Autoimmune Lymphoproliferative Syndrome Patients Research Articles

Study of the potential role of CASPASE-10 mutations in the development of autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is a primary disorder of lymphocyte homeostasis, leading to chronic lymphoproliferation, autoimmune cytopenia, and increased risk of lymphoma. The genetic landscape of ALPS includes mutations in FAS, FASLG, and FADD, all associated with apoptosis deficiency, while the role of CASP10 defect in the disease remains debated. In this study, we aimed to assess the impact of CASP10 variants on ALPS pathogenesis. We benefit from thousands of genetic analysis datasets performed in our Institute’s genetic platform to identify individuals carrying CASP10 variants previously suspected to be involved in ALPS outcome: p.C401LfsX15, p.V410I and p.Y446C, both at heterozygous and homozygous state. Clinical and laboratory features of the six included subjects were variable but not consistent with ALPS. Two individuals were healthy. Comprehensive analyses of CASP10 protein expression and FAS-mediated apoptosis were conducted and compared to healthy controls and ALPS patients with FAS mutations. Missense CASP10 variants (p.V410I and p.Y446C), which are common in the general population, did not disrupt CASP10 expression, nor FAS-mediated apoptosis. In contrast, homozygous p.C401LfsX15 CASP10 variant lead to a complete abolished CASP10 expression but had no impact on FAS-mediated apoptosis function. At heterozygous state, this p.C401LfsX15 variant lead to a reduced CASP10 protein levels but remained associated with a normal FAS-mediated apoptosis function. These findings demonstrate that CASPASE 10 is dispensable for FAS-mediated apoptosis. In consequences, CASP10 defect unlikely contribute to ALPS pathogenesis, since they did not result in an impairment of FAS-mediated apoptosis nor in clinical features of ALPS in human. Moreover, the absence of FAS expression up-regulation in subjects with CASP10 variants rule out any compensatory mechanisms possibly involved in the normal apoptosis function observed. In conclusion, this study challenges the notion that CASP10 variants contribute to the development of ALPS.

Combined germline and somatic human FADD mutations cause autoimmune lymphoproliferative syndrome

The autoimmune lymphoproliferative syndrome (ALPS) is a noninfectious and nonmalignant lymphoproliferative disease frequently associated with autoimmune cytopenia resulting from defective FAS signaling. We previously described germline monoallelic FAS (TNFRSF6) haploinsufficient mutations associated with somatic events, such as loss of heterozygosity on the second allele of FAS, as a cause of ALPS-FAS. These somatic events were identified by sequencing FAS in DNA from double-negative (DN) T cells, the pathognomonic T-cell subset in ALPS, in which the somatic events accumulated. We sought to identify whether a somatic event affecting the FAS-associated death domain (FADD) gene could be related to the disease onset in 4 unrelated patients with ALPS carrying a germline monoallelic mutation of the FADD protein inherited from a healthy parent. We sequenced FADD and performed array-based comparative genomic hybridization using DNA from sorted CD4+ or DN T cells. We found homozygous FADD mutations in the DN T cells from all 4 patients, which resulted from uniparental disomy. FADD deficiency caused by germline heterozygous FADD mutations associated with a somatic loss of heterozygosity was a phenocopy of ALPS-FAS without the more complex symptoms reported in patients with germline biallelic FADD mutations. The association of germline and somatic events affecting the FADD gene is a new genetic cause of ALPS.

Autoimmune Lymphoproliferative Syndrome (ALPS) Disease and ALPS Phenotype: Are They Two Distinct Entities?

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder of lymphocyte homeostasis classically due to mutation of FAS, FASL, and CASP10 genes (ALPS-FAS/CASP10). Despite recent progress, about one-third of ALPS patients does not carry classical mutations and still remains gene orphan (ALPS-U, undetermined genetic defects). The aims of the present study were to compare the clinical and immunological features of ALPS-FAS/CASP10 versus those of ALPS-U affected subjects and to deepen the genetic characteristics of this latter group. Demographical, anamnestic, biochemical data were retrieved from medical record of 46 ALPS subjects. An enlarged panel of genes (next-generation sequencing) was applied to the ALPS-U group. ALPS-U subjects showed a more complex phenotype if compared to ALPS-FAS/CASP10 group, characterized by multiorgan involvement (P = 0.001) and positivity of autoimmune markers (P = 0.02). Multilineage cytopenia was present in both groups without differences with the exception of lymphocytopenia and autoimmune neutropenia that were more frequent in ALPS-U than in the ALPS-FAS/CASP10 group (P = 0.01 and P = 0.04). First- and second-line treatments were able to control the symptoms in 100% of the ALPS-FAS/CASP10 patients, while 63% of ALPS-U needed >2 lines of treatment and remission in some cases was obtained only after target therapy. In the ALPS-U group, we found in 14 of 28 (50%) patients 19 variants; of these, 4 of 19 (21%) were known as pathogenic and 8 of 19 (42%) as likely pathogenic. A characteristic flow cytometry panel including CD3CD4-CD8-+TCRαβ+, CD3+CD25+/CD3HLADR+, TCR αβ+ B220+, and CD19+CD27+ identified the ALPS-FAS/CASP10 group. ALPS-U seems to represent a distinct entity from ALPS-FAS/CASP10; this is relevant for management and tailored treatments whenever available.

