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ALPS—Ten Lessons from an International Workshop on a Genetic Disease of Apoptosis

2010; Cell Press; Volume: 32; Issue: 3 Linguagem: Inglês

10.1016/j.immuni.2010.03.013

1097-4180

Michael J. Lenardo, João Bosco Oliveira, Lixin Zheng, V. Koneti Rao,

Lymphoma Diagnosis and Treatment

An international group of researchers investigating the molecular, cellular, immunological, and clinical aspects of the autoimmune lymphoproliferative syndrome (ALPS) met in Bethesda, Maryland on September 21-22, 2009 to discuss advances made over the past 15 years. Their discussions yielded ten broad messages applicable to genetic and immunological investigations of human disease. An international group of researchers investigating the molecular, cellular, immunological, and clinical aspects of the autoimmune lymphoproliferative syndrome (ALPS) met in Bethesda, Maryland on September 21-22, 2009 to discuss advances made over the past 15 years. Their discussions yielded ten broad messages applicable to genetic and immunological investigations of human disease. The autoimmune lymphoproliferative syndrome (ALPS) is one of the first and best understood genetic diseases involving defective apoptosis and autoimmunity (Rieux-Laucat et al., 1995Rieux-Laucat F. Le Deist F. Hivroz C. Roberts I.A. Debatin K.M. Fischer A. de Villartay J.P. Science. 1995; 268: 1347-1349Crossref PubMed Scopus (1139) Google Scholar, Fisher et al., 1995Fisher G.H. Rosenberg F.J. Straus S.E. Dale J.K. Middleton L.A. Lin A.Y. Strober W. Lenardo M.J. Puck J.M. Cell. 1995; 81: 935-946Abstract Full Text PDF PubMed Scopus (1253) Google Scholar). On September 21–22, 2009, an international group of investigators met at the National Institutes of Health (NIH) to discuss insights gained from extensive studies of several hundred ALPS families over the past 15 years. Jennifer Puck (University of California, San Francisco, USA) reviewed the disease definition, which comprises chronic lymphadenopathy and/or splenomegaly, defective lymphocyte apoptosis, and a striking elevation of CD4−CD8− (double negative), B220+ T cell receptor (TCR) αβ+ T cells (DNT cells). Typically, adults have <1% DNT cells, whereas ALPS patients exhibit expansions from 2% to 40%, which correlate with disease severity. Although not completely resolved, DNT cells apparently arise from CD8+ T cells by an unknown peripheral process rather than during thymic development. ALPS patients have autoantibody-mediated hematological deficiencies or "cytopenias" such as Coombs positive autoimmune hemolytic anemia, neutropenia, and thrombocytopenia. Deficiencies in Fas (also known as CD95, APO-1) apoptosis and related pathways constitute the genetic basis of the disease. Over the two days of talks and discussions, ten clear "take-home" lessons that can be applied to genetic and immunological diseases emerged. Michael Lenardo (NIH, USA) recounted initial experiments demonstrating an apoptosis defect in ALPS patients and the dominant-interfering effect of ALPS alleles in transfection assays. Most ALPS patients harbor heterozygous mutations in the Fas gene that encode defective, dominant-interfering Fas proteins (Type Ia) (Figure 1). The Fas receptor, similar to others in the TNF receptor (TNFR) superfamily, operates as a homotrimeric complex, which can be functionally "poisoned" by even a single defective subunit. The N-terminal preligand assembly domain (PLAD) is preserved so that the defective and normal receptor chains can associate (Figure 1). In nonconsanguineous humans, this dominant interference mechanism explains how heterozygous defects cause ALPS and may explain other diseases involving apoptosis pathways and TNFR superfamily receptors. Indeed, dominant mutations cause defective assembly of the TNF-R family member TACI subunits in common variable immunodeficiency (Garibyan et al., 2007Garibyan L. Lobito A.A. Siegel R.M. Call M.E. Wucherpfennig K.W. Geha R.S. J. Clin. Invest. 