Causes and consequences of the autoimmune lymphoproliferative syndrome.

Abstract Autoimmune lymphoproliferative syndrome (ALPS) is the first autoimmune hematological disease whose genetic basis has been defined. It is a disorder of apoptosis in which the inability of lymphocytes to die leads to lymphadenopathy, hypersplenism, and autoimmune cytopenias of childhood onset. More than 200 ALPS patients have been studied over the last 15 years and followed by our colleagues and ourselves at the Clinical Center of the National Institutes of Health. Based upon this experience we have determined that patients with germline mutations of the intracellular domain of Fas protein, the most frequent single genetic cause of ALPS, have a significantly increased risk of developing Hodgkin and non-Hodgkin lymphoma (NHL), underscoring the critical role played by cell surface receptor-mediated apoptosis in eliminating redundant proliferating lymphocytes with autoreactive and oncogenic potential. The major determinants of morbidity and mortality in ALPS are the severity of the autoimmune disease, hypersplenism, asplenia-related sepsis, and the risk of lymphoma, which in itself requires long-term surveillance. Though most episodes of cytopenias respond to courses of conventional immunomodulatory agents, some ALPS patients, especially those with massive splenomegaly and hypersplenism, may require splenectomy and/or ongoing immunosuppressive treatment. Thus, ALPS highlights the importance of cell death pathways in health and disease.


Introduction
The autoimmune lymphoproliferative syndrome (ALPS) is the first human immune disorder to be associated with defective lymphocyte apoptosis leading to accumulation of lymphoid mass and persistence of autoreactive cells as prominent nonmalignant lymphadenopathy and hepatosplenomegaly [1,2]. Many ALPS patients present to a hematologist in early childhood for further assessment and management of otherwise inexplicable autoimmune hemolytic anemia (AIHA), thrombocytopenia (ITP) and/or neutropenia and, in that background, some of them develop B-cell lymphoma.

Molecular pathogenesis of ALPS
Autoimmunity results from failure of self-tolerance, which can be further divided into central and peripheral tolerance. Central tolerance is fostered by apoptosis through elimination of unnecessary and immature effector cells in generative lymphoid organs (the bone marrow and thymus); while mechanisms of peripheral tolerance include anergy, deletion by apoptosis and suppression by regulatory T cells to avoid autoimmunity and tissue damage [3]. Apoptosis, the intrinsic death program of cells, is triggered by any of several effector molecules either through receptor-ligand interactions at the cell surface (the extrinsic pathway) or by the induction of mitochondrial enzymes (the intrinsic pathway). Lymphocyte apoptosis mediated by the cell surface receptor Fas plays a pivotal role in the termination of redundant immune responses. Originally characterized from a human diploid fibroblast cell line as the FS-7 associated surface antigen [4], Fas (also termed CD95 and APO-1) is a member of the tumor necrosis factor (TNF) receptor superfamily of proteins that directly trigger receptor mediated apoptosis to maintain lymphocyte homeostasis, peripheral immune tolerance and prevent autoimmunity. It is a membrane bound molecule that is highly expressed on activated B and T lymphocytes. Conformational change of Fas leading to its trimerization by the binding of FasL or agonistic antibodies triggers the rapid recruitment of the death domain (DD) of the adaptor protein FADD (Fasassociated death domain) to the homologous DD in the cytoplasmic tail of Fas. This is followed by the recruitment of procaspases 8 or 10 through the interaction of their death-effector domains with the amino termini of FADD. The resulting Fas/FADD/caspase complex is termed the death-inducing signaling complex (DISC) that incites a further cascade of caspase activation culminating in the death of cells [5][6][7].
The essential role that Fas exerts in maintaining lymphocyte homeostasis and peripheral immune tolerance to prevent autoimmunity was first elucidated by studies in Fas-deficient MRL/lpr 2 /2 mice. Mice homozygous for Fas mutations develop hypergammaglobulinemia, glomerulonephritis, massive lymphadenopathy and expansion of an otherwise rare and unique population of TCR ab cells that lack expression of both CD4 and CD8, and hence are known as double-negative (DN) T cells [6 -8]. This provided insights into the pathophysiology of a similar syndrome being observed in humans [8 -10].
ALPS, as this disorder was subsequently named, has been shown to represent a failure of apoptotic mechanisms. It is most often associated with heterozygous mutations in the gene encoding the Fas protein, TNFRSF6 (tumor necrosis factor receptor superfamily 6), and other related effector proteins that regulate lymphocyte survival. Most of these mutations are inherited in an autosomal dominant fashion with variable penetrance. The likelihood that an individual who carries a Fas mutation will manifest ALPS appears to depend on several factors, only some of which are known at this time [11,12].

