Long Term Follow-Up of Pediatric-Onset Evans Syndrome

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Long term follow-up of pediatric-onset Evans syndrome: broad immunopathological manifestations and high treatment burden by Thomas Pincez, Helder Fernandes, Thierry Leblanc, Gérard Michel, Vincent Barlogis, Yves Bertrand, Bénédicte Neven, Wadih Abou Chahla, Marlène Pasquet, Corinne Guitton, Aude Marie-Cardine, Isabelle Pellier, Corinne Armari-Alla, Joy Benadiba, Pascale Blouin, Eric Jeziorski, Frédéric Millot, Catherine Paillard, Caroline Thomas, Nathalie Cheikh, Sophie Bayart, Fanny Fouyssac, Christophe Piguet, Marianna Deparis, Claire Briandet, Eric Dore, Capucine Picard, Frédéric Rieux-Laucat, Judith Landman-Parker, Guy Leverger, and Nathalie Aladjidi

Haematologica 2021 [Epub ahead of print]

Citation: Thomas Pincez, Helder Fernandes, Thierry Leblanc, Gérard Michel, Vincent Barlogis, Yves Bertrand, Bénédicte Neven, Wadih Abou Chahla, Marlène Pasquet, Corinne Guitton, Aude Marie-Cardine, Isabelle Pellier, Corinne Armari-Alla, Joy Benadiba, Pascale Blouin, Eric Jeziorski, Frédéric Millot, Catherine Paillard, Caroline Thomas, Nathalie Cheikh, Sophie Bayart, Fanny Fouyssac, Christophe Piguet, Marianna Deparis, Claire Briandet, Eric Dore, Capucine Picard, Frédéric Rieux-Laucat, Judith Landman-Parker, Guy Leverger, and Nathalie Aladjidi. Long term follow-up of pediatric-onset Evans syndrome: broad immunopathological manifestations and high treatment burden. Haematologica. 2021; 106:xxx doi:10.3324/haematol.2020.271106

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Thomas Pincez,1,2 Helder Fernandes,1,3 Thierry Leblanc,4 Gérard Michel,5 Vincent Barlogis,5 Yves Bertrand,6 Bénédicte Neven,7,8,9 Wadih Abou Chahla,10 Marlène Pasquet,11 Corinne Guitton,12 Aude Marie-Cardine,13 Isabelle Pellier,14 Corinne Armari-Alla,15 Joy Benadiba,16 Pascale Blouin,17 Eric Jeziorski,18 Frédéric Millot,19 Catherine Paillard,20 Caroline Thomas,21 Nathalie Cheikh,22 Sophie Bayart,23 Fanny Fouyssac,24 Christophe Piguet,25 Marianna Deparis,26 Claire Briandet,27 Eric Dore,28 Capucine Picard,9,29 Frédéric Rieux-Laucat,8,9 Judith Landman-Parker,30 Guy Leverger,30 and Nathalie Aladjidi1,3 on the behalf of members of the French Reference Center for Pediatric Autoimmune Cytopenia (CEREVANCE) and of collaborators from the French Reference Center for Adult Autoimmune Cytopenia (CERECAI).

1 Centre de Référence National des Cytopénies Auto-immunes de l’Enfant (CEREVANCE), Bordeaux, France 2 Division of Pediatric Hematology-Oncology, Charles-Bruneau Cancer Center, Department of Pediatrics, Sainte-Justine University Hospital, Université de Montréal, Montréal, Québec, Canada 3 Pediatric Oncology Hematology Unit, University Hospital, Plurithématique CIC (CICP), Centre d’Investigation Clinique (CIC) 1401, INSERM Bordeaux, France 4 Pediatric Hematology Unit, Robert Debré University Hospital, AP-HP, Paris, France 5 Department of Pediatric Hematology, La Timone Hospital, Marseille University Hospital, Marseille, France 6 Institute of Pediatric Hematology and Oncology, Lyon University Hospital, Lyon, France 7 Pediatric Immuno-Hematology and Rheumatology Department, Necker-Enfants Malades University Hospital, AP-HP, Paris, France 8 Laboratory of Immunogenetics of Pediatric Autoimmune Diseases, Paris, France 9 Imagine Institute, UMR 1163 INSERM and Paris University, Paris, France 10 Department of Pediatric Hematology, Jeanne de Flandre Hospital, Lille University Hospital, Lille, France 11 Pediatric Oncology Immunology Hematology Unit, Children’s University Hospital, Toulouse, France 12 Department of Pediatrics, Bicêtre University Hospital, AP-HP, Le Kremlin-Bicêtre, France 13 Department of Pediatric Hematology and Oncology, Rouen University Hospital, Rouen, France 14 Pediatric Unit, Angers University Hospital, Angers, France 15 Pediatric Oncology Hematology Unit, Grenoble University Hospital, Grenoble, France 16 Department of Hemato-Oncology Pediatric, Nice University Hospital, Nice, France 17 Department of Pediatric Hematology-Oncology, Clocheville Hospital, Tours University Hospital, Tours, France 18 Pediatric Oncology Hematology Unit, Arnaud de Villeneuve University Hospital, Montpellier, France 19 Department of Pediatric Hematology, Poitiers University Hospital, Poitiers, France 20 Department of Pediatric Hematology and Oncology, Hautepierre University Hospital, Strasbourg, France 21 Pediatric Hematology Unit, Nantes University Hospital, Nantes, France 22 Department of Pediatric Hematology-Oncology, Besancon University Hospital, Besancon, France 23 Pediatric Hematology Unit, Rennes University Hospital, Rennes, France 24 Pediatric Hematology Unit, Nancy University Hospital, Nancy, France 25 Pediatric Oncology Hematology Unit, Limoges University Hospital, Limoges, France 26 Pediatric Oncology-Hematology Unit Department, Caen University Hospital, Caen, France 27 Department of Pediatrics, Dijon University Hospital, Dijon, France 28 Pediatric Unit, Clermont-Ferrand University Hospital, Clermont-Ferrand, France 1

29 Study Center for Primary Immunodeficiencies, Necker-Enfants Malades University Hospital, AP-HP, Paris, France 30 Pediatric Oncology Immunology Hematology Unit, Armand-Trousseau University Hospital, AP-HP, Paris, France

Running head: Pediatric-onset Evans syndrome outcomes

Correspondence: Nathalie Aladjidi, Pediatric Hematology Unit, CIC 1401, INSERM CICP, Centre de référence national des cytopénies auto-immunes de l’enfant CEREVANCE, Hôpital des Enfants, Hôpital Pellegrin, Place Amélie Raba Léon, 33000 Bordeaux, France. E-mail: [email protected], Tel: +335 56 79 59 39, Fax: +335 57 82 02 79

Word count Abstract: 250 Text: 3999 Tables: 2, Figures: 5 Supplemental data: 5 tables and 3 figures References: 33

Data sharing statement: Data are available on request to corresponding author.

Acknowledgments The complete list of collaborators is given in the appendix. The authors would like to thank all of the patients, families, medical and para-medical teams involved in the CEREVANCE prospective cohort study from 2004 onwards.

This work was supported from 2004 by the French Ministry of Health (Programme Hospitalier de Recherche Clinique [PHRC] 2005, Rare Disease Plan 2007 and 2017), the Association Bordelaise pour l’Avancement des Sciences Pédiatriques (ABASP) research charity, the Association pour la Recherche et les Maladies Hématologiques de l’Enfant (RMHE) research charity, the Association Française du Syndrome d’Evans (AFSE), the O-CYTO patients’ association, and partially by GlaxoSmithKline, AMGEN and Novartis. TP is a recipient of a Charles Bruneau fellowship award.

Author Contributions T.P., H.F., T.L., G.L. and N.A. designed the study, analyzed the data and drafted the paper. T.P. and H.F. performed statistical analyses. C.P. and F.R.-L. performed genetic analyses. All of the authors participated to prospective data collection and interpretation and revised the manuscript for critical content.

Conflict of Interest Disclosures The authors declare no competing financial interests.

