Myopathy related to chronic Graft‐Versus‐Host Disease: From clinic to histological &amp; immunological characterization by imaging mass cytometry
Authors: Amélie Bourhis, Marie Robin, Stéphanie Nguyen, Arnaud Uguen, Patrice Hemon, Nathalie Dhedin, Thierry Maisonobe, Adèle de Masson, Olivier Benveniste, Gérard Socié, Régis Peffault de La Tour, Sarah Leonard‐Louis, Baptiste Hervier
Journal: American Journal of Hematology (2024)

Chronic Graft-Versus-Host Disease (cGVHD) is a common complication of allogenic hematopoietic stem-cells transplantation, potentially affecting all organs.1, 2 Even if muscle involvement is not included in the National Institutes of Health (NIH) consensus criteria it may affect 0.5% to 3,4% of patients.3 We report here a complete clinical and histological series with an extensive characterization by imaging mass cytometry (IMC) of muscular GVHD (mGVHD), in order to precisely describe this entity. Between 2007 and 2021, all consecutive patients who underwent a muscular biopsy (MB) for muscular symptoms after an allogeneic transplantation were retrospectively included. Muscle weakness was evaluated according to MRC and reported for the most affected muscles. Severe myopathy included dysphagia, respiratory muscle involvement or MRC ≤3. Needle electroneuromyography was positive according to at least a myogenic pattern in one muscle. One T2 or STIR hypersignal on MRI (edema) defined muscle involvement. Change of immunosuppressant after 2 months due to insufficient response (no muscle strength improvement and/or persistence of anomalies on muscle MRI) or symptom relapse under treatment defined treatment line failure. Complete remission was obtained by the absence of muscle symptoms, normal creatin kinase (CK) and the absence of muscle oedema on MRI. All other cases were considered in partial remission. Analyses of all biopsies were centralized in a tertiary hospital by two pathologists. Formalin-fixed and paraffin-embedding muscle sections were used for imaging mass cytometry (IMC) analyses. Thirty-three antibodies were selected to stain inflammatory cells populations and muscular cells (Supplementary methods). Qualitative variables were compared using chi2 and quantitative variables using Kruskall-Wallis rank sum test. Twenty-one patients were included. Myalgias and muscle weakness were chronic manifestations of mGVHD (Table 1), being diagnosed on MB with a median time of 20 months (8–199) after allogenic transplantation (Supplementary Table 1). All but one patient (95%) had other cGHVD manifestations, with a median of 3 other involved organs (Supplementary Table 1). Treatments of cGVHD at the time of MB included at least one immunosuppressive drug in 2/3 of the cases, but no or only low doses of steroids. Myositis-specific antibodies were positive only once: patient 21 had anti-HMGCoA-reductase antibodies with a typical immune-mediated necrotizing myopathy on MB. He was excluded from subsequent analyses. The extended histological description (Table 1, Supplementary Table 1, Supplementary methods) revealed mild atrophy (Figure 1A) and no or few necrosis for 13/20 patients, whereas 7/20 had significant necrosis and atrophy. When present, both were distributed in the entire fascicule. MHC-I immunostaining was always diffusely positive, with a perifascicular enhancement in 70% (Figure 1B). MHC-II immunostaining was positive in 89.5% of cases with perifascicular location (Figure 1C). A mild capillary drop-out was observed in 45% of the cases, with C5b-9 capillary thrombi in only one case and negative MxA immunochemistry staining. Perimysial fibrosis was common (75%), whereas endomysial fibrosis was highly related to necrosis. Concerning inflammatory infiltrate: 4 patients experience no infiltrate, 13 patients had muscular inflammatory infiltrate (mild in six cases and significant in 7 Figure 1D.1-E.1); 3 were considered as an intense fasciitis in the foregraound (Figure 1F.1,F.2). Several degrees of fascial inflammation were also present in 69% of patients with muscular inflammation. Mild inflammatory infiltrate had a perimysial location, whereas significant ones were diffuse with endomysial involvement. All cases with significant inflammatory infiltrate had necrotic fibers, whereas other cases had none. Taken together, patients showed