Diagnostic evaluation of paediatric autoimmune lymphoproliferative immunodeficiencies (ALPID): a prospective cohort study.

BACKGROUND: Lymphoproliferation and autoimmune cytopenias characterise autoimmune lymphoproliferative syndrome. Other conditions sharing these manifestations have been termed autoimmune lymphoproliferative syndrome-like diseases, although they are frequently more severe. The aim of this study was to define the genetic, clinical, and immunological features of these disorders to improve their diagnostic classification. METHODS: In this prospective cohort study, patients were referred to the Center for Chronic Immunodeficiency in Freiburg, Germany, between Jan 1, 2008 and March 5, 2022. We enrolled patients younger than 18 years with lymphoproliferation and autoimmune cytopenia, lymphoproliferation and at least one additional sign of an inborn error of immunity (SoIEI), bilineage autoimmune cytopenia, or autoimmune cytopenia and at least one additional SoIEI. Autoimmune lymphoproliferative syndrome biomarkers were determined in all patients. Sanger sequencing followed by in-depth genetic studies were recommended for patients with biomarkers indicative of autoimmune lymphoproliferative syndrome, while IEI panels, exome sequencing, or genome sequencing were recommended for patients without such biomarkers. Genetic analyses were done as decided by the treating physician. The study was registered on the German Clinical Trials Register, DRKS00011383, and is ongoing. FINDINGS: We recruited 431 children referred for autoimmune lymphoproliferative syndrome evaluation, of whom 236 (55%) were included on the basis of lymphoproliferation and autoimmune cytopenia, 148 (34%) on the basis of lymphoproliferation and another SoIEI, 33 (8%) on the basis of autoimmune bicytopenia, and 14 (3%) on the basis of autoimmune cytopenia and another SoIEI. Median age at diagnostic evaluation was 9·8 years (IQR 5·5-13·8), and the cohort comprised 279 (65%) boys and 152 (35%) girls. After biomarker and genetic assessments, autoimmune lymphoproliferative syndrome was diagnosed in 71 (16%) patients. Among the remaining 360 patients, 54 (15%) had mostly autosomal-dominant autoimmune lymphoproliferative immunodeficiencies (AD-ALPID), most commonly affecting JAK-STAT (26 patients), CTLA4-LRBA (14), PI3K (six), RAS (five), or NFκB (three) signalling. 19 (5%) patients had other IEIs, 17 (5%) had non-IEI diagnoses, 79 (22%) were unresolved despite extended genetics (ALPID-U), and 191 (53%) had insufficient genetic workup for diagnosis. 16 (10%) of 161 patients with a final diagnosis had somatic mutations. Alternative classification of patients fulfilling common variable immunodeficiency or Evans syndrome criteria did not increase the proportion of genetic diagnoses. INTERPRETATION: The ALPID phenotype defined in this study is enriched for patients with genetic diseases treatable with targeted therapies. The term ALPID might be useful to focus diagnostic and therapeutic efforts by triggering extended genetic analysis and consideration of targeted therapies, including in some children currently classified as having common variable immunodeficiency or Evans syndrome. FUNDING: Deutsche Forschungsgemeinschaft under Germany's Excellence Strategy. TRANSLATION: For the German translation of the abstract see Supplementary Materials section.

Autoimmune lymphoproliferative immunodeficiencies (ALPIDs): A proposed approach to redefining ALPS and other lymphoproliferative immune disorders.

Chronic nonmalignant lymphoproliferation and autoimmune cytopenia are relevant manifestations of immunohematologic diseases of childhood. Their diagnostic classification is challenging but important for therapy. Autoimmune lymphoproliferative syndrome (ALPS) is a genetically defined inborn error of immunity combining these manifestations, but it can explain only a small proportion of cases. Diagnostic categories such as ALPS-like disease, common variable immunodeficiency, or Evans syndrome have therefore been used. Advances in genetics and increasing availablity of targeted therapies call for more therapy-oriented disease classification. Moreover, recent discoveries in the (re)analysis of genetic conditions affecting FAS signaling ask for a more precise definition of ALPS. In this review, we propose the term autoimmune lymphoproliferative immunodeficiencies for a disease phenotype that is enriched for patients with genetic diseases for which targeted therapies are available. For patients without a current molecular diagnosis, this term defines a subgroup of immune dysregulatory disorders for further studies. Within the concept of autoimmune lymphoproliferative immunodeficiencies, we propose a revision of the ALPS classification, restricting use of this term to conditions with clear evidence of perturbation of FAS signaling and resulting specific biologic and clinical consequences. This proposed approach to redefining ALPS and other lymphoproliferative conditions provides a framework for disease classification and diagnosis that is relevant for the many specialists confronted with these diseases.

Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing.

BACKGROUND: Elevated TCRαß+CD4-CD8- double-negative T cells (DNT) and serum biomarkers help identify FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified on standard exon sequencing (ALPS-undetermined: ALPS-U). OBJECTIVE: We sought to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. METHODS: Genetic analysis included whole FAS gene sequencing, copy number variation analysis, and sequencing of FAS cDNA and other FAS pathway-related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss of heterozygosity (sLOH). RESULTS: Nine of 16 patients with ALPS-U lacked FAS expression on CD57+DNT predicting heterozygous "loss-of-expression" FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7 of 9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT; 1 patient showed a FAS exon duplication. Three patients had reduced FAS expression, and 2 of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the 4 ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. CONCLUSION: A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.

Understanding the Spectrum of Immune Dysregulation Manifestations in Autoimmune Lymphoproliferative Syndrome and Autoimmune Lymphoproliferative Syndrome-like Disorders.

As a disorder of immune dysregulation, autoimmune lymphoproliferative syndrome (ALPS) stems from pathogenic variants in the first apoptosis signal-mediated apoptosis (Fas) and Fas-ligand pathway that result in elevations of CD3+ TCRαß+ CD4- CD8- T cells along with chronic lymphoproliferation, a heightened risk for malignancy, and importantly for the rheumatologist, increased risk of autoimmunity. While immune cytopenias are the most encountered autoimmune phenomena, there is increasing appreciation for ocular, musculoskeletal, pulmonary and renal inflammatory manifestations similar to more common rheumatology diseases. Additionally, ALPS-like conditions that share similar clinical features and opportunities for targeted therapy are increasingly recognized via genetic testing, highlighting the need for rheumatologists to be facile in the recognition and diagnosis of this spectrum of disorders. This review will focus on clinical and laboratory features of both ALPS and ALPS-like disorders with the intent to provide a framework for rheumatologists to understand the pathophysiologic drivers and discriminate between diagnoses.

Autoimmune lymphoproliferative syndrome: A disorder of immune dysregulation.

Autoimmune Lymphoproliferative Syndrome (ALPS) is an autoimmune disease that has been reported in over 2200 patients. It is a rare, genetic disease where pathogenic variants occur in the extrinsic pathway of apoptosis. Various mutations in different genes, such as FAS, FASL, and CASP10, can result in ALPS. Most commonly, pathogenic variants occur in the FAS receptor. This malfunctioning pathway allows for the abnormal accumulation of lymphocytes, namely CD3 + TCRαß+CD4 - CD8- (double negative (DN) T) cells, which are a hallmark of the disease. This disease usually presents in childhood with lymphadenopathy and splenomegaly as a result of lymphoproliferation. Over time, these patients may develop cytopenias or lymphomas because of irregularities in the immune system. Current treatments include glucocorticoids, mycophenolate mofetil, sirolimus, immunoglobulin G, and rituximab. These medications serve to manage the symptoms and there are no standardized recommendations for the management of ALPS. The only curative therapy is a bone marrow transplant, but this is rarely done because of the complications. This review serves to broaden the understanding of ALPS by discussing the mechanism of immune dysregulation, how the symptoms manifest, and the mechanisms of treatment. Additionally, we discuss the epidemiology, comorbidities, and medications relating to ALPS patients across the United States using data from Cosmos.

Severe RAS-Associated Lymphoproliferative Disease Case with Increasing αß Double-Negative T Cells with Atypical Features.