Abstract

Pediatric-onset Evans syndrome (pES) is defined by both immune thrombocytopenic purpura

(ITP) and autoimmune hemolytic anemia (AIHA) before the age of 18 years. There have been no comprehensive long-term studies of this rare disease, which can be associated to various immunopathological manifestations (IMs). We report outcomes of the 151 patients with pES and more than 5 years of follow-up from the nationwide French prospective OBS’CEREVANCE cohort. Median age at final follow-up was 18.5 (6.8–50.0) years and the median follow-up period was 11.3 (5.1–38.0) years. At 10 years, ITP and AIHA were in sustained complete remission in

54.5% and 78.4% of patients, respectively. The frequency and number of clinical and biological

IMs increased with age: at 20 years old, 74% had at least one clinical cIM. A wide range of cIMs occurred, mainly lymphoproliferation, dermatological, gastrointestinal/hepatic and pneumological IMs. The number of cIMs was associated with a subsequent increase in the number of second-line treatments received (other than steroids and immunoglobulins; hazard ratio, 1.4; 95% confidence interval, 1.15–1.60; p = 0.0002, Cox proportional hazards method).

Survival at 15 years after diagnosis was 84%. Death occurred at a median age of 18 (1.7–31.5) years, and the most frequent cause was infection. The number of second-line treatments and severe/recurrent infections were independently associated with mortality. In conclusion, long- term outcomes of pES showed remission of cytopenias but frequent IMs linked to high second- line treatment burden. Mortality was associated to drugs and/or underlying immunodeficiencies, and adolescents-young adults are a high-risk subgroup.

Introduction

The presence of both immune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (AIHA) defines Evans syndrome (ES). Pediatric-onset ES (pES) is a rare disease, and approximately 10 new cases are diagnosed every year in France, which has a population of 66 million.1 Since its first description in 1951 by Robert Evans,2 our understanding of pES has been based on small retrospective cohorts with limited follow-up.3–7 In 2004, the French Rare Disease

Center CEREVANCE set up the prospective national cohort OBS’CEREVANCE, which includes children with AIHA, chronic ITP persisting for more than 12 months (cITP), and pES.8

Preliminary reports from this cohort and previously published studies showed that pES is a chronic disease with a high rate of relapse for both types of cytopenias.1,3,4,7,9 Mortality rates across studies have ranged from 7–36%.1,3–7 In addition to cytopenias, immunopathological manifestations (IMs) such as autoimmune/autoinflammatory organ diseases, lymphoproliferation, and hypogammaglobulinemia have been reported in 70–80% of patients with pES.4,5,8,10 In an undetermined number of cases, pES is thought to be “secondary” and caused by an underlying disease, classically systemic lupus erythematosus (SLE) or autoimmune lymphoproliferative syndrome (ALPS).1,11,12 Recently, genetic analyses found a heterogeneous genetic background in up to 65% of a subset of 80 patients from the OBS’CEREVANCE cohort. These patients carried variants in genes that are linked to primary immunodeficiencies (PIDs) or involved in immune responses.13

Overall, outcomes and the long-term course of pES are poorly understood. There have been no comprehensive longitudinal studies including both cytopenia and IMs. In addition, the transition to adulthood is often particularly challenging for patients with chronic pediatric diseases.14 Adolescents–young adults (AYA) outcomes have not been investigated in patients with pES, and whether the disease improves with age is unknown. In a clinical setting, the 4 possibility to identify high-risk patients would be extremely helpful in the management of this complex disease. Here, we describe the long-term course of hematological, IMs and treatments received throughout childhood into adulthood in patients with pES from the OBS’CEREVANCE cohort. We aimed to identify clinically relevant factors associated to the occurrence of IMs, the number of second-line treatments received and mortality. Particularly, we investigated the impact of the number second-line treatment received and splenectomy on mortality.

Methods

OBS’CEREVANCE prospective national cohort

Inclusion and exclusion criteria are shown in Supplemental Table 1.1,8,15 Data collected have been previously detailed.1,8 Patients were included if < 18 years old at first cytopenia diagnosis. The coordinating center gathered and analyzed all data from the medical team in charge in real time, enabling prospective follow-up even after the pediatric-to-adult transition. The CEREVANCE group recommends scheduling clinical and biological follow up at least every 6-12 months.

Some patients underwent genetic analyses, as previously described.13 Written informed consent was obtained from parents and eligible patients. The cohort study was approved by the institutional ethics committee (CPPRB-A; Bordeaux, France) and the database was registered with the national data protection authority (CNIL, 1396823V0).

Patient selection

Patients with pES, defined as the simultaneous (within 1 month) or sequential association of ITP and AIHA, were included if at least 5 years of follow-up data were available after the first cytopenia diagnosis. To provide a complete mortality report, all patients, including those with

5 less than 5 years of follow-up data, were included in the survival analyses. The data were extracted on 21 June 2019.

Definitions (Supplemental Table 1)

Initial cytopenia refers to the onset of ITP or AIHA (whichever occurred first) and does not take autoimmune neutropenia (AIN) into account. The IM categories were separated in clinical (cIMs) and biological (bIMs). pES was defined as secondary if a diagnosis of SLE or PID was made during the follow-up period. SLE diagnosis was made according to the Systemic Lupus

International Collaborating Clinics Classification criteria (SLICC).16 ALPS diagnosis was based on international criteria.17 Second-line treatments were all immunomodulatory or immunosuppressive treatments, including splenectomy but excluding steroids and therapeutic intravenous immunoglobulins (IVIGs). Sustained complete remission (CR) was defined as remission persisting until final follow-up, regardless of ongoing treatments.

For analyses by age, patients were assessed annually from birth (for IMs and treatments) or from cytopenia onset (for AIHA and ITP), until final follow-up. Occasional treatments (e.g., splenectomy and rituximab) were considered as ongoing if these occurred during the previous year.

Statistical analyses

Continuous and categorical variables were compared using Wilcoxon–Mann–Whitney non- parametric test and Fisher’s exact test, respectively. Correlations were tested using the Pearson correlation coefficient. Survival and cumulative incidence estimates were based on the Kaplan–

Meier method and compared using log-rank test. Patients of OBS’CEREVANCE cohort with isolated cITP or AIHA were used for comparison in survival analyses (unpublished data). The 6

Cox proportional hazards method was used to analyze factors associated with time-dependent variables (i.e., time to CR, AIN, cIM, and second-line treatment, as well as survival). The potential cumulative and/or time-dependent nature of variables was taken in account.

Proportionality of hazard was assessed for each variable. Logistic regression was used to analyze factors associated with severe or recurrent infections. Variables that were statistically significant in the univariate analyses were included in the multivariate analyses. We investigated associations with the following characteristics and events: sex, consanguinity, cIMs/cancer in a first-degree relative, age at first and second cytopenia, sequence of cytopenia, AIN, hypogammaglobulinemia, time to ITP/AIHA CR, severe/recurrent infections, number of cIMs, number of second-line treatments. The 95% confidence intervals (CIs) for hazard ratios (HRs) and odds ratios (ORs) were not adjusted for multiple testing and should not be used to infer definitive effects. All tests were two-sided and a p-value < 0.05 was considered statistically significant. Statistical analyses were performed using R (ver. 4.0; R Development Core Team) and GraphPad Prism (ver. 8; GraphPad Software, Inc., San Diego, CA) software.

Results

Population

Of the 216 patients with pES, 151 were included in this study (Supplemental Figure 1). They were followed from 25 different centers. Patient characteristics are shown in Table 1. The median

(min–max) follow-up time from the first cytopenia diagnosis was 11.3 (5.1–38.0) years. Median age at final follow-up was 18.5 (6.8–50.0) years. In 20 cases (15%), follow-up was discontinued because the patient was considered cured (n = 11) or lost to follow-up (n = 9). Median age at loss to follow-up was 18.4 (6.8-25.1) years.

Hematological outcomes

AIN developed in 43 patients (28.5%). It was diagnosed 1 month before or after first cytopenia onset in 23/43 cases (53.5%), more than 1 month before in two cases (4.7%), and more than 1 month after in 18 cases (41.9%; maximal delay, 12.4 years). In all cases, the diagnosis was made before the age of 18 (median, 6.8; 0.6–16.2).