Case definition and classification
Criteria for the diagnosis of ALPS have been defined and appear to be highly sensitive and specific and explain a number of previously observed idiopathic clinical syndromes (Table I). Chronic lymphadenopathy and organomegaly simulating lymphoma has been recognized as a distinct clinical entity for nearly 40 years [13]. The clinical features of ALPS, as they are now understood, are due to the accumulation of lymphoid mass and persistence of autoreactive cells with an increase in characteristic circulating DNT cells.
Diagnostic criteria for ALPS include: chronic, nonmalignant lymphadenopathy and/or splenomegaly persisting for at least 6 months; defective (less than half normal) Fas-mediated lymphocyte apoptosis as assayed in vitro; and elevated ($ 1% or $ 20/ml) circulating CD4-/CD8-DNT cells expressing the ab T-cell receptor above their normal range of 0.1 -0.9% of all lymphocytes (with absolute counts exceeding 2-17/ml) ( Figure 1). Molecular studies have revealed that germline heterozygous mutations in the genes encoding apoptosis signaling proteins Fas, Fas ligand (FasL), Caspase 8, or Caspase 10 are present in most patients of ALPS [14 -16]. Mutations in the gene encoding Fas alone account for approximately 75% of all individuals with ALPS [2]. Currently patients are classified as ALPS Type Ia and Ib when they carry a mutation of the Fas and FasL gene, respectively, ALPS Type II when the patient carries a Caspase 10 or Caspase 8 gene mutation, and ALPS Type III when no mutation can be identified (Table II). More recently a subgroup of ALPS patients were also noted to have somatic mutations of Fas specifically detected in isolates of characteristic DNT lymphocytes [17,18]. Diagnostic work up includes evaluation of proband and family members through clinical and laboratory testing for required features of ALPS (Table I), which consists of immunophenotyping of peripheral blood for enumeration of DNT cells and in vitro apoptosis assay as well as detailed molecular genetic analysis for mutations in apoptosis pathway genes implicated in ALPS: Fas, Fas-ligand, and Caspases 10 and 8. Table I. Criteria for the diagnosis of ALPS.
Increase in circulating double negative T (DNT) cells that are CD4 2CD8 2 and express the alpha/beta T-cell receptor above the normal range of 0.1-0.9% of lymphocytes (absolute counts range normally from 2 to 17/ml) 3.
Demonstration of defective antigen-induced lymphocyte apoptosis on in vitro culture Supporting features 1.
Family history of autoimmune lymphoproliferative syndrome 2.
Typical findings on histopathologic analysis of lymph node or splenic tissue 3.
Mutations of genes encoding Fas or related apoptosis signaling proteins

Clinical features of ALPS
Prominent non-malignant lymphadenopathy in ALPS arises typically in early childhood and is often accompanied by hepatosplenomegaly and autoimmune cytopenias of one or more lineages. Lymphadenopathy and splenomegaly may be massive and distort anatomic landmarks, frequently affecting the neck and axillary regions and persist for two or more years [19]. Enlargement of abdominal and thoracic lymph nodes are also often noted by ultrasound and/or computerized tomography [20]. Splenomegaly may also manifest as cytopenias secondary to cellular sequestration due to hypersplenism. The clinical and laboratory features of ALPS do not appear to differ markedly between patients who have identified mutations and those who do not [2,19]. Several non-hematological autoimmune diseases have also been reported in association with ALPS including Guillian-Barré syndrome, glomerulonephritis, uveitis and autoimmune liver disease (Table III). Presentation of children with generalized adenopathy, splenomegaly and autoimmue mutilineage cytopenias represents a diagnostic challenge because their clinical and laboratory features overlap with and may manifest as those of other childhood hematological disorders including lymphoma, hereditary spherocytosis, Evans Syndrome [21] and Rosai -Dorfman Disease [22]. Other immunological disorders associated with autoimmune phenomena that must be differentiated from ALPS include common variable immunodeficiency, Wiskott -Aldrich syndrome, interleukin (IL)-2 receptor a-chain deficiency and the X-linked immunodysregulation -polyendocrinopathy -enteropathy (IPEX) syndrome.