Revisiting autoimmune lymphoproliferative syndrome caused by Fas ligand mutations

Fas ligand (FasL) is expressed by activated T cells and induces death in target cells upon binding to Fas. Loss-of-function FAS or FASLG mutations cause autoimmune-lymphoproliferative syndrome (ALPS) characterized by expanded double-negative T cells (DNT) and elevated serum biomarkers. While most ALPS patients carry heterozygous FAS mutations, FASLG mutations are rare and usually biallelic. Only 2 heterozygous variants were reported, associated with an atypical clinical phenotype. We revisited the significance of heterozygous FASLG mutations as a cause of ALPS. Clinical features and biomarkers were analyzed in 24 individuals with homozygous or heterozygous FASLG variants predicted to be deleterious. Cytotoxicity assays were performed with patient T cells and biochemical assays with recombinant FasL. Homozygous FASLG variants abrogated cytotoxicity and resulted in early-onset severe ALPS with elevated DNT, raised vitamin B12, and usually no soluble FasL. In contrast, heterozygous variants affected FasL function by reducing expression, impairing trimerization, or preventing Fas binding. However, they were not associated with elevated DNT and vitamin B12, and they did not affect FasL-mediated cytotoxicity. The dominant-negative effects of previously published variants could not be confirmed. Even Y166C, causing loss of Fas binding with a dominant-negative effect in biochemical assays, did not impair cellular cytotoxicity or cause vitamin B12 and DNT elevation. Heterozygous loss-of-function mutations are better tolerated for FASLG than for FAS, which may explain the low frequency of ALPS-FASLG.

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 TCRαβ+ 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.

Detection of Signature Double-Negative T Cells Is a Reliable Marker for ALPS Associated with FAS Mutation

Defects in FAS mediated apoptosis lead to a rare autoimmune disease called autoimmune lymphoproliferative syndrome (ALPS), which is manifested by a variety of symptoms including chronic lymphoproliferation, splenomegaly and autoimmune cytopenia. ALPS is mainly caused by germline or somatic FAS mutations, but gene analysis is time and cost intensive. Therefore, a tool is needed to screen for patients that are likely to harbor such a mutation to reduce costs and allow a faster diagnosis and treatment. One hallmark of the disease is the pathognomonic accumulation of TCRαβ+ CD4-/CD8- double-negative T (DNT) cells, which show high mitotic activity and unique expression profile. In this study, we compared the immune phenotype of ALPS patients with germline or somatic FAS mutation (ALPS-FAS/sFAS, n=11), patients who fulfill ALPS diagnostic criteria without FAS mutation (ALPS-U, n=11), and patients with ALPS-like disorders (two major diagnostic criteria without further criteria or at least one major criterion and autoimmune cytopenia, n=22). In addition, healthy donors, patients with chronic autoimmune cytopenia (AC, n=9), and patients with chronic non-malignant lymphoproliferation (LP, n=9) with no other major or minor criteria for ALPS diagnosis were used as controls. ALPS-FAS/sFAS patients showed accumulation of DNT cells with increased mitotic activity, terminal differentiation and expression of CD45RA/CD38 compared to healthy donors, AC and LP patients. Using receiver operating characteristic analyses, we could demonstrate that Ki67 and CD45RA/CD38 expression within DNT cells clearly distinguish ALPS-FAS/sFAS patients and ALPS-U and ALPS-like patients. To further strengthen specificity and sensitivity of the test, we assessed whether combination of DNT cell frequency with one additional marker is useful in supporting ALPS diagnosis. We found that a combination of DNT cell frequency with the expression of CD38 and CD45RA or Ki67 is most efficient to predict FAS mutation in ALPS patients. Taken together, flow cytometric analysis of these signature markers is a highly effective tool to discriminate patients with ALPS-FAS/sFAS and ALPS-related diseases and may also be useful for monitoring of treatment efficacy.

MO1033: Kidney and Bone Marrow Involvement in Ipex Syndrome with Atypical Presentation: the ‘Fil Rouge’ of Treg Between Ipex Features and Other Clinical Entities