ITP and AIHA flare rates at 5 years of follow-up were calculated for the 61 alive patients who did not received a second-line treatment during this period. Forty-eight patients (79%) had experienced an ITP flare and 7 (11%) an AIHA flare.

The proportion of patients achieving sustained CR for ITP and AIHA steadily increased after cytopenia onset (Figure 1A). At 5 and 10 years, ITP was in sustained CR in 40.5% and

62.3% of patients (p = 0.02) and AIHA was in sustained CR in 54.5% and 74.1% of patients (p =

0.001), respectively. Sustained CR was achieved earlier for AIHA than ITP (median time to CR,

4.0 vs. 7.0 years; p = 0.01). At the final follow-up of the 135 surviving patients, the numbers of patients in CR, partial remission, and no remission were 126 (83%), 5 (3%), and 1 (1%) for

AIHA and 119 (79%), 8 (5%), and 5 (3%) for ITP, respectively (missing data in three cases).

Forty-six patients (34%) had no treatment ongoing at last follow-up. No particular characteristic was associated with AIHA or ITP CR, including cIM and bIM.

Over the first 3 decades, the proportions of patients achieving sustained CR increased with age (Figure 1B). ITP and AIHA were in CR in 26% and 30% of cases at 10 years compared to 50% and 72% at 20 years, respectively (p < 0.001 for both comparisons).

Immunopathological manifestations

A total of 122/151 patients (81%) had at least one IM. The data for each category and specific diagnosis are shown in Supplemental Table 2. 8

cIMs developed in 100/151 patients (66%). A total of 47 patients (31%) had two or more

IMs and 22 (15%) patients had three or more IMs (Supplemental Figure 2A). Patients with no cIMs had shorter median follow-up times (9.7 vs. 13 years; p = 0.0002) and were younger when data were collected (15 vs. 20 years; p < 0.0001). A cIM was diagnosed before the first cytopenia in 21/100, simultaneously in 13, and after in 66 cases (median delay, 3.7 years [0.2–20.5]; Figure

2A). Among the 185 cIMs, 29 (16%) were diagnosed before any second-line treatment. No cIM category had a statistically significant difference in frequency before and after first second-line treatment. The number of cIMs increased with age. At 10 compared to 20 years old, 37% and

74% of patients had at least one cIM and 9% and 34% of patients had at least two cIMs, respectively (p < 0.001 for both comparisons; Figure 2B).

The most common cIM categories were lymphoproliferation (n = 71), dermatological (n =

26), gastrointestinal/hepatic (n = 23) and pneumological manifestations (n = 16, Figure 3,

Supplemental Figure 2B). The most frequent cIM diagnosis are shown in Table 2. Thirteen patients developed a neurological manifestation as previously described.10 Four patients had a hematological malignancy (age at diagnosis): Hodgkin’s lymphoma (16 years), juvenile myelomonocytic leukemia (20 years), large granular lymphocytic leukemia (21 years) and angioimmunoblastic T-cell lymphoma (29 years). Older age at ES diagnosis (HR, 1.09; 95% CI,

1.01–1.17; p = 0.02), cIMs/cancer in a first-degree relative (HR, 1.64; 95% CI, 1.1–2.4; p =

0.006), and the presence of AIN were independently associated with the number of cIMs (HR,

2.41; 95% CI, 1.5–3.8; p = 0.0002).

Biological IMs (bIMs) were diagnosed in 101/151 patients (67%), and the frequency of bIMs also increased with the age (Figure 2C). Hypogammaglobulinemia was the most frequently diagnosed bIM (n = 54), including 44 cases diagnosed prior to any anti-CD20 treatment. Among those 54 patients, 25 (46%) received immunoglobulin replacement therapy. SLE and ALPS 9 biomarkers were present (regardless of whether patients met the diagnostic criteria) in 42 and 24 patients, respectively. At 10 and 20 years of age, 39% and 75% of patients had at least one bIM, respectively (p < 0.001).

Patients with bIMs were more likely to have cIMs (79% vs. 40%; p < 0.001), and patients with cIMs were more likely to have bIMs (80% vs. 41%; p < 0.001) but the correlation between the number of bIMs and cIMs was low (r = 0.27; p < 0.001).

Secondary pES

In 37 patients (24.5%), pES eventually revealed a SLE or a PID unknown at cytopenia onset.

Eleven patients (7.3%) eventually met the SLE SLICC diagnostic criteria.16 These patients were older at first cytopenia (median age, 13 vs. 5 years; p = 0.007) and almost exclusively female (1/88 males [1%] and 10/63 females [16%]); p < 0.001).

Seven patients (4.6%) met the diagnostic criteria for ALPS after pES onset which prompted targeted genetic analysis.17 Overall, 66/151 patients (44%) underwent genetic analyses as previously described.13 Among them, 26 (39%) patients were considered to have a PID

(including the 7 with ALPS). They carried a heterozygous pathogenic variant in CTLA4 (n = 7),

TNFRSF6 (germline n = 6, somatic n = 1), STAT3 (n = 5), PIK3CD (n = 1), CBL (n = 1), and

KRAS (somatic n = 1) or a homozygous/compound heterozygous pathogenic variants in LRBA (n

= 3) and RAG1 (n = 1). Compared to the 40 other patients, the 26 with a PID had more cIMs (2

[1-5] vs. 1 [0-4], p = 0.008) and a trend toward more bIMs as shown by Wilcoxon–Mann–

Whitney sum ranks comparison but same medians (1 [0-3] vs. 1 [0-2], p = 0.029). There was no statistically significant difference in the median time to ITP CR (4.7 vs. 8.0 years, p = 0.26) and to AIHA CR (5.5 vs. 5.5 years, p > 0.9), the number of second-line treatment received (3 [0-9] vs. 2 [0-6], p = 0.057) and mortality (2/26 [7.7%] vs. 3/40 [7.5%], p > 0.9). 10

Treatments

All except two patients (98.6%) had received at least one first-line treatment course. Second-line treatments (regardless of the hematological and/or extra-hematological indication) were required in 117/151 (77%) patients (Supplemental Figure 3A). Patients who did not receive any second- line treatment had shorter median follow-up times (10.5 vs. 12.3 years; p = 0.017). The median number of second-line treatments received was two (0–9).

The number of second-line treatments received increased with the time elapsed since first cytopenia without reaching a plateau (Supplemental Figure 3B). After a sustained CR for both

ITP and AIHA achieved, the number of treatments received had continued to increase: at 5 years after CR of both cytopenias, 67% of patients who achieved CR for both ITP and AIHA had received a new first and/or second-line treatments and 31% had received a new second-line treatment (Supplemental Figure 3C).

The number of second-line treatments received increased with age, particularly after the first decade (Figure 4A). At 10 and 20 years, 47% and 88% of patients had received a second-line treatment, respectively (p < 0.001). The number of patients receiving ongoing treatments also increased with age (Figure 4B). At 10 and 20 years, 27% and 69% of patients had received an active second-line treatment, respectively (p < 0.001). At the final follow-up, patients with a cIM had received more second-line treatments (median, 3 vs. 1; p < 0.0001).

The most frequently used second-line treatments were rituximab (n = 79; 52%), azathioprine (n = 55; 36%), splenectomy (n = 36; 24%), and mycophenolate (n = 29; 19%;

Supplemental Table 3).

The number of cIMs was associated with a subsequent increase in the number of second- line treatments received (HR, 1.4; 95% CI, 1.15–1.60; p = 0.0002). On the contrary, the number 11 of second-line treatment was not associated to a subsequent increase in the number of cIMs in univariate analysis (HR, 1.09; 95% CI, 0.98–1.22; p = 0.11).

Infections

In total, 53 (35%) patients had severe or recurrent infections (Supplemental Table 4). The most frequent were herpes zoster (n = 17), sinusitis/otitis media (n = 15), pneumopathy (n = 12), and bronchiectasis (n = 11). Patients with infections had more cIMs (median, 2 vs. 1; p < 0.0001), a higher incidence of hypogammaglobulinemia (53% vs. 28%; p = 0.003), and received more second-line treatments (median, 3 vs. 1; p < 0.0001). Among the 16 patients with severe infection, 9 (63%) were receiving an active treatment at infection time.