two different patterns: fasciitis (group 1, n = 3) and inflammatory myopathy (mGVHD) from mild to intense, also divided into three additional groups (Table 1): pauci-inflammatory myopathy without inflammatory infiltrates or necrosis (group 2, n = 4), mild inflammatory myopathy (group 3, n = 6), intense inflammatory myopathy with necrosis (group 4, n = 7). Corroborating this way to classify the patients (Supplementary Figure 1A), there was a significant difference among the subgroups for the global inflammation score (0, 1.7 and 4 for piM, miM, and iiM respectively, p < 10−3). Interestingly, necrosis/regeneration score was also significantly higher in iiM as compared to the other groups (0, 0 and 1.4, respectively, p < 10−3), whereas, there was no significant difference between the groups concerning the atrophy score (1.8, 1.5, and 2.3, respectively, p = 0.37). Seven cases were studied by imaging mass cytometry (Figure 1): two fasciitis, two mild inflammatory myopathies and three high inflammatory myopathies. The distribution of inflammatory populations did not vary with the extent of the inflammatory infiltrate. The main population was always macrophage. Other well-represented populations were T lymphocytes (8%–22%), plasma cells (1%–23%), and granulocytes (1%–45%). B lymphocytes infiltrate was always low (<5%) and largely corresponded to germinal center B cells. The majority of all macrophages were pro-inflammatory M1 macrophages. In patients with fiber necrosis, M1 macrophages were mostly present around necrotic fibers, while M2 macrophages localized in the periphery. The majority of T lymphocytes were CD8+ (53%–82%). Extra-vascular granulocytes (eosinophils according to hematoxylin–eosin staining) were present in all cases and overrepresented in case of intense inflammation or fasciitis. MHC-II was strongly expressed by macrophages and weakly by perifascicular muscle fibers. On MB, patients of group 1 disclosed predominantly a fasciitis. Their clinical symptoms were suggestive of myopathy, neither CK, electromyography nor muscle MRI were abnormal. For the 17 remaining cases of mGVHD, clinical picture-associated myalgias (82%) and/or weakness (82%), with severity criteria in 7 (41%) cases. CK level was variable, but at least slightly elevated (>2 N) in 47% of the cases. Muscle MRI sensitivity for mGVHD diagnosis was low (60%), whereas, electromyography sensitivity was 93%. Whereas clinical symptoms were similar among the groups, CK increased and muscle edema on MRI discriminated the subgoups: iiM patients showed higher CK elevation (mean CK elevation of 8.9 compared to 3.3 and 1.25 N in iiM, miM, and piM respectively, p = 0.045) and muscle edema more frequently (proportion of: 100% in iiM, 20% in miM, and 50% in piM, p = 0.024). The time between the onset of muscular symptoms and mGVHD diagnosis was significantly longer in the lower inflammatory groups (mean of 708 days in piM, 108 days in miM, and 69 days in miM, p = 0.033 (Supplementary Figure 1,B–E). Regarding treatments, more than 10 different immunomodulatory drugs were given simultaneously or successively (median of 2 lines) in addition to oral steroids, among which ruxolitinib was recently more frequently used.4 The number of patients in complete remission at the end of the follow-up period (43 months, 5–156 months) tended to be higher in iiM (71.4%), the group with necrosis and the most important inflammation, the highest CKs, muscle edema on MRI and the shortest duration of symptoms. Interestingly, all the patients with increased CK treated within 1 month of symptom onset achieve complete remission. cGVHD is often a systemic and severe condition, for which diagnostic and therapeutic issues are challenging. As part of cGVHD manifestations, mGVHD is rare and its clinical diagnosis is difficult because of associated confounding factors as denutrition, adverse events of previous treatments or other cGVHD manifestations.2 Our large series thus provides important information on the diagnosis approach. Electromyography is the cornerstone of the diagnosis (showing a myogenic pattern in 93% of the patients with mGVHD), followed by CK measurement and muscle MRI, which have a lower sensitivity. Finally, the muscle biopsy is necessary for positive diagnosis, providing qualitative and quantitative