Abstract BACKGROUND AND AIMS The transcription factor Forkhead box protein P3 (FOXP3) is central to the function of regulatory T cells (Treg). Mutations in the FOXP3 gene lead to a systemic disease called immune dysregulation, polyendocrinopathy and enteropathy, X-linked syndrome (IPEX). Some FOXP3 mutations were associated with atypical presentation including rare diseases such as IgG4 related disease (IgG4 RD) and autoimmune lymphoproliferative syndrome (ALPS). IgG4 RD is characterized by fibro-inflammatory tissue damage and often by elevated serum IgG4, whereas ALPS is presented by early-onset, chronic, nonmalignant lymphoproliferation, splenomegaly and autoimmune manifestations due to defective lymphocyte apoptosis, due mainly to mutation of genes involved in the apoptosis pathway. Kidney involvement in ALPS is uncommon, whereas in IPEX and IgG4 RD is more frequent, with different renal manifestations. METHOD We reported two atypical cases of IPEX characterized by the kidney and haematologic involvement. These cases are anecdotal for the wide spectrum of possible phenotypes associated with FOXP3 mutations. RESULTS Case 1. A 16-year male with clinical diagnosis of ALPS was treated with an mTOR inhibitor. Due to the onset of proteinuria until 650 mg/day and estimated glomerular filtration rate (eGFR) 66 mL/min, a kidney biopsy was performed and light microscopy showed a membranous pattern. The M-type phospholipase A2 receptor (PLA2R) on serum was negative as well as immunofluorescence (IF) on tissue. IgG-IgG4 immunohistochemical (IHC) staining was negative. We decided to re-evaluate the genetics of the patient, implementing the HPO (Human Phenotype Ontology) code. We found a mutation of the FOXP3 gene (NM_014009.3): c.779T &amp;gt; A (p.L260Q), never reported before on the GnomAD database and predicted to be ‘Likely Pathogenic’ by the suite of variant annotation. The patient does not present the classical triad of IPEX and Treg resulted normal. He was treated with steroids and continues mTOR inhibitor with good control of proteinuria and stable kidney function. Case 2. A 2-year boy was diagnosed with a trilinear bone marrow failure, genetic investigations were negative. He presented elevated serum levels of IgG4 subclass and kidney failure with tubular acidosis. A renal biopsy showed MGP associated with TIN. IF and IHC for PLA2R resulted positive. IHC staining for IgG4 showed overlapping positivity for IgG and IgG4. After the diagnosis of IgG4 RD, steroid therapy was started, without clinical response. Thereafter the patient underwent a bone marrow transplant from his brother (HLA-identical). In consideration of the rarity of IgG4 RD in children, we decided to perform a new genetic exam. A whole-exome sequencing on the proband and relatives was done. A hemizygous mutation of the FOXP3 gene (NM_014009.3), c.1087A &amp;gt; G (p.I363V), was found. This variant, inherited by the mother and found also in the proband's brother, was already described in the literature, thus a diagnosis of IPEX was done. Therefore, Treg resulted normal for brother and mother but not in our patient (0.3%). The patient underwent kidney transplantation, and after 1 year he presents normal kidney function. CONCLUSION MGP and IgG4 RD are rare in paediatrics, and they encouraged us to perform further investigations and reconsider the previous diagnosis. MGP pathogenesis in IPEX is consistent with recent evidence of unbalance of Th17/Treg in idiopathic MGP, with a significant reduction of Treg cell and FOXP3 expression. Th17/Treg may be implicated also in the pathogenesis of IgG4 RD as sources of cytokines production responsible for fibrosis and IgG4-producing B cells. FOXP3 + Treg and FAS-ligand are crucial for autoimmunity and self-tolerance. In ALPS patients a reduced expression of FOXP3 + Treg subsets but with the normal suppressive function was found. IPEX poses a diagnostic challenge considering the spectrum of different phenotypes, but to recognize kidney involvement, together with the growing use of wide genomic analysis, could play a central role.

Rheumatologic diseases in patients with inborn errors of immunity in the USIDNET registry

There is a gap in clinical knowledge regarding associations between specific inborn errors of immunity (IEIs) and rheumatologic diseases. This study reports the frequency of rheumatologic conditions in a large cohort of patients with IEI using the USIDNET (United States Immunodeficiency Network) registry. We used the USIDNET registry to conduct the analysis. We included all IEI patients within the registry for whom a diagnosed rheumatologic disease was reported. The total number of patients with IEI in our query was 5058. Among those, 278 (5.49%) patients had a diagnosis of rheumatologic disease. This cohort included 172 (61.8%) female and 106 (38.2%) male patients. Rheumatologic complications were highest in the interferonopathies (66.6%), autoimmune lymphoproliferative syndrome (ALPS) (13.7%), and immunoglobulin G subclass deficiency (IgGSD) (11.11%). Additionally, disease patterns were noted to be different in various IEI disease groups. Inflammatory myopathies were the most common rheumatologic condition in patients with X-linked agammaglobulinemia (1.65%), Sjogren's syndrome was the most common rheumatologic disease reported in ALPS patients (6.85%), and systemic lupus erythematosus was the most common rheumatologic disease in patients with chronic mucocutaneous candidiasis (CMC) (7.41%). Rheumatoid arthritis (RA) report rate was highest in patients with IgGSD (3.70%), specific antibody deficiency (SAD) (3.66%), and ALPS (2.74%). This study reports that rheumatologic diseases are frequently observed in patients with IEI. The frequency of different rheumatologic conditions was variable based on the underlying diagnosis. Clinicians caring for patients with IEI should be vigilant to monitor for rheumatologic complications. Key Points • The rates of reported rheumatologic diseases in the USIDNET registry are different in individual IEIs. • Further studies are needed to guide clinicians for detecting rheumatologic conditions earlier in patients with IEI.