Severe/recurrent infections were independently associated with hypogammaglobulinemia

(OR, 2.4; 95% CI, 1.10–5.33; p = 0.03) and the number of second-line treatments (OR, 1.34; 95%

CI, 1.13–1.71; p = 0.002).

Mortality

Sixteen of the 151 patients followed for more than 5 years (10.6%) died, and seven other patients died before the fifth year of follow-up (23 deaths in total, 22 with available data). Patient survival at 5, 10, and 15 years after the first cytopenia was 97%, 92%, and 84%, respectively (Figure 5A).

Mortality rates in patients with pES were higher than those in patients with cITP or AIHA alone

(p < 0.0001 for both comparisons).

Deaths occurred regularly throughout the follow-up period (median delay after first cytopenia diagnosis, 8.9 [0.1–24.3] years) and at a median age of 18.0 (1.7–31.5) years (Figure

5B). In the majority of these patients, cytopenia was under control at the time of death: 15 (65%) and 19 (83%) patients had CR or partial remission from ITP and AIHA, respectively (Figure 5C). 12

Mortality was linked to the disease, the treatment, or both in 8 (36%), 2 (9%), and 12 (55%) cases, respectively. The most frequent cause of death was infections (n = 12 [52%]; Supplemental

Table 5). Four patients (18%) died of a hemorrhage, and all were less than 13 years of age. The patients who died from a hemorrhage were younger than those who died from an infection

(median, 10 vs. 18; p = 0.03). All of these patients, except for one who died in the first month of a cerebral hemorrhage, had at least one cIM. Eight of the patients (36%) had hypogammaglobulinemia.

The patients who died had received more second-line treatments than the others in the cohort (median, 3 vs. 2; p = 0.02), including splenectomy, which was more common in this subgroup (56% vs. 20%; p = 0.003). Patients who had received more than two second-line treatments had a three-fold increase in the risk of death compared to those who had received two or less (11/65 [16.9%] vs. 5/86 [5.8%], p = 0.03). At death, 81% of patients were receiving ongoing second-line treatment. The number of second-line treatments (HR, 1.3; 95% CI, 1.1–1.6; p = 0.004) and severe/recurrent infections (HR, 3.4; 95% CI, 1.2–9.7; p = 0.02) were independently associated with a higher risk of mortality after 5 years of follow-up.

Discussion

This large follow-up study of pES patients included more than 1,900 patient-years. Over the long term, AIHA and ITP were sustainably controlled in the majority of patients. Conversely, clinical and biological IMs increased in frequency and number with increasing patient age, finally affecting almost all adult patients. The number of cIMs was associated with a subsequent increase in the number of second-line treatments received. Mortality was high, frequently occurred while cytopenias were in remission, and most deaths concerned AYA. Two characteristics were associated to mortality: severe or recurrent infections and the number of second-line treatments

13 received. Overall, the age-related clinical picture showed similar trends for all patients, shifting from cytopenia to increased IMs, a greater treatment burden, and an increased risk of mortality.

In setting up a nationwide cohort, the CEREVANCE group tried to ensure unbiased patient inclusion in this study. Omitting patients with less than 5 years of follow-up data limited any bias due to short-term follow-up, which probably accounts for many of the discrepancies between previous studies. Indeed, our median follow-up period was more than twice as long as in previous studies (median, 4.8 years [3–7]).1,3–7 However, although the trends reported here are clear, some factors may also influence the estimates. The loss to follow-up mainly concerned

AYA and few patients were followed after 20 years old. As well, the CEREVANCE group recommends clinical and biological follow-up at least every 6-12 months but local practice or patients’ phenotype (such as the presence of cIM) may have influenced biological testing.

Sustained CR was eventually achieved for both types of cytopenia in the vast majority of patients, although this often took many years, especially for ITP (> 10 years for one-third of our patients). Because active treatments are used to treat most AYAs (notably because of cIMs), hematological CR may be drug induced and it is not possible to determine whether an underlying hematological autoimmunity is still present. The higher rate of sustained CR in ITP among patients with pES compared to patients with cITP alone may be due to more patients with pES receiving treatment.18

One of the most striking findings in this study was the progressive increase in the frequency and number of IMs. A range of cIMs, affecting almost every organ, were identified and developed independently of cytopenias. These findings clearly show that pES is a marker for a more general tendency toward immunodeficiencies despite we cannot exclude a contribution of the second-line treatments received to some IMs. The underlying etiology is not completely understood and may vary among patients, with both genetic and environmental factors being 14 important. Consequently, pES may be considered a composite syndrome with several overlapping subgroups of secondary pES. One of these subgroups includes patients with PIDs. Classically,

ALPS has been associated with pES.12 In this study, only 4% of patients were diagnosed with

ALPS based on well-defined criteria, despite evocative biological “ALPS-like” abnormalities in a larger proportion of patients.17 This observation is consistent with our previous study,13 which showed that more immune-response genes are potentially involved in pES than initially suspected.13,19–21 However, pES rarely comports as a Mendelian disease,1 and some of these variants may be predisposing rather than disease-causing alleles. Even in patients carrying a variant in a monogenic PID gene (e.g., TNFRFS6 or CTLA4),13,22 the altered genes show incomplete penetrance.23,24 We were unable to evaluate the proportion of patients who met common variable immunodeficiency disorders diagnostic criteria,25 as vaccine responses were not evaluable in all cases due to second-line treatments received. A second subgroup includes patients with SLE, although the prevalence of this subgroup is controversial.11,26,27 Our cohort suggests that SLE eventually occurs almost exclusively within the known at-risk population of female adolescents and is frequent in this subgroup, as it developed in 7/15 (47%) of the females

> 12 years.26 Despite its heterogeneity, the course of pES, in terms of age-related changes and trends, was similar for the majority of patients. The spectrum of IMs described here is probably influenced by the underlying etiology, and further analyses are needed to understand the determinant of IMs. The long-term follow-up of the present study confirms that the subgroup of patients with identified PID had more cIMs.13

Most patients required second-line treatments. These treatments reflect local practices and we cannot draw conclusions regarding their efficacy. We were unable to investigate the risk associated to specific treatments given the high heterogeneity in second-line treatments combination and duration as well as the changes in management practices since the cohort onset 15 in 2004. The rapid initial increase in second-line treatments is partly due to the high rate of early relapse and the current practice of administering steroid-sparing agents to treat pES.28 However, the presence of cytopenia is not the only reason for using these drugs and first- and second-line treatments were also used after CR of both cytopenias. cIMs are important in determining the number of second-line treatments used, but bIMs may also play a role, particularly in patients with SLE biomarkers, who are frequently given hydroxychloroquine. Nevertheless, second-line treatments are rarely selected based on a single factor. Patients with pES often have biological and clinical IMs, and the whole clinical picture needs to be assessed before selecting a treatment strategy. As previously reported,13 approximately one-third of patients may carry alterations in genes that are potentially accessible to targeted therapy.29–31 Given the high burden of second-line treatments and their association with infections and mortality, the CEREVANCE network has proposed implementing genetic analyses for all patients with pES to limit the use of immunosuppressive and toxic drugs.

Comprehensively, the pES clinical picture changes as patients age. From 10 to 20 years of age, cytopenia tends to be controlled but IMs are more prevalent, and active second-line treatments are used in more than two-thirds of patients during the pediatric-to-adult transition.

Overall, as patients age, the illness becomes more severe and the risk of mortality increases. Both

IMs and treatment burden contribute to the infection-related mortality peak observed at the end of the second decade. The patients who died had received more second-line treatments, including splenectomy. Because these two parameters are correlated (r = 0.60; p < 0.0001), the number of deaths was too low to determine whether splenectomy alone was a risk factor of mortality per se or a marker of severity.

In conclusion, pES must now be considered a complex multisystemic disease in which cytopenias frequently present fewer challenges than IMs and infections in long-term follow-up. 16

Adult patients with pES form a specific subgroup, distinct from older adults with ES.32

Multidisciplinary follow-up of patients with pES is needed and must focus on IMs screening, genetic diagnosis, infections prevention, patient-tailored drugs development, and AYA management. Specifically, the infection burden may be reduced by ensuring up-to-date vaccinations, eradicating chronic infections, and using adequate antimicrobial prophylaxis or immunoglobulin replacements. As in several chronic pediatric diseases,33 dedicated child-to-adult transition programs are warranted to improve outcomes in patients with pES.