2007; 117: 1550-1557Crossref PubMed Scopus (71) Google Scholar). Lesson #1: in outbred human populations, dominant-interfering rather than loss-of-function gene mutations may predominate in pathways that require protein complexes. Frederic Rieux-Laucat (Hôpital Necker, INSERM, France) described his discovery that ALPS patients can exhibit somatic Fas mutations selectively in the DNT cells and other minor lymphocyte subpopulations (ALPS Type 1 m) (Holzelova et al., 2004Holzelova E. Vonarbourg C. Stolzenberg M.C. Arkwright P.D. Selz F. Prieur A.M. Blanche S. Bartunkova J. Vilmer E. Fischer A. et al.N. Engl. J. Med. 2004; 351: 1409-1418Crossref PubMed Scopus (205) Google Scholar). These patients are a phenocopy of ALPS patients with germline mutations, indicating that DNT cells may be the main pathogenetic catalyst for the disease. Type 1 m accounts for up to 30% of the patients without germline mutations. Lesson #2: in immune diseases involving the emergence of unexpected cell populations, somatic pathogenic gene mutations limited to a subset of cells that confer a selective survival advantage may be found. Tom Fleisher (NIH, USA) discussed how ALPS involves more than a single Mendelian trait. Gene mutations leading to ALPS have been designated subtypes I to IV (Figure 1). The majority of mutations affect the Fas signaling apparatus, called the "death-inducing signaling complex" (DISC). Type Ia represents mutations in the Fas (TNFRSF6) gene on chromosome 10q24. Most Fas mutations (more than 70%) in ALPS Type Ia patients alter the intracytoplasmic death domain and cause the prototypical clinical phenotype. Type Ib involves mutations in the Fas ligand (TNFSF6, M5) gene on chromosome 1 (map position 1 H2.1|1 85.0 cM). These also generate dominant-interfering proteins because the ligand also operates as a trimeric complex. Type II ALPS is due to dominant-interfering mutations in caspase-10 (CASP10; chromosome 2, map position 626.70). A disease related to ALPS, called caspase-8 deficiency state (CEDS), is caused by loss-of-function mutations in caspase-8 (CASP8; chromosome 2, map position 626.70), a close paralog occupying the same genomic locus as caspase-10. CEDS shares the lymphoproliferation and apoptosis defect observed with ALPS but exhibits immunodeficiency rather than autoimmunity (Chun et al., 2002Chun H.J. Zheng L. Ahmad M. Wang J. Speirs C.K. Siegel R.M. Dale J.K. Puck J. Davis J. Hall C.G. et al.Nature. 2002; 419: 395-399Crossref PubMed Scopus (533) Google Scholar). Caspase-8 has been shown to play a dual role in the induction of the NF-κB transcription factor during lymphocyte activation as well as in apoptosis mediated by the Fas DISC (Su et al., 2005Su H. Bidère N. Zheng L. Cubre A. Sakai K. Dale J. Salmena L. Hakem R. Straus S. Lenardo M. Science. 2005; 307: 1465-1468Crossref PubMed Scopus (344) Google Scholar). Type III designates ALPS of unknown molecular etiology, which is a potentially exciting source for new discoveries. Umberto Dianzani (University of Eastern Piedmont, Italy) discussed ALPS variants with severe autoimmunity, lymphoproliferation, and defects in Fas apoptosis, but normal DNTs. Gene defects in osteopontin and perforin may underly these variant syndromes (Clementi et al., 2006Clementi R. Chiocchetti A. Cappellano G. Cerutti E. Ferretti M. Orilieri E. Dianzani I. Ferrarini M. Bregni M. Danesino C. et al.Blood. 2006; 108: 3079-3084Crossref PubMed Scopus (50) Google Scholar). Finally, ALPS type IV patients harbor somatic activating mutations in oncogenic Ras genes (for example, a G13D mutation in N-Ras) that also engender a propensity to hematopoietic malignancies (Oliveira et al., 2007Oliveira J.B. Bidère N. Niemela J.E. Zheng L. Sakai K. Nix C.P. Danner R.L. Barb J. Munson P.J. Puck J.M. et al.Proc. Natl. Acad. Sci. USA. 2007; 104: 8953-8958Crossref PubMed Scopus (163) Google Scholar). Joao Bosco Oliveira (NIH, USA) described how activated Ras markedly decreases Bim protein expression leading to impaired lymphokine withdrawal and TCR-induced apoptosis. Interestingly, a subpopulation of patients with a disease resembling juvenile myelomonocytic leukemia (JMML), which is typically fatal in infancy, have a markedly milder condition with features of ALPS Type IV. Lesson #3: as more genes are identified in the pathogenesis of a disease, the clinical heterogeneity of the disease broadens to reflect the physiological effects of the involved genes. Elaine Jaffe (NIH, USA) described the characteristic histopathology of ALPS that affects multiple immune cell compartments including lymphocytes, macrophages (histiocytes), and dendritic cells (DCs) (Lim et al., 1998Lim M.S. Straus S.E. Dale J.K. Fleisher T.A. Stetler-Stevenson M. Strober W. Sneller M.C. Puck J.M. Lenardo M.J. Elenitoba-Johnson K.S. et al.Am. J. Pathol. 