Clinical, immunological, and genetic features in 780 patients with autoimmune lymphoproliferative syndrome (ALPS) and ALPS‐like diseases: A systematic review

AbstractBackgroundAutoimmune lymphoproliferative syndrome (ALPS) is a group of genetic disorders characterized by early‐onset lymphoproliferation, autoimmune cytopenias, and susceptibility to lymphoma. The majority of ALPS patients carry heterozygous germline mutations in the TNFRSF6 gene. In this study, we conducted a systematic review of patients with ALPS and ALPS‐like syndrome.MethodsThe literature search was performed in Web of Science, Scopus, and PubMed databases to find eligible studies. Additionally, the reference list of all included papers was hand‐searched for additional studies. Demographic, clinical, immunological, and molecular data were extracted and compared between the ALPS and ALPS‐like syndrome.ResultsTotally, 720 patients with ALPS (532 genetically determined and 189 genetically undetermined ALPS) and 59 cases with ALPS‐like phenotype due to mutations in genes other than ALPS genes were assessed. In both ALPS and ALPS‐like patients, splenomegaly was the most common clinical presentation followed by autoimmune cytopenias and lymphadenopathy. Among other clinical manifestations, respiratory tract infections were significantly higher in ALPS‐like patients than ALPS. The immunological analysis showed a lower serum level of IgA, IgG, and lymphocyte count in ALPS‐like patients compared to ALPS. Most (85%) of the ALPS and ALPS‐like cases with determined genetic defects carry mutations in the FAS gene. About one‐third of patients received immunosuppressive therapy with conventional or targeted immunotherapy agents. A small fraction of patients (3.3%) received hematopoietic stem cell transplantation with successful engraftment, and all except two patients survived after transplantation.ConclusionOur results showed that the FAS gene with 85% frequency is the main etiological cause of genetically diagnosed patients with ALPS phenotype; therefore, the genetic defect of the majority of suspected ALPS patients could be confirmed by mutation analysis of FAS gene.

Next Generation Sequencing for Detecting Somatic FAS Mutations in Patients With Autoimmune Lymphoproliferative Syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is a primary immune regulatory disorder clinically defined by chronic and benign lymphoproliferation, autoimmunity and an increased risk of lymphoma due to a genetic defect in the FAS-FASL apoptotic pathway. Genetic defects associated with ALPS are germinal and somatic mutations in FAS gene, in addition to germinal mutations in FASLG, FADD, CASP8 and CASP10 genes. The accumulation of CD3+TCRαβ+CD4-CD8- double negative T-cells (DNT) is a hallmark of the disease and 20-25% of ALPS patients show heterozygous somatic mutations restricted to DNT in the FAS gene (ALPS-sFAS patients). Nowadays, somatic mutations in the FAS gene are detected through Sanger sequencing in isolated DNT. In this study, we report an ALPS-sFAS patient fulfilling clinical and laboratory ALPS criteria, who was diagnosed through NGS with a targeted gene panel using DNA from whole blood. Data analysis was carried out with Torrent Suite Software and variant detection was performed by both germinal and somatic variant caller plugin. The somatic variant caller correctly detected other six ALPS-sFAS patients previously diagnosed in the authors’ laboratories. In summary, this approach allows the detection of both germline and somatic mutations related to ALPS by NGS, avoiding the isolation of DNT as the first step. The reads of the somatic variants could be detected even in patients with DNT in the cut off limit. Thus, custom-designed NGS panel testing may be a faster and more reliable method for the diagnosis of new ALPS patients, including those with somatic FAS mutations (ALPS-sFAS).

The challenge of early diagnosis of autoimmune lymphoproliferative syndrome in children with suspected autoinflammatory/autoimmune disorders.

To test the usefulness of an extended panel of lymphocyte subsets in combination with Oliveira's diagnostic criteria for the identification of autoimmune lymphoproliferative syndrome (ALPS) in children referred to a paediatric rheumatology centre. Patients referred from 2015 to 2018 to our rheumatology unit for an autoimmune or autoinflammatory condition were retrospectively analysed. Oliveira's required criteria [chronic lymphoproliferation and elevated double-negative T (DNT)] were applied as first screening. Flow cytometry study included double-negative CD4-CD8-TCRαβ+ T lymphocytes (DNT), CD25+CD3+, HLA-DR+CD3+ T cells, B220+ T cells and CD27+ B cells. Data were analysed with a univariate logistic regression analysis, followed by a multivariate analysis. Sensitivity and specificity of the Oliveira's required criteria were calculated. A total of 264 patients were included in the study and classified as: (i) autoimmune diseases (n = 26); (ii) juvenile idiopathic arthritis (JIA) (35); (iii) monogenic systemic autoinflammatory disease (27); (iv) periodic fever, aphthous stomatitis, pharyngitis and adenitis syndrome (100); (v) systemic undefined recurrent fever (45); (vi) undetermined-systemic autoinflammatory disease (14); or (vii) ALPS (17). Oliveira's required criteria displayed a sensitivity of 100% and specificity of 79%. When compared with other diseases the TCRαβ+B220+ lymphocytes were significantly increased in ALPS patients. The multivariate analysis revealed five clinical/laboratory parameters positively associated to ALPS: splenomegaly, female gender, arthralgia, elevated DNT and TCRαβ+B220+ lymphocytes. Oliveira's required criteria are useful for the early suspicion of ALPS. TCRαβ+B220+ lymphocytes should be added in the diagnostic work-up of patients referred to the paediatric rheumatology unit for a suspected autoimmune or autoinflammatory condition, providing a relevant support in the early diagnosis of ALPS.