References

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Table 1. Patient characteristics Number of patients 151

Sex ratio (Male/Female) 1.40 (88/63) Consanguinity, n (%) 12 (7.9) cIM/cancer in first-degree relative, n (%) 43 (28) Median age (years) at

First cytopenia (min–max) 5.4 (0.2–16.0)

ITP diagnosis (min–max) 6.7 (0.2–17.1)* AIHA diagnosis (min–max) 7.8 (0.2–21.5)*

ES diagnosis (min–max) 8.9 (0.2–21.5) Sequence of ES

Simultaneous, n (%) 52 (34.4) ITP then AIHA, n (%) 62 (41.1)

AIHA then ITP, n (%) 37 (24.5) Time between first and second cytopenia (years)

Median (min–max)** 2.5 (0.1-15.8) Direct antiglobulin test at AIHA diagnosis

IgG, n (%) 73 (48.3) IgG + C3, n (%) 61 (40.4)

Unspecified, n (%) 11 (7.3) C3, n (%) 4 (2.6)

IgA + C3, n (%) 1 (0.7) IgM then IgG, n (%) 1 (0.7) Duration of follow-up after first cytopenia (years)

Median (min–max) 11.3 (5.1–38.0)

Mean ± SD 12.5 ± 6.0 Age at last follow-up (years)

Median (min–max) 18.5 (6.8–50.0) Mean ± SD 19.1 ± 6.8

* p = 0.0076 **Considering the 99 patients with sequential cytopenias. Abbreviations: cIM, clinical immunopathological manifestation; Ig, immunoglobulin; SD, standard deviation; ITP, immune thrombocytopenic purpura; AIHA, autoimmune hemolytic anemia; ES, Evans syndrome.

Table 2. Most frequent clinical immunopathological manifestations (cIMs) diagnosis cIM n (%) cIM n (%)

Superficial (palpable) adenopathies 61 (40) Lymphoid enteropathy 5 (3)

Splenomegaly 49 (33) Chronic gastritis 5 (3)

Deep (abdominal or thoracic) adenopathies 16 (11) Polyarthritis 5 (3)

Granulomatous–lymphocytic interstitial lung disease 16 (11) Vitiligo 4 (3)

Cutaneous lupus erythematosus involvement 8 (5) Eczema 4 (3)

Autoimmune hepatitis 7 (5) Keratitis 4 (3)

Subtentorial inflammatory lesions 7 (5) Uveitis 4 (3)

Diagnosis present in at least four patients are shown and ordered by frequency. Complete diagnosis list is provided in Supplemental Table 2.

Figure Legends

Figure 1. Hematological outcomes (A) Cumulative incidence of patients achieving a sustained complete remission (CR). Among the 23 patients without sustained CR for AIHA, four (17%) had achieved sustained CR for ITP. Conversely, among the 32 patients without sustained CR for ITP, 13 (40%) had achieved sustained CR for AIHA. (B) Percentage of patients with a sustained complete remission according to age. Abbreviations: ITP, immune thrombocytopenic purpura; AIHA, autoimmune hemolytic anemia.

Figure 2. Immunopathological manifestations (IMs) (A) Age at first clinical IM (cIM) diagnosis and at first cytopenia. Pearson correlation coefficient r = 0.42, p < 0.0001. There was no difference in the median age at first cIM and at first cytopenia in terms of the number of cIMs (data not shown). (B) Cumulative incidence of cIMs according to age. Half of the patients had developed a cIM by the age of 13.5 years and a second IM by the age of 27 years. (C) Cumulative incidence of any biological IM (bIM), as well as each category. Half of the patients had at least one bIM diagnosed by 13.2 years of age. The biological workup was not standardized and was made at the clinician’s discretion.

Figure 3. Immunopathological manifestations (IMs) and other associated manifestations Individual occurrence of autoimmune neutropenia, clinical IMs (cIM), biological IMs (bIM), atopy, severe or recurrent infections, and malignancies. Each column represents a patient. The patients are ordered according to their cIMs, from the most (lymphoproliferation) to the least (hematological, other) frequent. Abbreviations: Hypoγ, hypogammaglobulinemia; SLE, systemic lupus erythematosus; ALPS, autoimmune lymphoproliferative syndrome.

Figure 4. Second-line treatments (A) Total number of second-line treatments received according to the age. (B) Number of second- line treatments ongoing according to age.

Figure 5. Long-term survival (A) Survival estimate of patients in terms of time from first cytopenia. At 10-year follow-up, survival rates among patients with cITP alone, AIHA alone and pES were 100%, 99% and 92%, respectively. (B) Mortality is shown in terms of time from first cytopenia, as well as age. Individual values are shown as dots with medians and interquartile ranges shown as lines. (C) Hematological status at death. Abbreviations: (c)ITP, (chronic) immune thrombocytopenic purpura; AIHA, autoimmune hemolytic anemia; ES, Evans syndrome; CR, complete remission; PR, partial remission; NR, no remission.

Long term follow-up of pediatric-onset Evans syndrome: broad immunopathological manifestations and high treatment burden

Supplemental data Page Supplemental Table 1. Patient selection and definitions 2 Supplemental Table 2. Immunopathological and other manifestations 3 Supplemental Table 3. Second-line treatments received 6 Supplemental Table 4. Severe or recurrent infections 7 Supplemental Table 5. Characteristics of patients who died 8 Supplemental Figure 1. Selection of patients within the OBS’CEREVANCE database 9 Supplemental Figure 2. Clinical immunopathological manifestations 10 Supplemental Figure 3. Second-line treatments 11 Appendix 12

Supplemental Table 1. Patient selection and definitions Inclusion criteria Evans syndrome (ES) is defined as the simultaneous (less than 1 month) or sequential association of immune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (AIHA). Patients are included in the cohort if they are less than 18 years old at first cytopenia diagnosis, regardless of the age at inclusion. Follow-up is calculated from first cytopenia diagnosis. ITP, AIHA and ES definitions ITP is defined according to the international working group criteria (Rodeghiero et al., Blood 2009). AIHA is defined as Hb < 110 g/L with a positive direct antiglobulin test (DAT) and at least one of the following hemolysis criteria: reticulocyte count > 120 G/L, free bilirubin > 17 mmol/L, or haptoglobin < 10 mg/dL. Autoimmune neutropenia (AIN) is defined as peripheral neutropenia < 1 G/L persisting for more than 6 months that is neither infection nor drug driven, regardless of the presence or absence of neutrophil autoantibodies. Patients with AIHA and autoimmune neutropenia (AIN) or with ITP and AIN are not classified as ES in the present analysis; patients with isolated DAT or anti-platelet antibodies, or compensated hemolysis without AIHA were only considered as having ES when cytopenia was present. Exclusion criteria Inherited red cell or platelet disease. Autoimmune cytopenia secondary to human immunodeficiency virus infection. Chemotherapy before ES. Bone marrow or organ transplantation before ES. Known PIDs before ES onset. Systemic lupus erythematosus known before ES onset. Patients living outside metropolitan France. Immune thrombocytopenic purpura and autoimmune hemolytic anemia status ITP and AIHA status were separately classified as follows: no remission (NR) = platelet count < 30 G/L or Hb < 70 g/L; partial remission (PR) = platelet count 30–100 G/L or Hb 70–110 g/L, with reticulocytes > 120 G/L; complete remission (CR) = platelet count > 100 G/L or Hb ≥ 110 g/L, with reticulocytes ≤ 120 G/L. Cytopenia flare was defined as change of status from CR or PR to NR, whether the CR or PR has been reached spontaneously or after a first-line treatment. Immunopathological manifestations Lymphoproliferation: significant splenomegaly without hemolysis or significant persistent lymphadenopathy (> 1 cm), eventually requiring a biopsy. Clinical IMs: lymphoproliferation or autoimmune / autoinflammatory organ disease : pulmonary (granulomatous-lymphocytic interstitial lung disease), liver and digestive tract