1998; 153: 1541-1550Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Increased DNT cells in the lymph node cause follicular hyperplasia and prominent expansion of the paracortex, which can be mistaken for T cell lymphoma. In fact, caution should attend a diagnosis of T cell lymphoma in ALPS because DNT expansions typically remain polyclonal and nonmalignant. The DNT cells are CD57+, possibly suggesting cell senescence, and usually express high amounts of Fas and Fas ligand despite the apoptosis defect in ALPS. S-100+ histiocytes can also be increased amidst the DNT cells. The S100+ histiocytes show evidence of emperipolesis (penetration of lymphocytes and plasma cells within the histiocytes). These features are characteristic of Rosai-Dorfman syndrome, a rare pediatric disease characterized by cervical lymphadenopathy and autoimmunity. This condition, also referred to as sinus histiocytosis with massive lymphadenopathy, may represent a forme fruste of ALPS. Histiocytes and DCs in ALPS apparently share the same apoptotic defects as lymphocytes, and a single case of histiocytic sarcoma has been encountered. Lesson #4: although autoimmunity is generally thought to arise from lymphocyte disturbances, abnormalities of dendritic cell or macrophage homeostasis and/or function can also play an important role. Stefania Pittaluga (NIH, USA) reviewed the 50-fold and 14-fold increased incidence of Hodgkin and non-Hodgkin lymphoma, respectively, in ALPS (Straus et al., 2001Straus S.E. Jaffe E.S. Puck J.M. Dale J.K. Elkon K.B. Rösen-Wolff A. Peters A.M. Sneller M.C. Hallahan C.W. Wang J. et al.Blood. 2001; 98: 194-200Crossref PubMed Scopus (342) Google Scholar). Defective T cell surveillance, Epstein Barr virus (EBV) infection, and defective B cell apoptosis may all predispose to lymphoma. Also, high levels of IL-10 due to a skewing of peripheral T cells to a Th2 cell phenotype or from expression of the BCRF1 gene in EBV (viral IL-10) may similarly contribute. Studies of sporadic lymphomas have also uncovered Fas mutations (Takahashi et al., 2006Takahashi H. Feuerhake F. Kutok J.L. Monti S. Dal Cin P. Neuberg D. Aster J.C. Shipp M.A. Clin. Cancer Res. 2006; 12: 3265-3271Crossref PubMed Scopus (34) Google Scholar). Louis Staudt (NIH, USA) described how mRNA expression microarrays can delineate molecular subtypes of lymphoma that have distinct treatment susceptibilities and prognoses. Nevertheless, except for the general notion that a failure of apoptosis in ALPS can lead to B cell expansions, there is a limited understanding of the propensity to lymphomas. Lesson #5: the Fas pathway plays a general role in preventing B cell lymphomas. The defining characteristics of the disease and the variability among individuals and subtypes of ALPS were discussed. Importantly, individuals in the same pedigree who harbor an identical Fas gene mutation can manifest a wide range of disease symptoms. This variable penetrance has not been explained molecularly, but might stem from as-yet-ill-defined "background" genetic differences or environmental factors. It is notable that backcrossing the lpr or gld mutations into different inbred strains of mice also yields heterogeneous disease phenotypes. The pleiotropy of single-gene defects in genetically diverse individuals is emerging as a recurrent theme in human diseases, in contradistinction to mice or other inbred experimental organisms. The marked phenotypic variation in humans raised questions about what clinical findings should define ALPS. The classic triad required for a diagnosis of ALPS includes chronic lymphadenopathy and splenomegaly, a functional defect in lymphocyte apoptosis, and