Unmasking Evans Syndrome: T Cell Phenotype and Apoptotic Response Reveal Autoimmune Lymphoproliferative Syndrome (ALPS).
Authors: Teachey David T. , Manno Catherine S. , Axsom Kelly , Andrews Timothy , Choi John K. , Greenbaum Barbara M. , Sullivan Kathleen , Travis Susan F. , Grupp Stephan A. 
Published: April 2020

Abstract
ALPS is a recently described disorder of disrupted lymphocyte homeostasis. Patients with ALPS have mutations in the Fas apoptotic pathway, leading to abnormal lymphocyte survival. Clinical manifestations in patients with ALPS vary but typically include autoimmune cytopenias, organomegaly, lymphadenopathy, and increased risk of development of lymphoma and certain leukemias. A similar constellation of findings can be seen in Evans Syndrome (ES), a hematologic disorder defined by autoimmune destruction of at least two hematologic cell types. We hypothesized that a subset of patients diagnosed with ES may have ALPS. We have designed a study to screen children with the diagnosis of ES syndrome for ALPS with a three tiered approach. First, a retrospective chart review was performed on all patients treated at CHOP in the past five years for ES who are still followed by the Division of Hematology. Next, patients with a clinical diagnosis of ES without another underlying diagnosis were screened for ALPS by flow cytometric analysis for double negative T cells (DNTs) (CD3+, CD4−, CD8−, TCRαβ+). DNTs are elevated in patients with ALPS and may provide the best current screening assay for this disease. DNTs analysis is easy to perform at low cost. Finally, patients were tested for ALPS by the gold standard test, defective in-vitro Fas mediated apoptosis. This test is labor intensive and not clinically available. 21 patients met entry criteria for the study, and, of these, 19 consented to enrollment. 16/19 had immune mediated destruction of all three hematologic cell lines. 2/19 had only had destruction of two cell types and 1 patient had immune thrombocytopenia with a positive DAT but no hemolysis or neutropenia. 14/19 had chronic disease with frequent hematologic exacerbations. The other 5 had occasional flares with illness. 10/19 had significant lymphadenopathy and 11/19 had organomegaly at some point in their history. 14/19 had either lymphadenopathy or organomegaly. 17/19 patients were screened for DNTs and 10 had elevated DNTs. Thus far, 7 patients have been tested for defective Fas mediated apoptosis. Every patient was tested in tandem with a normal control who demonstrated normal Fas mediated apoptosis. Five of the patients, all of whom had elevated DNTs, had defective Fas mediated apoptosis, confirming the diagnosis of ALPS. The other two patients did not have defective Fas mediated apoptosis: one did not have elevated DNTs and the other had a borderline result (2.7%; upper limit normal 2.6%). One of the patients with defective Fas mediated apoptosis and elevated DNTs had no history of lymphadenopathy or organomegaly. The lack of clinically identifiable lymphoproliferation in a patient with defective apoptosis is an unexpected finding, in apparent contradiction of the accepted NIH definition of ALPS which lists lymphoproliferation as a mandatory diagnostic criterion. This patient is three years old and may develop clinically identifiable lymphoproliferation with age. In summary, in the group of 17 ES patients screened to date, 59% had elevated DNTs suggestive of ALPS, with functional confirmation in 5/5 tested. Our data suggest that DNTs may be a sensitive first line-screening test and may serve as a marker that identifies patients who require definitive testing. Our preliminary findings suggest a high prevalence of ALPS among ES patients, a novel finding with important implications.