problems (lymphoïd enteropathy, coeliac-like disease, inflammatory bowel disease, chronic gastritis, autoimmune or giant cell hepatitis, cirrhosis), neurologic (autoimmune or autoinflammatory or granulomatous or vascular manifestations), endocrinologic (autoimmune thyroiditis, Grave’s disease or isolated specific autoantibodies), dermatologic (psoriasis, vitiligo, alopecia, vasculitis) and other autoimmune or autoinflammatory manifestations (ophthalmologic, cardiac, renal or hematological). Biological IMs*: hypogammaglobulinemia, systemic lupus erythematosus (SLE) biomarkers, or autoimmune lymphoproliferative syndrome (ALPS) first line biomarkers. Hypogammaglobulinemia: Immunoglobulin (Ig)G levels below the mean for the subject’s age (at least 2 SD) on two separate samples, associated or not with IgA levels below the mean for the subject’s age (at least 2 SD) or with defects in vaccine immunizations. SLE biomarkers*: antinuclear antibodies titer > 1/160; anti double-stranded DNA or Smith, hypocomplementemia, and lupus anticoagulant (all in two separate samples). ALPS biomarkers*: persistent hypergammaglobulinemia (at least 2 SD over the mean for age), or high counts of circulating TCRαβ CD4- CD8- double negative T lymphocytes. Severe or recurrent infections Bacteremia, pneumonia, bronchiectasis, recurrent pyogenes infections, severe herpes virus infections, or opportunistic infections. Second-line treatments All immunomodulatory or immunosuppressive treatments (including splenectomy), except steroids or intravenous immunoglobulins (considered as first-line treatments). Loss to follow-up A patient is classified “lost to follow-up” if no data are available for more than 2 years and no future appointments are scheduled. * The biological workup is made at the clinician’s discretion, which is annually in most cases.

Supplemental Table 2. Immunopathological and other manifestations Median age (years) Number (%) at onset (min–max) Clinical IMs Lymphoproliferation 71 (47) 7.8 (0–41) Superficial (palpable) adenopathies 61 (40.4) 8 (0–41) Deep (abdominal or thoracic) adenopathies 16 (10.6) 10.9 (2.7–19.1) Splenomegaly 49 (32.5) 7.6 (0–19) Associated hepatomegaly 17 (11.3) 5.0 (0.3–19) Pneumological 16 (10.6) 14.3 (4.6–27.7) Granulomatous–lymphocytic interstitial lung disease 16 (10.6) 14.3 (4.6–27.7) Gastrointestinal/hepatic 23 (15.2) 13 (0.7–25.9) Coeliac disease 3 (2) 1 (0.7–14.9)

Autoimmune hepatitis 7 (4.6) 11.3 (1.6–14.7) Lymphoid enteropathy 5 (3.3) 16.4 (6–25.9) Chronic gastritis 5 (3.3) 16.5 (6.5–19.4) Inflammatory bowel disease 2 (1.3) 14.4 (9.8–18.9) Rosai–Dorfman 1 (0.7) 7.3 Primary sclerosing cholangitis 1 (0.7) 13 (13–13) Neurological 13 (8.6) 16.3 (7.8–27.9) Vasculitis 3 (2) 13.1 (12.1–17.8) Lymphoproliferation 1 (0.7) 7.8 Myelitis 2 (1.3) 26.7 (25.4–27.9) Infratentorial inflammatory lesions 2 (1.3) 18.2 (11.9–24.6) Subtentorial inflammatory lesions 7 (4.6) 14.2 (7.8–27.9) Sensitive ganglionopathy 1 (0.7) 16.3 Endocrinological 5 (3.3) 12.2 (3–41.1) Type 1 diabetes 2 (1.3) 3.2 (3–3.3) Grave's hyperthyroidism 2 (1.3) 29.1 (17.1–41.1) Hashimoto thyroiditis 1 (0.7) 12.2 Dermatological 26 (17.2) 15.5 (0–27.3) Vitiligo 4 (2.6) 3 (0–15.5) Chronic urticaria 2 (1.3) 8.2 (0.8–15.6) Psoriasis 3 (2) 21.1 (15.6–27.3) Bullous pemphigoid 1 (0.7) 15.5 Eczema 4 (2.6) 10.9 (0–17.2) Cutaneous lupus erythematosus involvement 8 (5.3) 17.2 (12.1–21.5) Granuloma 2 (1.3) 7.8 (0–15.6) Immunoglobulin A vasculitis 2 (1.3) 11.8 (5.7–17.9) Sjögren's syndrome 1 (0.7) 15.5 Livedo reticularis 1 (0.7) 17.6 Rheumatological 12 (7.9) 15.1 (2.6–41.1) Monoarthritis 3 (2) 13.7 (2.6–15.4) Oligoarthritis 2 (1.3) 16.6 (16.3–16.9) Polyarthritis 5 (3.3) 14.4 (5.9–41.1) Polyarticular pain without arthritis 3 (2) 15 (13.7–19.3) Cardiological 4 (2.6) 15.9 (12.7–20.9) Pericarditis 3 (2) 15.2 (12.7–20.9) Granulomatous aortitis 1 (0.7) 16.6 Ophthalmological 8 (5.3) 9.6 (6.1–15.6) Keratitis 4 (2.6) 8.7 (6.1–9.7) Uveitis 4 (2.6) 12 (7.9–15.6) Ischemic optic neuropathy 1 (0.7) 12.1

Nephrological 3 (2) 14.2 (10.6–16.4) Tubulopathy 1 (0.7) 10.6 Granulomatous infiltration 2 (1.3) 15.3 (14.2–16.4) Hematological, other 2 (1.3) 6.9 (2.9–10.9) Myelofibrosis 1 (0.7) 10.9 Acquired thrombotic thrombocytopenic purpura 1 (0.7) 2.9 Biological IMs Hypogammaglobulinemia 54 (35.8) 10 (2.3–23) Low IgG before anti-CD20 44 (29.1) 10.5 (2.3–22.4) Low IgG persisting after anti-CD20 8 (5.3) 9.5 (5–23) Low IgA 5 (3.3) 11 (6.5–15.8) Immunoglobulin replacement therapy 24 (15.9) 9.4 (2.3–22.4) SLE biomarkers 42 (27.8) 10.2 (0.3–19.5) Antinuclear antibodies 38 (25.2) 9.8 (0.3–19.5) Anti double-stranded DNA or anti Smith 6 (4) 14.7 (6–19.5) Hypocomplementemia 10 (6.6) 12.6 (0.3–19.5) Lupus anticoagulant 6 (4) 10.6 (0.8–16) ALPS biomarkers 24 (15.9) 8.4 (2.8–26.7) Hypergammaglobulinemia 14 (9.3) 7.8 (2.8–26.7) Elevated Fas ligand 5 (3.3) 8 (4–10.8) Defective Fas-mediated apoptosis 3 (2) 9.6 (4–13.5) Increase IL-10 and/or vitamin B12 2 (1.3) 13.8 (13.5–14) Elevated alpha beta double-negative T cells 10 (6.6) 9.2 (5.2–15) Other manifestations Granuloma on pathology, any localization 9 (6) 16.57 (0–26.2) Lymph node 3 (2) 14.6 (2.7–17.8) Lung 3 (2) 10.1 (7.4–26.2) Cutaneous 2 (1.3) 7.8 (0–15.6) Another organ 4 (2.6) 15.4 (2.7–18) Malignancies 4 (2.6) 20.5 (16.3–28.8) Angioimmunoblastic T-cell lymphoma 1 (0.7) 28.8 Juvenile myelomonocytic leukemia 1 (0.7) 20 EBV-negative Hodgkin Lymphoma IVBb 1 (0.7) 16.3 Large granular lymphocytic leukemia 1 (0.7) 21 A given patient may have several manifestations within the same category. Abbreviations: IMs, immunopathological manifestations; EBV, Epstein–Barr Virus; Ig, immunoglobulin; SLE, systemic lupus erythematosus; ALPS, autoimmune lymphoproliferative syndrome; IL, interleukin.

