44%)

2 (25%)

0 (0%)

2 (22%)

2 (25%)

0 (0%)

1 (11%)

IVMP < 2 weeks of diagnosis

5 (63%)

5 (100%)

4 (44%)

Necrosis grades

2 (1.3 – 2.5)

2 (2 – 2.5)

1.5 (1 – 2)

MAC sarcoplasmic grades

1.3 (0.5 – 1.8)

2 (1.5 – 2)

1 (0 – 1)

CD8+ count/HPF

0.3 (0.2 – 10.2)

3.6 (2.1 – 6.3)

2 (0.8 – 5)

CD45+ count/HPF

3.5 (2.0 – 23.3)

31.7 (16.6 – 33.7)

7.4 (4 – 12.3)

CD68+ count/HPF

20.5 (0.2 – 44.8)

38.8 (32.2 – 43.2)

26.3 (22.2 – 32.7)

MHC-I grades

0.5 (0 – 2)

3 (2 – 3)

1 (1 – 2)

Age (years)

a

Female Symptom duration (days)

a

Symptoms > 2 years Statin Exposure

Dysphagia

b

a

Extramuscular features Cancer

a

c

Neuromuscular outcome at one year

a

Death

3 (38%)

1 (20%)

0 (0%)

Severe deficit

2 (25%)

3 (60%)

1 (11%)

Mild-Moderate deficit

2 (25%)

0 (0%)

6 (67%)

Normal power

1 (13%)

0 (0%)

2 (22%)

Unknown

0 (0%)

1 (20%)

0 (0%)

at presentation; b at symptom onset; c within 3 years of IIM diagnosis.

38