The ITK Inhibitor Cpi-818 Blocks Activation of T Cells from Autoimmune Lymphoproliferative Syndrome (ALPS) Patients and Is Active in a Murine Model of ALPS

Introduction We present preclinical evidence confirming the expression of ITK in double negative (DN) T cells of ALPS patients and demonstrate that CPI-818 inhibits the TCR-induced activation of these cells ex vivo. Furthermore, CPI-818 effectively suppressed the accumulation of DN T cells in the MRL/lpr mice, leading to dramatic improvements in lymphadenopathy, splenomegaly and other pathologies associated with lymphocyte accumulation due to Fas-deficiency. ALPS is a disorder of lymphocyte apoptosis resulting in childhood onset lymphadenopathy, multilineage cytopenias, hypersplenism and autoimmune peripheral destruction. Most ALPS cases are associated with a loss of function mutation in the Fas gene resulting in immune dysregulation such as a marked elevation of abnormal TCR+CD4-CD8-double negative (DN) T cells. ALPS patients are currently treated with immunosuppressive and cytotoxic agents, hence targeted, less toxic treatments are imperative. The critical role of Fas protein in maintaining lymphocyte homeostasis and peripheral immune tolerance to prevent autoimmunity was initially discovered with studies in Fas-deficient MRL/LpJ-Tnfrsf6lpr (MRL/lpr) mice. These mice have dramatically elevated levels of DN T cells and develop many clinical manifestations similar to those observed in ALPS patients. Thus, MRL/lpr mice have been a useful model to assess compounds and therapeutic strategies for the treatment of ALPS. Interleukin-2-inducible T cell kinase (ITK) is a tyrosine kinase critical to T cell receptor (TCR) signal transduction, which modulates T cell proliferation and differentiation. We sought to test whether the effect of selective inhibition of TCR signaling through ITK inhibition could be a strategy to suppress the DN T cell expansion and disease manifestation in MRL/lpr mice. CPI-818 is an orally bioavailable, covalent inhibitor of ITK that potently inhibits TCR signal transduction. It is currently being evaluated in a phase 1/1b clinical trial in T-cell lymphoma (NCT03952078) where it has been well tolerated and resulted in anti-tumor activity. Methods PBMC samples were obtained from healthy donors or ALPS patients collected under NIAID IRB-approved protocol 93-I-0063. Cells were cultured with or without anti-CD3/CD28 activation. The impact of CPI-818 on viability, expression of ITK, and expression of the surface markers CD 25 and CD69 was assessed by flow cytometry. For in vivo studies, MRL/lpr mice received a control diet or a CPI-818 formulated diet (300 mg/kg/day). Lymph nodes were calipered weekly over the course of 22 weeks of disease development. Spleens, lungs and kidneys were harvested at 22 weeks to assess the impact of CPI-818 treatment on T cell subsets and histology. Results Ex vivo studies with PBMC preparations from normal donors and ALPS patients confirmed the expression of ITK in CD4+, CD8+, and DN T cells from ALPS patients. When these T cells were stimulated with anti-CD3/CD28, CPI-818 potently inhibited the upregulation of the activation markers CD25 and CD69 in DN T cells assessed by flow cytometry. In companion experiments, CPI-818 was not cytotoxic to DN T cells when activated through the TCR signaling pathway. Treatment of MRL/lpr mice with CPI-818 prevented the onset or, in a therapeutic design, led to the regression of lymphadenopathy and splenomegaly comparable to the treatment effects observed with cyclophosphamide. Unlike the effects seen with the cytotoxic agent cyclophosphamide which suppressed all T cells, as well as B, NK and myeloid cells in the spleen, CPI-818 selectively depleted DN T cells (see Figure). CPI-818 treatment also resulted in decreased levels of DN T cells in the kidneys and lungs of these mice without impacting the level of normal CD4 and CD8 T cells. CPI-818 limited the increased proteinuria measured in the control animals during the 22-week treatment window. Histological assessment of the kidney revealed reduced inflammatory damage in CPI-818 treated animals. Conclusions The selective, covalent ITK inhibitor, CPI-818, significantly reduced DN T cell numbers, lymphadenopathy and other disease end points in the MRL/lpr murine model of ALPS. This suggests that the dysregulated growth of Fas-deficient DN T cells requires functional TCR signaling. Further, our data suggests that blocking TCR signaling by targeting ITK may be an effective strategy for treating adult and pediatric ALPS. Figure Disclosures Ng: Corvus Pharmaceuticals: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Hill:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Miller:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Buggy:Corvus Pharmaceuticals: Consultancy, Current Employment, Current equity holder in publicly-traded company. Janc:Corvus Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.

Lymphoproliferative disorders and inborn errors of immunity

Lymphoproliferative disease (LPD) is a comprehensive concept covering diseases ranging from transient lymphadenopathy to lymphoma. LPD is frequently associated with Epstein-Barr virus (EBV) infections and tends to occur in patients with inborn errors of immunity (IEI) and in patients after organ transplantation. Most patients with severe combined immunodeficiency or X-linked lymphoproliferative disease develop LPD. Autoimmune lymphoproliferative syndrome (ALPS), a typical LPD disease, is caused by germline mutations in FAS, FASL, CASP10, CASP8 and FADD, which are involved in the apoptosis pathway. ALPS patients develop autoimmune diseases and LPDs such as hepatosplenomegaly and lymphadenopathy. On the other hand, RAS-associated ALPS-like syndrome and CTLA4 haploinsufficiency also belong to ALPS-associated diseases. EBV-associated LPD is a clinical condition that should be noted in patients with IEI. Patients with genetic defects in SH2D1A, XIAP, CD27, CD70, CD137, ITK, CTPS, RASGRP1, and MAGT1 are prone to EBV-associated LPD.