Supplemental Table 3. Second-line treatments received At treatment start (years) Number Percentage Median time after Second-line treatment of of patients first cytopenia (min– Median age (min–max) patients max) Rituximab 79 52.3 5.6 (0.0–35.5) 14.1 (0.8–47.4)

Azathioprine 55 36.4 3.8 (0.0–27.8) 12.4 (0.5–39.8)

Splenectomy 36 23.8 6.7 (0.1–16.0) 11.1 (1.5–19.8)

Mycophenolate 29 19.2 6.1 (0.1–29.1) 11.5 (2.4–14.7)

Ciclosporin 28 18.5 4.0 (0.2–15.0) 10.8 (0.8–19.8)

Hydroxychloroquine 27 17.9 5.9 (0.0–17.5) 15.8 (5.6–39.8)

Mercaptopurine 12 7.9 4.8 (0.2–11.9) 13.9 (2.8–18.3)

Romiplostim 12 7.9 14.7 (0.5–38.1) 19.4 (5.3–50.0)

Vinblastine 11 7.3 5.5 (0.3–24.3) 13.8 (1.8–36.5)

Cyclophosphamide 10 6.6 3.2 (0.2–18.5) 11.6 (1.8–23.8)

Colchicine 8 5.3 4.3 (0.9–20.9) 15.3 (9.3–30.6) Sirolimus 8 5.3 6.7 (3.2–11.4) 11.4 (8.0–19.8) Vincristine 7 4.6 7.9 (0.1–16.6) 12.3 (0.5–20.8) Eltrombopag 6 4.0 14.7 (6.1–35.4) 22.6 (9.0–47.3) Abatacept 5 3.3 12.5 (7.5–20.5) 20.0 (9.7–26.5) Dapsone 4 2.6 6.7 (1.6–11.2) 11.9 (4.3–19.0) Anti-D immunoglobulin 3 2.0 0.2 (0.0–7.1) 2.9 (2.4–14.7) Allogenic hematopoietic 3 2.0 8.7 (1.2–14.2) 11.5 (2.5–18.2) stem-cell transplantation Danazol 2 1.3 6.7 (2.9–10.5) 12.7 (5.7–19.7) Everolimus 1 0.7 11.7 14.3 For treatment start assessments only, each separate course has been included for a given patient.

Supplemental Table 4. Severe or recurrent infections Infection type Number (%) Infection type Number (%)

Oral or genital herpes Herpes zoster 17 (11.3) 4 (2.6) simplex infection Sinusitis/otitis media 15 (9.9) Aphthous stomatitis 4 (2.6)

Pneumopathy 12 (8.0) Cellulitis 3 (2.0)

Nontuberculous Bronchopathy 11 (7.3) 2 (1.3) mycobacteria Bacteremia* 8 (5.3) Meningitis*** 2 (1.3)

Impetigo/furuncle/cutaneous Chronic blood EBV 7 (4.6) 2 (1.3) abscesses replication Other 7 (4.6) Cutaneous warts 2 (1.3)

Gastrointestinal infections** 5 (3.1) Molluscum contagiosum 2 (1.3)

Other (once each): cerebral toxoplasmosis, pityriasis versicolor, oral abscess, anogenital warts, bacterial arthritis, CMV disseminated infection, parotitis. * Microorganisms identified: Staphylococcus aureus, Pasteurella, Streptococcus pneumococcus, Pseudomonas aeruginosa ** Microorganisms identified: Campylobacter, Salmonella *** Microorganisms identified: HSV, Streptococcus pneumococcus The onset times of recurrent infections could not be determined. Therefore, age data were not collected. Abbreviations: HSV, herpes simplex virus; EBV, Epstein–Barr virus; CMV, cytomegalovirus.

Supplemental Table 5. Characteristics of patients who died Second-line treatments

Delay Year Age at after first Sex of death IM Nb Sequence Cause of death cytopenia death (years) (years) M 2006 0.1 1.7 No 1 VBL Cerebral hemorrhage CsA, RTX, SPX, AZT, VCR, CPX, F 2002 1.8 3.8 Yes 7 Sepsis HSCT F 2018 0.9 3.9 Yes 1 CD20 Fulminant hepatitis M 2001 1.6 5.0 Yes 3 CsA, AZT, CD20 Sepsis M 1996 3.0 8.3 Yes 4 AZA, VCR, DAP, SPX Cerebral hemorrhage F 2017 5.2 10.7 Yes 1 AZT HLH and cerebral vasculitis M 2001 1.3 11.6 Yes 2 CD20, CsA Pancreatitis M 2010 10.3 11.9 Yes 3 CsA, AZT, CD20 Gastrointestinal hemorrhage M 1995 8.1 12.7 Yes 3 CsA, SPX, VBL Cerebral hemorrhage M 1995 8.8 15.5 Yes 2 SPX, CsA Sepsis F 2007 12.9 17.8 Yes 1 CD20 Sepsis M 2010 16.4 18.2 Yes 4 AZT, SPX, MMF, CD20 Sepsis F 2014 8.9 18.4 Yes 4 AZT, CD20, CsA, 6MP EBV-related lymphoproliferation SPX, CPX, HCQ, AZT, CD20, F 2002 14.6 18.5 Yes 7 Post-HSCT CMV infection CsA, HSCT F 2016 12.4 19.3 Yes 4 CD20, AZT, CPX, MMF Fulminant pneumococcal infection F 2017 6.0 19.4 Yes 0 Unknown

M 2013 15.1 20.0 Yes 3 CD20, SPX, 6MP JMML F 2013 5.1 21.1 Yes 4 CD20, HCQ, SPX, CPX Vasculitis, SLE F 2012 12.9 24.5 Yes 3 SPX, CD20, AZT Probably sepsis M 2011 24.2 28.1 Yes 5 SPX, CsA, AZT, HCQ, MMF Sepsis M 2017 24.3 28.9 Yes 1 DAP Probably sepsis MMF, CsA, HCQ, CD20, SPX, M 2015 18.3 31.5 Yes 6 Probably sepsis TPO-RA Patients are ordered by age at death. Among the 22 patients with available data, 16 were being followed by pediatric teams, and six patients (aged 20–31.5 years) were being followed by adult teams. Abbreviations: IM, immunopathological manifestation; Nb, number; DAP, dapsone; AZT, azathioprine; CD20, rituximab; CPX, cyclophosphamide; MMF, mycophenolate; CsA, ciclosporine; HCQ, hydroxychloroquine; TPO-RA, thrombopoietin receptor agonist; VBL, vinblastine; SLE, systemic erythematosus lupus; SPX, splenectomy; HSCT, hematopoietic stem-cell transplantation; HLH, hemophagocytic lymphohistiocytosis; EBV, Epstein–Barr virus; CMV, cytomegalovirus. JMML: Juvenile myelomonocytic leukemia

Total number of patients included in OBS’CEREVANCE cohort n = 1,505

Evans syndrome Isolated AIHA Isolated cITP n = 216 n = 444 n = 845

Known secondary Evans syndrome at inclusion n = 6* Secondary cytopenia Isolated AIHA n = 23 Isolated cITP n = 15 Less than 5 years of follow-up from first cytopenia n = 59

Patients included n = 151

Patients included as comparators in the survival analysis Patients included in the survival analysis Isolated AIHA n = 421 n = 210 Isolated cITP n = 830

*post allogeneic hematopoietic stem cell transplantation (n = 3), Autoimmune lymphoproliferative syndrome (n = 2), 22q11 microdeletion (n = 1).

Supplemental Figure 1. Selection of patients within the OBS’CEREVANCE database The numbers shown are those recorded on the day of data extraction (21 June 2019). Among the 151 patients with Evans syndrome with more than 5 years of follow-up, 115 were alive and actively followed by a pediatric hematologist (n = 73), an adult internist (n = 37), an adult hematologist (n = 4), or an adult gastroenterologist (n = 1). Abbreviations: AIHA, autoimmune hemolytic anemia; cITP, chronic immune thrombocytopenic purpura.