Autoimmune lymphoproliferative syndrome (ALPS) is a disease of lymphocyte homeostasis caused by FAS-mediated apoptotic pathway dysfunction and is characterized by non-malignant lymphoproliferation with an increased number of TCRαß+CD4-CD8- double-negative T cells (αßDNTs). Conversely, RAS-associated leukoproliferative disease (RALD), which is caused by gain-of-functional somatic variants in KRAS or NRAS, is considered a group of diseases with a similar course. Herein, we present a 7-year-old Japanese female of RALD harboring NRAS variant that aggressively progressed to juvenile myelomonocytic leukemia (JMML) with increased αßDNTs. She eventually underwent hematopoietic cell transplantation due to acute respiratory distress which was caused by pulmonary infiltration of JMML blasts. In general, αßDNTs have been remarkably increased in ALPS; however, FAS pathway gene abnormalities were not observed in this case. This case with RALD had repeated shock/pre-shock episodes as the condition progressed. This shock was thought to be caused by the presence of a high number of αßDNTs. The αßDNTs observed in this case revealed high CCR4, CCR6, and CD45RO expressions, which were similar to Th17. These increased Th17-like αßDNTs have triggered the inflammation, resulting in the pathogenesis of shock, because Th17 secretes pro-inflammatory cytokines such as interleukin (IL)-17A and granulocyte-macrophage colony-stimulating factor. The presence of IL-17A-secreting αßDNTs has been reported in systemic lupus erythematosus (SLE) and Sjögren's syndrome. The present case is complicated with SLE, suggesting the involvement of Th17-like αßDNTs in the disease pathogenesis. Examining the characteristics of αßDNTs in RALD, JMML, and ALPS may reveal the pathologies in these cases.

Novel Germline TET2 Mutations in Two Unrelated Patients with Autoimmune Lymphoproliferative Syndrome-Like Phenotype and Hematologic Malignancy.

Somatic mutations in the ten-eleven translocation methylcytosine dioxygenase 2 gene (TET2) have been associated to hematologic malignancies. More recently, biallelic, and monoallelic germline mutations conferring susceptibility to lymphoid and myeloid cancer have been described. We report two unrelated autoimmune lymphoproliferative syndrome-like patients who presented with T-cell lymphoma associated with novel germline biallelic or monoallelic mutations in the TET2 gene. Both patients presented a history of chronic lymphoproliferation with lymphadenopathies and splenomegaly, cytopenias, and immune dysregulation. We identified the first compound heterozygous patient for TET2 mutations (P1) and the first ALPS-like patient with a monoallelic TET2 mutation (P2). P1 had the most severe form of autosomal recessive disease due to TET2 loss of function resulting in absent TET2 expression and profound increase in DNA methylation. Additionally, the immunophenotype showed some alterations in innate and adaptive immune system as inverted myeloid/plasmacytoid dendritic cells ratio, elevated terminally differentiated effector memory CD8 + T-cells re-expressing CD45RA, regulatory T-cells, and Th2 circulating follicular T-cells. Double-negative T-cells, vitamin B12, and IL-10 were elevated according to the ALPS-like suspicion. Interestingly, the healthy P1's brother carried a TET2 mutation and presented some markers of immune dysregulation. P2 showed elevated vitamin B12, hypergammaglobulinemia, and decreased HDL levels. Therefore, novel molecular defects in TET2 confirm and expand both clinical and immunological phenotype, contributing to a better knowledge of the bridge between cancer and immunity.