A Spectrum of Clinical Findings from ALPS to CVID: Several Novel LRBA Defects.

Autosomal recessively inherited lipopolysaccharide-responsive beige-like anchor (LRBA) protein deficiency was shown to be responsible for different types of inborn errors of immunity, such as common variable immunodeficiency (CVID) and autoimmune lymphoproliferative syndrome (ALPS). The aim of this study was to compare patients with LRBA-related ALPS and LRBA-related CVID, to describe their clinical and laboratory phenotypes, and to prepare an algorithm for their diagnosis and management. Fifteen LRBA-deficient patients were identified among 31 CVID and 14 possible ALPS patients with Western blotting (WB), primary immunodeficiency disease (PIDD) gene, next-generation panel screening (NGS), and whole exome sequencing (WES). The median age on admission and age of diagnosis were 7years (0.3-16.5) and 11years (5-44), respectively. Splenomegaly was seen in 93.3% (14/15) of the patients on admission. Splenectomy was performed to 1/5. Recurrent upper respiratory tract infections (93.3% (14/15)), autoimmune cytopenia (80% (12/15)), chronic diarrhea (53.3% (8/15)), lower respiratory tract infections (53.3% (8/15)), lymphoma (26.6% (4/15)), Evans syndrome (26.6% (4/15)), and autoimmune thyroiditis (20% (3/15)) were common clinical findings and diseases. Lymphopenia (5/15), intermittant neutropenia (4/15), eosinophilia (4/15), and progressive hypogammaglobulinemia are recorded in given number of patients. Double negative T cells (TCRαβ+CD4-CD8-) were increased in 80% (8/10) of the patients. B cell percentage/numbers were low in 60% (9/15) of the patients on admission. Decreased switched memory B cells, decreased naive and recent thymic emigrant (RTE) Thelper (Th) cells, markedly increased effector memory/effector memory RA+ (TEMRA) Th were documented. Large PD1+ population, increased memory, and enlarged follicular helper T cell population in the CD4+ T cell compartment was seen in one of the patients. Most of the deleterious missense mutations were located in the DUF1088 and BEACH domains. Interestingly, one of the two siblings with the same homozygous LRBA defect did not have any clinical symptom. Hematopoietic stem cell transplantation (HSCT) was performed to 7/15 (46.6%) of the patients. Transplanted patients are alive and well after a median of 2years (1-3). In total, one patient died from sepsis during adulthood before HSCT. Patients with LRBA deficiency may initially be diagnosed as CVID or ALPS in the clinical practice. Progressive decrease in B cells as well as IgG in ALPS-like patients and addition of IBD symptoms in the follow-up should raise the suspicion for LRBA deficiency. Decreased switched memory B cells, decreased naive and recent thymic emigrant (RTE) Th cells, and markedly increased effector memory/effector memory RA+ Th cells (TEMRA Th) cells are important for the diagnosis of the patients in addition to clinical features. Analysis of protein by either WB or flow cytometry is required when the clinicians come across especially with missense LRBA variants of uncertain significance. High rate of malignancy shows the regulatory T cell's important role of immune surveillance. HSCT is curative and succesful in patients with HLA-matched family donor.

PF351 ALPS DISEASE AND ALPS PHENOTYPE: DISTINCT ENTITIES?

Background:Autoimmune lymphoproliferative syndrome (ALPS) defined according to the clinical/biochemical criteria (NIH 2009) is known to be mainly caused by FAS, FASL, FADD and CASP10 gene mutations. Nevertheless, several patients carrying a clear ALPS phenotype are not associated with these mutationsAims:Describe the clinical, biochemical and therapeutic characteristic in ALPS patients with and without the classical mutations and correlate them with genetics.Methods:The demographic, clinical, biochemical, genetic informations and details about treatment are derived from the ALPS Italian Network. Search of mutations was performed with Sanger PCR and/or Next Generation sequencing techniques (extended to immunodeficiency genes panel).Results:Among 68 ALPS patients registered in the data base (Males, 60%), 42 subjects (62%) received a genetic definition: 14 pts (33%) carried classical mutations in FAS/CASP genes, 16 pts (37%) showed mutation in genes other than FAS/CASP (LRBA, STAT, CTLA4, CECR, BAFF, TACI, RAG, NEMO, IKBKG, Gaucher) and 12 pts (28%) were negative for FAS/CASP.Clinical phenotype in the two groups appeared rather different being “more complex“ in the ALPS FAS negative group if compared with the FAS/CASP one. (p = 0.003) (Table 1).The multi‐organ involvment was often associate with positivity of autoimmune markers (p = 0.002) (Table 1).Cytopenia affecting one or more haematopoietic lineages was present in both groups (69% and 82%) without difference; while leucopenia/lymphocytopenia was shown more frequently in FAS/CASP mutated group rather than in the ALPS FAS negative one (p = 0.03) (Table 1). As for lymphocyte subclasses and immunoglobulin dosage no differences were shown within the two groups. Vitamin B12 and IL‐10 were more frequently raised in ALPS FAS/CASP mutated group than in the non mutated FAS/CASP genes (p = 0.01, p = 0.001).Four of 42 (9%) patients did not require any treatment, while the remaining 38 received one or more lines or drug combinations. First line treatment (steroid or intravenous immunoglobulins) controlled the disease in 4/38 (10%). In the ALPS FAS/CASP POS group response rate to second line treatment (mostly MMF and rapamycin) was 100%, while in the ALPS FAS/CASP negative group MMF and rapamycin were not able to control symptoms in the majority of subjects (response rate 40%). Drug combinations or target therapies were more commonly applied in patients belonging to FAS/CASP negative group rather than in the ALPS FAS/CASP POS group (p = 0.002, p = 0.02) (Table 1).Summary/Conclusion:Patients carryng ALPS phenotype without FAS or CASP mutations probably represent a distinct entity as compared to those with classical ALPS phenotype and genotype regarding clinical appearance, biochemical characteristic and genetic pattern. Moreover genes frequently associated with immunodeficiency/disimmunity patterns are more represented in FAS/CASP negative group. These findings are relevant for therapeutic management, especially in the cases when target therapies are available. In ALPS FAS/CASP POS group, MMF or rapamycin are highly effective to manage disease. Their use would allows steroid‐sparing strategy and in some cases should be proposed even as a first line choice.image