A B 60 s

t 50

51 53 n e

(34%) (35%) i t

40 a 40

f n

25 o

i 30 t

(17%) e 30

14 t

f n o 20 20

(9%)

c e

4 r b

10 (3%) 2 2 e 10

m P

u (1%) (1%) N 0 0 0 1 2 3 4 5 6 0 5 10 15 20 25 30 Number of cIM Age (years) Number at risk Lymphoproliferation 131 100 66 37 16 7

Dermatological 147 133 101 52 19 8 Pneumological 151 134 103 58 23 8 Gastrointestinal/Hepatic 149 132 97 55 23 9

Neurological 151 137 106 60 25 9

Rheumatological 151 136 106 58 24 9

Ophthalmological 151 137 108 63 27 10

Cardiological 151 136 106 62 26 10

Endocrinological 150 137 110 62 26 10

Nephrological 151 139 110 64 27 10 Hematological, other 150 138 110 64 27 10

Supplemental Figure 2. Clinical immunopathological manifestations (cIMs) (A) Number of cIMs across the cohort. (B) Cumulative incidence of each cIM and numbers at risk.

A B

100 Number of treatments 35 40 34 1 (23%)

(23%) s 80 t

n 2

i t

n 26

30 a 3

p i

(17%) 60

f a

o 4

18 e

f 16 g 5

(12%) a

o 20 t

(11%) 40 r

n 6

e e

9 c

b r

6 e 7 m (6%) 5 10 P

u 20

(4%) (3%) 2 8 N (1%) 9 0 0 0 1 2 3 4 5 6 7 8 9 0 5 10 15 20 25 Number of treatments Time after first cytopenia onset (years)

100 All treatments

Second-line only

s t

n 80

f 60

e g

a 40

c r

e 20 P

0 0 3 6 9 12 Time after complete remission Number at risk All treatments 19 8 5 3 Second-line only 28 18 11 6

Supplemental Figure 3. Second-line treatments (A) Number and percentage of second-line treatments in the 151 patients with Evans syndrome (ES). Among these patients, 34 (23%) had none, 35 (23%) had one, and 82 (54%) had two or more second-line treatments. The median number of treatments received (2 [0–9]) was higher for ES than for cITP (1 [0–14]; p < 0.0001) but not AIHA (2 [0–7]; p = 0.7) alone. (B) Cumulative number of second-line treatments after first cytopenia onset. Half of the patients had received at least one, two, and three different treatments at 2.7, 10.5, and 14.7 years after first cytopenia diagnosis, respectively. (C) Cumulative incidence of treatments received after complete hematological remission, including the 61 patients with > 1 year of follow-up after complete remission from cytopenia. Second-line treatments are shown in red. All treatments (i.e., second- line, steroid, and therapeutic intravenous immunoglobulin treatments) are shown in blue. a

Appendix

Collaborators: Louis Terriou,1 Jean-François Viallard,2 Pierre Duffau,3 Arnaud Hot,4 Isabelle Durieu,5 Lionel Galicier,6 Claire Fieschi,6 Pierre Cougoul,7 Françoise Sarrot-Reynauld,8 Bertrand Godeau,9 Marc Michel,9 Gaëtan Sauvetre,10 Mikael Ebbo,11 Felipe Suarez,12 Cyrille Hoarau,13 Mohamed Hamidou,14 Agathe Masseau,14 Christian Lavigne,15 Frederic Bauduer,16 Dominique Bordessoule,17 Robert Navarro,18 Alexis Mathian,19 Olivier Fain,20 Frédérique Roy-Peaud,21 Guillaume Denis,22 and Anne-Sophie Korganow.22

1 Department of Internal Medicine and Immunology, Claude-Huriez University Hospital, Lille, France 2 Department of Internal Medicine, Haut-Lévêque Hospital, Bordeaux University Hospital, Pessac, France 3 Department of Internal Medicine, Saint-André Hospital, Bordeaux University Hospital, Bordeaux, France 4 Department of Internal Medicine, Edouard Herriot University Hospital, Lyon, France 5 Department of Internal Medicine, Lyon University Hospital, and Équipe d’Accueil Health Services and Performance Research (HESPER) 7425, Lyon University, Lyon, France 6 Department of Clinical Immunology, Saint Louis University Hospital, AP-HP, and EA3518, Paris University, Paris, France 7 Department of Internal Medicine, Cancer University Institute of Toulouse-Oncopole, Toulouse, France 8 Department of Internal Medicine, University Hospital of Grenoble, Grenoble, France 9 Department of Internal Medicine, National Referral Center for Adult's Immune Cytopenias (CERECAI), Henri Mondor University Hospital, AP-HP, Créteil, France 10 Department of Internal Medicine, Charles Nicolle University Hospital, Rouen, France 11 Department of Internal Medicine, La Timone Hospital, Marseille University Hospital, Marseille, France 12 Department of Hematology, French National Reference Center for Primary Immune Deficiencies, Necker Hospital for Sick Children University Hospital, Imagine Institute, Paris, France 13 Department of Allergology and Immunology, Clocheville Hospital, CHRU de Tours, Tours, France 14 Department of Internal Medicine, Hôtel Dieu University Hospital, Nantes, France 15 Internal Medicine and Vascular Diseases Department, Angers University Hospital, Angers, France 16 Department of Hematology, Côte Basque Hospital, Bayonne, France 17 Department of Hematology, Limoges University Hospital, Limoges, France 18 Department of Hematology, Montpellier University Hospital, Montpellier, France 19 Department of Internal Medicine 2, Pitié-Salpêtrière University Hospital, AP-HP, Paris, France 20 Department of Internal Medicine, Saint Antoine University Hospital, AP-HP, and Sorbonne University, Paris, France 21 Department of Internal Medicine and Infectious Diseases, Poitiers University Hospital, Poitiers, France 22 Department of Internal Medicine and Hematology, Rochefort Hospital, Rochefort, France 23 Department of internal Medicine and Clinical immunology, Strasbourg University Hospital, Strasbourg, France

We thank the following pediatricians and their teams in charge of the patients at the pediatric age: G. Leverger (AP-HP A.Trousseau, n = 22); N. Aladjidi (Bordeaux, n = 15); V. Barlogis (AP-HM, Marseille, n = 12); Y. Bertrand (IHOP Lyon, n = 12); B. Neven (AP-HP Necker, n = 12); W.

Abou Chahla (Lille, n = 11); M. Pasquet (Toulouse, n = 9); T. Leblanc (AP-HP R.Debré, n = 7); C. Guitton (AP-HP Bicêtre, n = 6); A. Marie-Cardine (Rouen, n = 5); I. Pellier (Angers, n = 5); C. Armari-Alla (Grenoble, n = 4); J. Benadiba (Nice, n = 4); P. Blouin (Tours, n = 4); E. Jeziorski (Montpellier, n = 3); F. Millot (Poitiers, n = 3); C. Paillard (Strasbourg, n = 3); C. Thomas (Nantes, n = 3); N. Cheikh (Besançon, n = 2); S. Bayart (Rennes, n = 2); F. Fouyssac (Nancy, n = 2); C. Piguet (Limoges, n = 2); M. Deparis (n = 1, Caen); C. Briandet (Dijon, n = 1); and E. Dore (Clermont-Ferrand, n = 1).

In total, 51 of the 151 patients with Evans syndrome were actively followed by an adult team. We thank the following adult clinicians and their teams: L.Terriou (Lille, n = 7); J.F Viallard, P. Duffau (Bordeaux, n = 5); A. Hot, I. Durieu (Lyon, n = 4); L. Galicier, C. Fieschi (AP-HP, Saint Louis, n = 4); P. Cougoul (Toulouse, n = 4); F. Sarrot-Reynauld (Grenoble, n = 3); B. Godeau, M. Michel (AP-HP, H. Mondor, n = 3); G. Sauvetre (Rouen, n = 3); M. Ebbo (Marseille, n = 3); F. Suarez (AP-HP, Necker, n = 2); C. Hoarau (Tours , n = 2); M. Hamidou, A Masseau (Nantes , n = 2); C. Lavigne (Angers, n = 1); F. Bauduer (Bayonne, n = 1); D. Bordessoule (Limoges, n = 1); R. Navarro (Montpellier, n = 1); A. Mathian (APHP, Pitié Salpêtrière, n = 1); O. Fain (APHP, St Antoine, n = 1); F. Roy-Peaud (Poitiers, n = 1); G. Denis (Rochefort, n = 1); and A.S Korganow (Strasbourg, n = 1).