elevation of DNT cells. It was also noteworthy that despite the many cell types and tissues in which Fas apoptosis has been studied, the clinical abnormalities in ALPS patients are largely confined to the immune system. Koneti Rao (NIH, USA) summarized the clinical spectrum of ALPS observed at NIH Clinical Center over the last 15 years in more than 250 patients. He showed that disseminated lymphoproliferation can be readily visualized by CT scan. When hematologists see young patients with cytopenias and enlarged lymph nodes and splenomegaly, they must be circumspect in raising the issue of possible cancer until ALPS or related nonmalignant causes have been excluded. ALPS patients generally exhibit hypergammaglobulinemia, monocytosis, and eosinophilia, and bone marrow biopsies often reveal the characteristic histopathology as well as the pathognomonic DNT cells. Lesson #6: Fas pathway defects chiefly cause immunological abnormalities and humans, unlike inbred mouse strains, exhibit substantial pleiotropy and variable penetrance associated with gene defects, presumably because of background genes or environmental differences that exist between individuals even within the same family. Most problematic for ALPS diagnosis is the functional apoptosis assays for peripheral blood lymphocytes. The participants discussed two challenges. First, what should the defining apoptosis defect be? Thus far, apoptosis abnormalities in phytohemagglutinnin A (PHA) and IL-2 activated T cells from ALPS patients have been identified in direct Fas-triggered death (Type Ia and II), TCR-induced death (called restimulation-induced cell death [RICD]) (Type Ia, Ib and II), and lymphokine withdrawal death (Type IV). Hence, the relevant apoptosis "defect" for ALPS actually differs according to the precise molecular defect. Thus, a Fas apoptosis defect is too narrow to define all cases of ALPS. Second, how can assay standardization and efficiency be achieved across medical centers that currently have widely varying procedures and results? This variability is less in prototypical Type Ia patients and perhaps greatest in type III patients, suggesting that it also relates to the nature of the gene defect involved. Richard Siegel (NIH, USA) called attention to the variations in PHA+IL-2-stimulated T cell preparations stemming from culture incubation times (which can be days to weeks). Consensus emerged that there was a need for a rapid assay on fresh cells. Siegel related that particular CD4+ T cell subsets are selectively susceptible to Fas killing in normal individuals. Thus, a tailored approach to specific subsets might yield a more effective assay. Certain medical centers have established well-controlled assays, which generated optimism that consensus assays could be developed as standards for the field. Lesson #7: human clinical testing for uncommon diseases can lead to great variability between medical centers, and such variability can only be overcome by rigorous cross-comparison of testing protocols and standards. As with many genetic disorders, genomic DNA sequencing without functional assessment is increasingly being used for diagnosis. Most single-nucleotide changes in ALPS can be detected by commercial testing services. This can be useful in local centers especially when the mutation has been previously confirmed to be functionally deleterious. However, demonstrating a functional apoptosis abnormality is important for a rigorous diagnosis of ALPS. Tabulating known ALPS-associated mutations around the world in a widely accessible format can now be achieved through the Leiden Online Variant Database (LOVD) described by Lisa Forman (National Library of Medicine, USA). This is a new open-source Web site capable of collating mutations in any genetic disease. The National Center for Biotechnology Information at NIH will establish an ALPS site linking LOVD Web pages for each of the genes encoding Fas, FasL, Casp-10, etc., which will be managed by Joao Bosco Oliveira and Julie Niemela (NIH, USA). Locus-specific internet databases linked by disease portals will probably become a foundation for genetic disease research and