Case report of a molar-root incisor malformation in a patient with an autoimmune lymphoproliferative syndrome

BackgroundMolar-root incisor malformation (MRIM) is a novel dental phenotype likely related to a patient’s past medical history. This case aimed to confirm MRIM by histological and scanning electron microscopy (SEM) examination for the first time in a patient diagnosed with autoimmune lymphoproliferative syndrome (ALPS) and to propose a possible link between ALPS and MRIM that could be attributable to abnormally proliferated bone marrow.Case presentationA 12.5-year-old boy with an extensive medical history, including diagnosis of ALPS, was examined clinically and radiologically to elucidate the reason for pain primarily originating from the area of the lower left permanent first molar tooth (PFM; tooth 36). Dental examination and radiographic survey revealed abnormal pulp cavity morphology of all four PFMs, and these were extracted, resolving the dental pain in the patient. The extracted PFMs were subjected to light microscopy, SEM evaluation and mineral density and elemental composition analyses. Histology of two PFMs revealed the presence of dentin-, bone- and cartilage-like tissues with abundant blood vessels occupying the majority of the pulp chamber. The root canals were obliterated with mineralized structures resembling pulp stones. Two different, highly mineralized abnormal tissues filling the majority of the pulp chamber revealed by SEM and confirming the diagnosis of MRIM displayed a mineral density and elemental composition similar to those of enamel and dentin, respectively.ConclusionsIt appears likely that in addition to the complex medical history during early childhood in the present case, extensive lymphoid infiltrates that are possible in ALPS patients can be regarded as a cofactor in the development of MRIM by exerting considerable pressure on the developing tooth bud and providing cells capable of differentiating into diverse cell types.

The Autoimmune Lymphoproliferative Syndrome with Defective FAS or FAS-Ligand Functions.

The autoimmune lymphoproliferative syndrome (ALPS) is a non-malignant and non-infectious uncontrolled proliferation of lymphocytes accompanied by autoimmune cytopenia. The genetic etiology of the ALPS was described in 1995 by the discovery of the FAS gene mutations. The related apoptosis defect accounts for the accumulation of autoreactive lymphocytes as well as for specific clinical and biological features that distinguish the ALPS-FAS from other monogenic defects of this apoptosis pathway, such as FADD and CASPASE 8 deficiencies. The ALPS-FAS was the first description of a monogenic cause of autoimmunity, but its non-Mendelian expression remained elusive until the description of somatic and germline mutations in ALPS patients. The recognition of these genetic diseases brought new information on the role of this apoptotic pathway in controlling the adaptive immune response in humans.

Autoimmune Lymphoproliferative Syndrome-FAS Patients Have an Abnormal Regulatory T Cell (Treg) Phenotype but Display Normal Natural Treg-Suppressive Function on T Cell Proliferation.

Autoimmune lymphoproliferative syndrome (ALPS) with FAS mutation (ALPS-FAS) is a nonmalignant, noninfectious, lymphoproliferative disease with autoimmunity. Given the central role of natural regulatory T cells (nTregs) in the control of lymphoproliferation and autoimmunity, we assessed nTreg-suppressive function in 16 patients with ALPS-FAS. The proportion of CD25highCD127low Tregs was lower in ALPS-FAS patients than in healthy controls. This subset was correlated with a reduced CD25 expression in CD3+CD4+ T cells from ALPS patients and thus an abnormally low proportion of CD25highFOXP3+ Helios+ T cells. The ALPS patients also displayed a high proportion of naïve Treg (FOXP3lowCD45RA+) and an unusual subpopulation (CD4+CD127lowCD15s+CD45RA+). Despite this abnormal phenotype, the CD25highCD127low Tregs' suppressive function was unaffected. Furthermore, conventional T cells from FAS-mutated patients showed normal levels of sensitivity to Treg suppression. An abnormal Treg phenotype is observed in circulating lymphocytes of ALPS patients. However, these Tregs displayed a normal suppressive function on T effector proliferation in vitro. This is suggesting that lymphoproliferation observed in ALPS patients does not result from Tregs functional defect or T effector cells insensitivity to Tregs suppression.