Detection and evolutionary dynamics of somatic FAS variants in autoimmune lymphoproliferative syndrome: Diagnostic implications.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare primary immune disorder characterized by impaired apoptotic homeostasis. The clinical characteristics include lymphoproliferation, autoimmunity (mainly cytopenia), and an increased risk of lymphoma. A distinctive biological feature is accumulation (>2.5%) of an abnormal cell subset composed of TCRαß+ CD4-CD8- T cells (DNTs). The most common genetic causes of ALPS are monoallelic pathogenic variants in the FAS gene followed by somatic FAS variants, mainly restricted to DNTs. Identification of somatic FAS variants has been typically addressed by Sanger sequencing in isolated DNTs. However, this approach can be costly and technically challenging, and may not be successful in patients with normal DNT counts receiving immunosuppressive treatment. In this study, we identified a novel somatic mutation in FAS (c.718_719insGTCG) by Sanger sequencing on purified CD3+ cells. We then followed the evolutionary dynamics of the variant along time with an NGS-based approach involving deep amplicon sequencing (DAS) at high coverage (20,000-30,000x). Over five years of clinical follow-up, we obtained six blood samples for molecular study from the pre-treatment (DNTs>7%) and treatment (DNTs<2%) periods. DAS enabled detection of the somatic variant in all samples, even the one obtained after five years of immunosuppressive treatment (DNTs: 0.89%). The variant allele frequency (VAF) range was 4%-5% in pre-treatment samples and <1.5% in treatment samples, and there was a strong positive correlation between DNT counts and VAF (Pearson's R: 0.98, p=0.0003). We then explored whether the same approach could be used in a discovery setting. In the last follow-up sample (DNT: 0.89%) we performed somatic variant calling on the FAS exon 9 DAS data from whole blood and purified CD3+ cells using VarScan 2. The c.718_719insGTCG variant was identified in both samples and showed the highest VAF (0.67% blood, 1.58% CD3+ cells) among >400 variants called. In summary, our study illustrates the evolutionary dynamics of a somatic FAS mutation before and during immunosuppressive treatment. The results show that pathogenic somatic FAS variants can be identified with the use of DAS in whole blood of ALPS patients regardless of their DNT counts.

[Autoimmune lymphoproliferative syndrome: a case report]. / Syndrome lymphoprolifératif avec autoimmunité: à propos d'un cas.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disorder of lymphocyte homeostasis, resulting from mutations in the Fas apoptotic pathway. It is characterized by non-infectious and non-malignant chronic lymphoproliferation and an increased risk of lymphoid malignancy. The diagnosis of this condition usually combines chronic lymphadenopathy and/or splenomegaly exceeding 6 months, autoimmune cytopenias, with an elevated level of CD3+CD4-CD8- Tαß lymphocytes, known as "double-negative" T cells. Differential diagnosis includes infections, autoimmune diseases or malignancies. Although clinical examination and laboratory tests are highly suggestive, this disease goes widely unrecognized. We here report, for the first time, the case of ALPS, a Moroccan patient, and aged 8 years, with recurrent fever, splenomegaly and adenopathies. Paraclinical examinations revealed chronic pancytopenia, higher than normal TαÎ2 double negative lymphocytes, hypergammaglobulinemia, and elevated serum levels of soluble FAS ligand. The diagnosis of ALPS was made. First-line treatment included corticosteroids and immunoglobulins. Then the patient received mycophenolate followed by Sirolimus. This treatment resulted in better clinical and laboratory tests results. Our aim is to raise awareness of this rare condition, which may be under-diagnosed, among physicians.

Lipopolysaccharide Responsive Beige-like Anchor Protein Deficiency in a Patient with Autoimmune Lymphoproliferative Syndrome-like Disease Phenotype: A Case Report and Literature Review.

LPS-responsive beige-like anchor protein (LRBA) deficiency is a primary immunodeficiency caused by a mutation in the LRBA gene. Affected individuals present with a variety of clinical symptoms including hypogammaglobulinemia, recurrent infections, splenomegaly, hepatomegaly, and autoimmune cytopenias. Except for hypogammaglobulinemia, the remaining features resemble autoimmune lymphoproliferative syndrome (ALPS). Here, we report the case of a 14-year-old boy with the ALPS phenotype, eventually diagnosed with LRBA deficiency. He presented with lymphadenopathy and hepatosplenomegaly, along with autoimmune cytopenia. Due to recurrent infections and worsening gastrointestinal symptoms, whole-exome sequencing was conducted and revealed a novel homozygous pathogenic variant in the LRBA gene (c.534del; p.9Asp179IIef*16). The patient recently suffered from clinical deterioration due to SARS-COV-2 which appears to have triggered an acute worsening of his existing Cytomegalovirus colitis leading to an eventual demise. A literature search for reported LRBA deficient patients with ALPS-like phenotype revealed 11 patients. The most common clinical presentations in LRBA patients with ALPS-like phenotype included autoimmunity (100%), splenomegaly (91%), lymphadenopathy (36.4%), and respiratory tract infections (63.6%). LRBA deficiency is unique in the fact that it encompasses immune deficiency, autoimmunity, and lymphoproliferation. In children with multiple symptoms related to these domains, a genetic diagnosis is necessary to ensure tailored and precise medical therapy.