information. Myopathy, in the context of cGVHD is rare and, therefore, poorly understood. The majority of histologic descriptions indifferently classified these cases as polymyositis or dermatomyositis.1, 5 We described mGVHD as a perimysial inflammatory myopathy characterized by diffuse MHC-I immunostaining with perifascicular enhancement and perifascicular MHC-II immunostaining, associated with inflammatory infiltrate of various intensity, which has a perimysial distribution, but becomes more diffuse in highly inflammatory cases with necrosis. This pattern differs from other idiopathic inflammatory myopathies. "Idiopathic inflammatory myopathy" in the context of cGVHD are possible (like patient 21), but diagnostic criteria are regularly missing. In addition, mGVHD is commonly associated with a fasciitis. Considering the cases of predominant fasciitis, it suggests a continuum between these forms: from isolated fasciitis, to myo-fasciitis with more or less intense inflammation in link with necrosis of the muscle fibers. Imaging mass cytometry allowed us to better characterize inflammatory infiltrate. It did not vary according to its density, but differs from other idiopathic inflammatory myopathies by the presence of eosinophils. Eosinophils are known to be involved in cGVHD pathogenesis.6 Thus, their presence in muscle biopsy could be an argument for mGVHD compared to other diagnoses. These extensive immuno-histologic analyses argue for a specific entity different from other idiopathic inflammatory myopathies. We were able to distinguish different patients' profile according to the duration of muscular symptoms, CK increase, T2/STIR hypersignal on MRI, that were quite in accordance with the intensity of the inflammatory infiltrate on MB. mGVHD is difficult to treat, with most patients requiring ≥2 treatment lines. In the absence of homogenous treatment strategy, this retrospective series do not allow us to recommend any immunosuppressant. As approved, however, in cGVHD, recent mGVHD patients have received ruxolitinib, with promising results (yet to be rigorously confirmed). However, several parameters appear to be linked with mGVHD prognosis and with complete remission: short delay between first muscular symptoms and treatment onset, increased CK as well as intensity of muscular inflammation on MB should guide clinicians in an early management of mGVHD as they seemed to be good prognosis factors. In conclusion, mGVHD is a specific inflammatory myopathy, with diffuse MHC-I and perifascicular MHC-II staining, predominant fascial and perimysial inflammatory infiltrates with eosinophils, eventually associated with necrotic fibers in the cases of significant inflammation. Intensity of the muscular infiltrate seems linked with short duration of mGVHD, increased CK and muscle edema on MRI, all being arguments for early treatment of patients with mGVHD to allow complete remission. Amélie Bourhis, Marie Robin, Stéphanie Nguyen, Nathalie Dhedin, Thierry Maisonobe, Adèle de Masson, Olivier Benveniste, Gérard Socié, Régis Peffault de La Tour, Sarah Leonard-Louis, & Baptiste Hervier contributed to conception and designed of the study. Amélie Bourhis, Marie Robin, Stéphanie Nguyen, Arnaud Uguen, Patrice Hemon, Nathalie Dhedin, Thierry Maisonobe, Adèle de Masson, Olivier Benveniste, Gérard Socié, Régis Peffault de La Tour, Sarah Leonard-Louis, & Baptiste Hervier contributed to acquisition and analyses of the data, in particular Amélie Bourhis, Arnaud Uguen, Patrice Hemon, & Sarah Leonard-Louis performed the histological and immunological analyses. Adèle de Masson, Sarah Leonard-Louis, & Baptiste Hervier wrote the manuscript and prepared the figures. Yves Allenbach, Homa Biassette, Jean-David Bouaziz, Florian Chevillon, Raymonde Fahd, Alienor Xhaard, Pierre Lozeron, David Michonneau, Quentin Monzani, Jean-Herlé Raphalen, Flore Sicre de Fontbrune, Gérard Socié, Laetitia Souchet. The authors acknowledge the cytometry flow core facility Hyperion (Brest, France) for its technical assistance, as well as the European FEDER grant program Progos RU 000950. None of the authors have a conflict of interest regarding this manuscript. Data available on request due to privacy/ethical restriction. Data S1. Supplementary materials. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.