clinical management. Lesson #8: cataloging mutations associated with rare diseases has become more effective with open-access, Web-based tools such as the Leiden Online Variant Database. Jack Bleesing (University of Cincinnati, USA) described a flow cytometry panel used at Cincinnati Children's Hospital Medical Center as a diagnostic screen for ALPS. It defines a quantitative immunophenotype including the number of DNT cells, the ratio of CD25+CD3+ to HLA−DR+CD3+ cells, increased B220+ T cells, and decreased CD27+ memory B cells. This panel facilitates ALPS patient classification. Nevertheless, pleiotropy is also observed in various ALPS markers in specific patients and the normal ranges for the markers vary with the age of the patient. It was discussed that the abnormal lymphocyte populations, as well as disease severity, decline as patients age past puberty and the thymus involutes. Other biomarkers that are dramatically increased in ALPS include soluble Fas ligand, circulating IL-10, and, surprisingly, vitamin B-12, whose biological provenance is unknown. Combinations of these markers can be highly diagnostic of ALPS in lieu of Fas sequencing and Joao Bosco Oliveira and Tom Fleisher have been working on a diagnostic algorithm for ALPS utilizing flow cytometry and serological markers. A new consensus ALPS classification and revised diagnostic criteria will expedite collaborations and standardization between different centers. ALPS diagnosis has been beneficial to families with this disorder. Although the genetic lesion and biochemical pathway cannot be directly corrected, the medical importance of knowing the molecular etiology of ALPS should not be underestimated. In many cases, knowledge of the gene defect prevents misdiagnosis, especially confusion with lymphoid malignancies, and the administration of unnecessary and potentially toxic treatments. The natural history of the disease is variable and many patients can live with no therapy despite their clinical abnormalities. Most patients receive immunomodulatory agents such as corticosteroids, rituximab, mycophenolate mofetil (MMF, Cellcept), intravenous immunoglobulin, and, most recently, rapamycin (sirolimus). It is a telling insight into the current state of immune therapeutics that corticosteroids, despite substantial metabolic and immunological side effects, remain the initial and perhaps most reliable treatment for ALPS. Other aspects of clinical management include CT scans (every 2 years), the application of spleen guards (because children are prone to traumatic rupture of their enlarged spleens), and antibiotic prophylaxis postsplenectomy for pneumococcal sepsis. Avoidance of splenectomy is increasingly successful by treatment with mycophenolate mofetil and rapamycin, which remains the corner stone of managing these patients today. David Teachey (University of Pennsylvania, USA) discussed the therapeutic effectiveness of rapamycin and other targeted therapies in ALPS. His group first showed that rapamycin, an mTOR kinase inhibitor, was highly effective in lprcg mice, which have disease manifestations similar to ALPS due to Fas mutations. Studies in the clinic revealed that rapamycin in some cases could dramatically resolve the disease manifestations of ALPS with renormalization of DNTs and resolution of lymphadenopathy, splenomegaly, and autoimmunity (Teachey et al., 2006Teachey D.T. Obzut D.A. Axsom K. Choi J.K. Goldsmith K.C. Hall J. Hulitt J. Manno C.S. Maris J.M. Rhodin N. et al.Blood. 2006; 108: 1965-1971Crossref PubMed Scopus (72) Google Scholar). This agent probably resolves disease symptoms with its ability to efficiently eliminate DNT cells; such a finding is not observed with other agents. However, termination of the therapy resulted in the return of disease manifestations. There were also significant concerns regarding side effects of rapamycin especially in children. Some patients do not respond to rapamycin and these might be managed by drug combinations, for example, mTOR inhibitors, mycophenolate mofetil, and methotrexate. Teachey and colleagues also have targeted notch signaling in ALPS by using gamma-secretase inhibitors. These agents had promising effects on disease manifestations in preclinical studies (Teachey et al., 2008Teachey D.T. Seif A.E. Brown V.I. Bruno M. Bunte R.M. Chang Y.J. Choi J.K. Fish J.D. Hall J. Reid G.S. et al.Blood. 