Laboratory findings in ALPS
Lymphocyte dysregulation in ALPS. Lymphocyte dysregulation in ALPS is revealed by an elevated percentage of DNT cells in the peripheral blood, bone marrow, spleen and lymphoid tissues of patients producing a fairly characteristic histopathological picture [23,24]. Lymph node biopsy often shows both follicular hyperplasia and paracortical expansion with a mixed infiltrate containing TCRa/b DNT cells, a feature unique to ALPS, and helps to distinguish this syndrome from other benign and malignant lymphoproliferative lesions ( Figure 3). Study of lymph node tissue for clonality through immunoglobulin and T-cell receptor gene rearrangements as well as cytogenetic evaluation for chromosomal aneuploidy are important to help diagnose lymphoma in patients with a background of chronic lymphadenopathy and other features resembling ALPS. Patients with ALPS also demonstrate a lymphocytosis with a skewed Th-2 pattern of immune responses, one manifestation of which is elevated circulating and tissue interleukin-10 (IL-10) levels [25].
Other associated abnormalities noted in peripheral blood, bone marrow. Features of ALPS include hypergamma globulinemia and circulating autoantibodies, most commonly manifesting as Coomb's direct antiglobulin test (DAT) positive hemolytic anemia, autoimmune thrombocytopenia and neutropenia [26 -28]. Eosinophilia [29] and monocytosis are frequent findings in ALPS patients. Bone marrow showing some degree of dyserythropoiesis [30] and depleted iron stores are also not uncommon.
In vitro apoptosis defect. All patients with ALPS, by definition, exhibit defective lymphocyte apoptosis assayed in vitro by specialized laboratories using peripheral blood lymphocytes cultured in the presence of IL-2 for a few days and stimulated with phytohemagglutinin. Apoptosis is induced in these assays by a monoclonal antibody that binds to activated-Fas on the lymphocyte cell surface. The majority of cells from normal controls are killed, while lymphocytes from patients with clinical features of ALPS are relatively spared [31,32] (Figure 2).
Natural history: Hypersplenism, lymphadenopathy, autoimmune cytopenias and lymphoma transformation The first clinical expression of ALPS may be recognized by the pediatrician as chronically enlarged lymph nodes and spleen in infants who are otherwise asymptomatic. However, a large proportion of patients eventually proven to have ALPS may present initially with episodes of pallor and icterus associated with hemolytic anemia, spontaneous bruises and mucocutaneous hemorrhages due to thrombocytopenia or bacterial infections associated with neutropenia. The potential for multiple autoimmune diseases involving different organ systems such as uveitis, hepatitis, glomerulonephritis,and encephalomyelitis in a single patient with ALPS is being recognized increasingly as patients are followed for many years. The median age of initial presentation with florid lymphadenopathy and splenomegaly is about 24 months. The extent of adenopathy and splenomegaly may decrease through adolescence. Similarly, clinical manifestations of refractory, chronic cytopenias are most severe in childhood; however symptoms related to cytopenias can first appear at any age.
Genetic counseling is an integral part of the evaluation of a family with one or more ALPS patients. Assessment of relatives of ALPS probands with mutations in genes encoding Fas or caspase 10 usually identifies a parent, sibling or a more distant relative with identical heterozygous mutations inherited in an autosomal dominant fashion. Many such subjects manifest few if any clinical features of ALPS, although they all share in vitro apoptosis defects. Elevation in DN T cell numbers and IL-10 levels are typically not present in the absence of the clinical stigmata of ALPS [33]. Patient and family members are educated to seek timely help for any systemic symptoms, cytopenias, unexpected fluctuations in lymph node and spleen size. Patients with mutations involving the intracellular death domain of the Fas molecule are significantly more likely than ones with extracellular domain mutations to manifest ALPS features and they have more severe clinical phenotypes.
ALPS patients with germline mutation of the intracellular domain of Fas have a significantly increased (14 and 51-fold) risk of developing non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL) [34,35], respectively, underscoring the critical role played by receptor-mediated apoptosis in eliminating proliferating lymphocytes with oncogenic potential. Among the cohort of 200 patients seen at NIH, 11 have developed lymphomas (6 Hodgkins and 5 B-cell NHL) with a median age of presentation with lymphoma of 17 years (range 2 -50 years). More recently TNFRSF6 gene encoding Fas was proposed to act as a tumor suppressor as it is silenced in many tumors [36]. In one report 20% of B-cell lymphomas derived from (post) germinal center B-cells carried somatic mutations in the exon 9 of this gene, which encodes the Fas intracellular death domain [37].
Inherited CASP10 gene mutations also underlie defective lymphocyte and dendritic cell apoptosis in some patients with ALPS. CASP10 gene was analyzed for the detection of somatic mutations in 117 human NHLs in one recent study. Overall, 17 NHLs (14.5%) were found to have CASP10 mutations. When the tumor-derived caspase 10 mutants were expressed in cells, apoptosis was suppressed. These data suggest that the inactivating mutations of the CASP10 gene might lead to the loss of its apoptotic function and contribute to the pathogenesis of some cases of NHL [38].