Utility of Fluorodeoxyglucose Positron Emission Tomography(FDG-PET) and Biomarkers, Serum Vitamin B12 and HDL for Assessing Lymphoproliferation in Children and Adults with Autoimmune Lymphoproliferative Syndrome Due to FAS Mutations (ALPS-FAS)

Introduction/Background:The autoimmune lymphoproliferative syndrome (ALPS) is a disorder of impaired lymphocyte apoptosis commonly associated with defects in the FAS gene in the CD95 signaling cascade. Childhood onset lymphadenopathy, splenomegaly and autoimmune cytopenias are characteristic clinical presentation of ALPS. The immunophenotypical hallmark of ALPS is the expansion of "double negative" TCR-__+T lymphocytes (DNTs) that lack both CD4 and CD8 markers and when these cells comprise more than 1.5% of all circulating lymphocytes. Patients with ALPS are at increased risk for B-cell lymphoma.Patients and Methods:We sought to evaluate the lymphoproliferative burden of patients referred to our institution and meeting the diagnostic criteria for ALPS using FDG-PET scans over a 10year period. Readily available biomarkers like serum vitamin B12 and HDL have also been very useful in diagnosis and follow up of patients with ALPS-FAS.Results:We have performed 91 FDG-PET scans in 76 ALPS patients. Whole body PET scans were performed, extending from the pelvis up to the cervical nodal regions, starting at 90 minutes after the FDG injection. The longer uptake times were chosen to account for nonmalignant nature of the condition under study. This included patients with ALPS-FAS (n=71) who had a median standardized uptake value (SUV) of 7.87 (range: 3.8-13.73) and non-FAS patients (n=20) with a median SUV of 7.47 (range: 4.21-14.41); none of these patients had lymphoma on clinical evaluation and further follow-up. In addition, 3 ALPS-FAS patients with lymphoma who underwent an FDG-PET scan prior to therapy for lymphoma had SUVs of 11.78, 15.24, and 26.86, respectively. Here we share the SUVMax data from PET imaging and associated biomarker data (serum vitamin B12, n=64 and serum HDL, n=21) available from 64 patients with ALPS-FAS (Table 1). FDG-PET SUVMax did not statistically correlate with high serum vitamin B12 levels and low serum HDL (n=21) (Correlation coefficient -0.14 and -0.02 respectively). Younger ALPS-FAS patients with more prolific lymphoproliferation were noted to have higher serum vitamin B12 values and more FDG avid lymphadenopathy in our series (Correlation coefficient Ð 0.56 and 0.29 respectively).Conclusions:1. FDG accumulates in the lymph nodes of patients affected by ALPS, but we postulate that FDG may accumulate in higher concentrations in lymph nodes from ALPS patients that have undergone malignant transformation.2. Clinical correlation including history of sudden onset focal enlargement of nodes and systemic symptoms is essential to suspect a diagnosis of lymphoma in ALPS-FAS patients as FDG-PET scans by themselves have limited utility in patients with background of nonmalignant lymphoproliferative disorders.3. FDG-PET/CT may aid the early detection of lymphoma in this at-risk population, as ALPS patients have a predisposition to develop malignancies, and choosing the most FDG avid lymph nodes for biopsy may reduce the need for repeat biopsies in response to lymph node size changes commonly observed in ALPS patients.4. For diagnosis as well as assessing lymphoproliferative burden during long term follow up of ALPS-FAS patients both high serum vitamin B12 levels and lipid profile (low serum HDL) can be used as surrogate biomarkers even though they do not correlate very well with FDG avidity of their lymph nodes (Figure1) in the limited series presented here*.*Reference: Moraitis AG, Freeman LA, Shamburek RD, Wesley R, Wilson W, Grant CM, Price S, Demosky S, Thacker SG, Zarzour A, Hornung RL, Pucino F, Csako G, Yarboro C, McInnes IB, Kuroiwa T, Boumpas D, Rao VK, Illei GG, Remaley AT. Elevated interleukin-10: a new cause of dyslipidemia leading to severe HDL deficiency.J Clin Lipidol. 2015 Jan-Feb;9(1):81-90. PMID: 25670364. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ocular Inflammatory Disorders in Autoimmune Lymphoproliferative Syndrome (ALPS)

ABSTRACTPurpose: To describe inflammatory ocular findings in patients with autoimmune lymphoproliferative syndrome (ALPS).Methods: A retrospective review of medical records for ALPS patients seen at the National Eye Institute between 2003 and 2013.Results: A total of 29 ALPS patients previously referred for ocular or visual symptoms or with a history of prolonged corticosteroid use, were identified. Mean age was 20 years (range: 4–66 years). The majority were male (n = 21, 72.4%) and Caucasian (n = 24, 82.8%). Ten (34.5%) had abnormal ocular findings, the most common of which was an ocular inflammatory disorder (n = 4, 13.8%). Uveitis was seen in two patients with ALPS-FAS and one with ALPS-U, all of whom required long-term systemic immunosuppression. One patient with ALPS-FAS had a history of optic neuritis.Conclusions: ALPS can have intraocular inflammatory manifestations that require routine follow-up to ensure appropriate and timely treatment of intraocular disease. Long-term immunosuppression may be needed for patients with ALPS-associated uveitis.