2008; 111: 705-714Crossref PubMed Scopus (63) Google Scholar); however, they are not yet currently available for clinical testing. Thus, the future of ALPS therapy may portend agents that are more disease-specific and potentially less toxic than currently available agents. Lesson #9: for autoimmune complications arising from abnormal lymphocyte homeostasis, corticosteroids remain the initial drug of choice, although newer steroid-sparing agents are being developed and tested. Further insights into what lies ahead in ALPS research were provided by the final talks. From the clinical side, Nichola Cooper (Institute of Child Health, UK) reported that increased DNT cells are found in some immune thrombocytopenic purpura (ITP) patients, suggesting a syndrome overlap with Fas function and ALPS. Stefan Muljo (NIH, USA) described microRNA research indicating that genetic diseases such as ALPS deserve further analysis of microRNAs as potentially causative agents, biomarkers, risk factors, or even determinants of disease penetrance. Lorraine O'Reilly (Walter and Eliza Hall Institute, Australia) described studies in the laboratory of Andreas Strasser in mice genetically engineered to selectively express either the membrane-bound or the soluble form of Fas ligand (O' Reilly et al., 2009O' Reilly L.A. Tai L. Lee L. Kruse E.A. Grabow S. Fairlie W.D. Haynes N.M. Tarlinton D.M. Zhang J.-G. Belz G.T. et al.Nature. 2009; 461: 659-663Crossref PubMed Scopus (284) Google Scholar). She has determined that membrane-bound Fas ligand is required for T cell-receptor-induced death and the lack of this form of Fas ligand can lead to lymphoproliferation, autoimmunity, glomerulonephritis, dermatitis, and an increased incidence of histiocytic sarcoma. Hao Wu (Cornell University, USA) described a new structure of Fas: Fas-associated death domain (FADD) protein death domain (DD) association that has an interesting symmetry of interlocking DDs that reveals how Fas DD mutations in ALPS actually map to interfaces and disrupt function. The oligomeric nature of the Fas and FADD DD complex may facilitate cooperativity in signaling. The structure provided the participants with a visual representation of how specific mutations could generate the dominant-interference that constitutes the genetic basis of ALPS. Lesson #10: structural and biochemical examinations of the signaling protein complex are key to illustrating how gene mutations cause disease. In summary, the conference stimulated discussion that coalesced around these lessons that are germane to ALPS and other human genetic disorders. There is important overlap in the features of ALPS and common variable immunodeficiency (CVID), familial hemophagocytic lymphohistiocytosis (FHLH), and X-linked lymphoproliferative disease (XLP), especially because XLP involves defective apoptosis (Snow et al., 2009Snow A.L. Marsh R.A. Krummey S.M. Roehrs P. Young L.R. Zhang K. van Hoff J. Dhar D. Nichols K.E. Filipovich A.H. et al.J. Clin. Invest. 2009; 119: 2976-2989PubMed Google Scholar). Perhaps the final lesson for the participants was that the intensive molecular, cellular, clinical, and therapeutic investigation of rare genetic diseases such as ALPS provides a wealth of insights into normal immune system functions. We thank R. Siegel, T. Fleisher, H. Su, and E. Jaffe for their contributions to this manuscript as well as all of the other speakers including J. Bleesing, N. Cooper, U. Dianzani, L. Forman, S. Muljo, S. Pittaluga, L. O' Reilly, J. Puck, F. Rieux-Laucat, A. Snow, L. Staudt, D. Teachey, and H. Wu together with the participants of the ALPS workshop. This meeting and associated intramural research were supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases and the Office of Rare Diseases, the National Cancer Institute, the National Institute of Arthritis and Musculoskeletal and Skin Diseases, and the Clinical Center, National Institutes of Health.