Differential Expression of Proteins in an Atypical Presentation of Autoimmune Lymphoproliferative Syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare disease defined as a defect in the lymphocyte apoptotic pathway. Currently, the diagnosis of ALPS is based on clinical aspects, defective lymphocyte apoptosis and mutations in Fas, FasL and Casp 10 genes. Despite this, ALPS has been misdiagnosed. The aim of this work was to go one step further in the knowledge of the disease, through a molecular and proteomic analysis of peripheral blood mononuclear cells (PBMCs) from two children, a 13-year-old girl and a 6-year-old boy, called patient 1 and patient 2, respectively, with clinical data supporting the diagnosis of ALPS. Fas, FasL and Casp10 genes from both patients were sequenced, and a sample of the total proteins from patient 1 was analyzed by label-free proteomics. Pathway analysis of deregulated proteins from PBMCs was performed on the STRING and PANTHER bioinformatics databases. A mutation resulting in an in-frame premature stop codon and protein truncation was detected in the Fas gene from patient 2. From patient 1, the proteomic analysis showed differences in the level of expression of proteins involved in, among other processes, cell cycle, regulation of cell cycle arrest and immune response. Noticeably, the most down-regulated protein is an important regulator of the cell cycle process. This could be an explanation of the disease in patient 1.

The contribution of rare copy number variants in FAS toward pathogenesis of autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by chronic nonmalignant lymphadenopathy, splenomegaly, cytopenias, and other autoimmune manifestations. ALPS is caused by lymphocyte accumulation from defects in FAS-mediated apoptosis. Heterozygous germline or somatic pathogenic single nucleotide variants in FAS are the most common molecular etiology of ALPS. Through the Centralized Sequencing Program at the National Institute of Allergy and Infectious Diseases, we performed exome sequencing on subjects with a clinical diagnosis of ALPS, with a subset receiving copy number variant (CNV) analysis. In this cohort, we identified 3 subjects from unrelated families with CNVs at the FAS locus. One subject had a de novo ∼0.828 Mb copy number loss encompassing all of FAS. The second subject had a maternally inherited ∼1.004 Mb copy number loss encompassing all of FAS. The third subject had a paternally inherited ∼0.044 Mb copy number loss encompassing exons 7 through 9 of FAS. Subjects with deletions in FAS had clinical presentations and biomarker profiles similar to those with ALPS and with germline and somatic FAS variants. We demonstrate that CNV analysis should be pursued if there is clinical and biomarker evidence of ALPS because it can lead to a molecular diagnosis and appropriate treatment when FAS sequencing is inconclusive.

A unique phenotype of longitudinal extensive transverse myelitis in autoimmune lymphoproliferative syndrome.

A 49-year-old patient with a history of lymphoproliferation and autoimmune cytopenias presented with unexplained longitudinal extensive transverse myelitis. Flow cytometry on peripheral blood showed an elevated level of double negative T lymphocytes, a finding typical for autoimmune lymphoproliferative syndrome (ALPS). Inborn error of immunity (IEI) gene panel demonstrated a heterozygous variant in the FAS gene (c.857G > A, p.(Gly286Glu)), formally confirming the diagnosis. Autoimmune neurological conditions in a context of predisposition for infection and lymphoproliferation should raise suspicion of IEI. Specific testing for ALPS should be considered in patients with a history of non-malignant lymphoproliferation, multilineage cytopenias and unexplained autoimmune (neurological) manifestations.