Management of lymphoproliferation and hypersplenism
Although massive adenopathy in growing children can incite considerable anxiety and may be socially isolating, treatment is not specifically indicated to reduce lymph nodes for cosmetic reasons alone. Neither corticosteroids nor immunosuppressive drugs like azathioprine, cyclosporin or mycophenolate mofitil reliably shrink the spleen or lymph nodes in patients with ALPS. Though the use of Fansidar first initiated for pneumocystis carinii prophylaxis was reported in a small series of patients with ALPS and ALPS-like conditions to be associated with reductions in lymphoproliferation [39], our experience with this drug as well as its active component pyrimethamine has failed to show regression of adenopathy or splenomegaly.
Approximately half of the 200 ALPS patients being followed in our clinic have had a splenectomy in order to manage recurring and chronic cytopenias, nearly 75% of who achieved long-term remission. However, five asplenic ALPS patients have had fatal opportunistic infections and many others have presented with pneumococcal sepsis following splenectomy. Thus, splenectomy should be avoided unless it is the only remaining measure to control chronic, refractory, lifethreatening cytopenias. Spleen-guards made of fiberglass have been used with apparent success in protecting active children with large spleens from splenic rupture, allowing them to participate in usual school sports programs.

Asplenia care in ALPS patients
Asplenic ALPS patients require vigilance for septicemia due to pneumococcal bacteremia, which can be fatal, and has been in some of our patients. We have found that ALPS patients may be unable to mount or sustain protective levels of antibodies directed against pneumococcal polysaccharide antigens following vaccination. Based upon our experience, we advise that all asplenic ALPS patients be maintained on long-term antibiotic prophylaxis against pneumococal sepsis using penicillin V or fluoroquinolones. We further advise our asplenic patients to wear alert bracelets; we also educate them and their parents about the importance of seeking medical care promptly for a significant febrile illness requiring IV antibiotics until bacterial sepsis is ruled out.
Our recommendations also include maintenance of daily antibiotic prophylaxis as well as periodic surveillance and reimmunization against pneumococci using a combination of both 7-valent conjugate (Prevnar) and 23-valent polysaccharide (Pneumovax) vaccines [40].

Treatment of ALPS related cytopenias:
The initial management of patients with ALPS-related autoimmune cytopenias (AIHA, ITP, AIN) is similar to that used for immune cytopenias in other patient populations: 1. Immune-suppression with corticosteroids: Highdose pulse therapy with IV methylprednisolone (5-30 mg/kg) followed by low-dose oral prednisone (1 -2 mg/kg) maintenance that can often be successfully tapered over several months.  [42].

Surveillance for lymphoma
Chronic adenopathy in ALPS patients can fluctuate over time and create some concern of evolving lymphoma if one or more nodes enlarge unusually. Hence these patients need close clinical observation and serial CT scans, while some undergo repeated biopsies. Most ALPS patients with lymphoma respond to conventional multi-agent chemotherapy and radiation. They present additional diagnostic challenges once therapy is discontinued as ALPS associated adenopathy recurs and must be distinguished from relapsing lymphoma. Non-invasive tests are desirable in ALPS to help determine if a biopsy is warranted and which of many enlarged nodes will likely yield informative tissue. Positron emission tomography (PET) using [ 18 F] fluoro-2-deoxy-Dglucose (FDG) uptake, as a measure of cellular glucose metabolism, has become a standard in the staging and follow up evaluation of cancers, including lymphomas [43,44]. We have undertaken a formal, prospective study in which we are exploring the potential value of whole body FDG -PET scan to determine whether qualitative or quantitative FDG localization can help differentiate ALPS patients with benign, albeit prominent, adenopathy from those with ALPS-associated lymphomas.

Role of allogeneic BMT in ALPS
The short-term prognosis of most of the patients with ALPS appears to be very good. The chronic cytopenias seen in many ALPS patients may improve with age and does continue to respond to conventional immunosuppressive treatment. Thus, there is no need to entertain allogeneic stem cell transplantation in many patients with ALPS. Stem cell transplantation has been successful in patients with very severe and refractory cytopenias [45,46]. Of course, a sibling who carries the same apoptosis mutation as the proband is not an appropriate marrow donor. Mortality due to matched unrelated donor derived allogenic BMT is too high (35-45%) to justify this procedure for the vast majority of patients with a chronic disease like ALPS in which a near normal lifespan can otherwise be expected. At present, stem cell transplantation should be reserved for those ALPS patients with a severe phenotype, such as that associated with the rare occurrence of a homozygous Fas mutation [47][48][49].

Future directions and implications of ALPS
The major determinants of morbidity and mortality in ALPS depend on the severity of the autoimmune disease, hypersplenism and asplenia related sepsis as well as development of lymphoma. Novel and nontoxic therapeutic interventions are necessary to control the lymphoproliferative process in patients with ALPS who have chronic lymphadenopathy and splenomegaly. However, patients with mutations abrogating function of the intracellular domain of the Fas protein are at risk of developing lymphomas and they need diligent long-term surveillance through monitoring of their adenopathy. Paradigms derived from managing a large cohort of patients with a rare disease like ALPS may also help understand lymphoma biology, streamline care of asplenic patients of diverse etiology, while exploring treatment approaches for more widely prevalent yet refractory immune-mediated hematological cytopenias like chronic ITP [50].