Non-malignant, non-infectious lymphoproliferation: challenges in the diagnosis and treatment of autoimmune lymphoproliferative syndrome / Nem malignus, nem infectiosus lymphoproliferatio: kihívások az autoimmun lymphoproliferativ szindróma diagnosztikájában és kezelésében.

Összefoglaló. Az autoimmun lymphoproliferativ szindróma egy ritka, immundeficientiával járó genetikai betegség. Hátterében az extrinszik apoptotikus útvonal génjeinek örökletes vagy szerzett mutációi és a következményesen kialakuló, aktivált lymphocyták negatív szelekciójának a defektusa áll. Az autoimmun lymphoproliferativ szindróma klinikai megjelenésére jellemzo a jóindulatú lymphocytaburjánzás következtében kialakuló lymphadenopathia és lépmegnagyobbodás. Gyakran társul olyan autoimmun kórképekkel, mint az autoimmun haemolyticus anaemia vagy az autoimmun thrombocytopenia. A betegségben jellemzo laboratóriumi eltérések a következok: az αß+ CD4-/CD8- kettos negatív T-sejtek szaporulata, a szolúbilis Fas-ligand, az interleukin-10 és interleukin-18, valamint a B12-vitamin szérumszintjének emelkedése. A kórkép diagnózisához hozzátartozik az in vitro Fas-mediált apoptózis funkciójának vizsgálata, valamint a genetikai vizsgálat. Differenciáldiagnosztikai szempontból fontos elkülöníteni a lymphomáktól, valamint az autoimmun lymphoproliferativ szindrómaszeru betegségektol. A kezelés alapja a társuló autoimmun kórképek tüneteinek csökkentése immunszuppresszív terápiával. Orv Hetil. 2022; 163(4): 123-131. Summary. The autoimmune lymphoproliferative syndrome is a rare genetic disorder causing immunodeficiency. In the background of the disease, germline or somatic mutations of genes participating in the extrinsic apoptotic pathway and the consequential defect in the negative selection of activated lymphocytes were discovered. The clinical appearance of autoimmune lymphoproliferative syndrome consists of non-malignant lymphoproliferation, lymphadenopathy and splenomegaly, it is frequently accompanied by autoimmune disorders such as autoimmune haemolytic anaemia or autoimmune thrombocytopenia. The main diagnostic laboratory findings of this disease are the following: an elevation in αß+, CD4-/CD8- double-negative T cell count, elevated serum levels of soluble Fas-ligand, interleukin-10, interleukin-18 and vitamin B12. Other useful laboratory tests are the in vitro Fas-mediated apoptotic functional assay and the genetic screening for gene mutations. Differential diagnosis should exclude malignant lymphoproliferation in lymphomas and non-malignant autoimmune lymphoprolipherative syndrome-like diseases. The main aim of the treatment is the amelioration of the accompanying autoimmune disease with immunosuppressive therapy. Orv Hetil. 2022; 163(4): 123-131.

ALPS, FAS, and beyond: from inborn errors of immunity to acquired immunodeficiencies.

Autoimmune lymphoproliferative syndrome (ALPS) is a primary immune regulatory disorder characterized by benign or malignant lymphoproliferation and autoimmunity. Classically, ALPS is due to mutations in FAS and other related genes; however, recent research revealed that other genes could be responsible for similar clinical features. Therefore, ALPS classification and diagnostic criteria have changed over time, and several ALPS-like disorders have been recently identified. Moreover, mutations in FAS often show an incomplete penetrance, and certain genotypes have been associated to a dominant or recessive inheritance pattern. FAS mutations may also be acquired or could become pathogenic when associated to variants in other genes, delineating a possible digenic type of inheritance. Intriguingly, variants in FAS and increased TCR αß double-negative T cells (DNTs, a hallmark of ALPS) have been identified in multifactorial autoimmune diseases, while FAS itself could play a potential role in carcinogenesis. These findings suggest that alterations of FAS-mediated apoptosis could trespass the universe of inborn errors of immunity and that somatic mutations leading to ALPS could only be the tip of the iceberg of acquired immunodeficiencies.