Autoimmune Lymphoproliferative Syndrome (ALPS) Disease and ALPS Phenotype: Are They Two Distinct Entities?
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
This study was performed in line with the principles of the Declaration of Helsinki.
Informed consent was obtained from the parents and/or individuals participants included in the study.
Abstract
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.
INTRODUCTION
Autoimmune lymphoproliferative syndrome (ALPS) is a rare inherited disorder due to the dysregulation of the FAS apoptotic pathway, usually manifested in childhood, although symptoms may occur at any age. As a result of disturbed lymphocyte homeostasis, autoreactive cells persist, leading to the development of chronic nonmalignant lymphadenopathy, splenomegaly, multilineage cytopenias, and increased risk of lymphoma.1 Currently, the diagnosis of ALPS is based on phenotypical criteria since it is determined by the combination of different clinical and laboratory findings as from reported in 2009 by a panel of NIH (National Institutes of Health) experts.2 Lymphoproliferation is the most common clinical manifestation of ALPS followed by autoimmunity that appears in 70% of cases.3, 4 Many patients have isolated or variable combination of immune cytopenia such as autoimmune hemolytic anemia (AIHA), neutropenia (AIN), and immune thrombocytopenia (ITP).5
The typical biomarker is an increased number of a characteristic T-cell population named “double-negative T cells” (DNTs); other characteristic laboratory abnormalities include elevated levels of interleukin (IL) IL-10, IL-18, vitamin B12, and soluble FAS-ligand (sFASL).6
Elevated DNTs lymphocytes expressing B220,7-9 together with an expansion of HLA-DR+ T cells10 and a decrease of CD4+CD25+ T cells and CD27+ B cells10, 11 were also reported in ALPS patients and generated the proposal of a flow cytometry panel as a diagnostic screening tool for ALPS including: CD3CD4-CD8-+TCRαβ+, CD3+CD25+/CD3HLADR+, TCRαβ+ B220+, and CD19+CD27+.
Management of ALPS patients aims to control clinical manifestations and specific complications such as cytopenia are often difficult to handle because stable remission may be difficult to achieve after the first (steroids and immunoglobulins) and even the second line of treatment (mychopenolate-mophetil [MMF] and rapamycin).12-16
Mutations in FAS, FASL, and CASP 10 genes are found to cause the disease in about 70% of patients, but in one-third of ALPS subjects, no pathogenic genetic lesion is found (ALPS-U, undetermined genetic defects).2, 14, 17
Discovery of novel defects causing immunodeficiencies or immune dysregulation would be identified as potential drivers in ALPS-U patients.18 ALPS, which is primarily a “phenotypic definition” according to the NIH criteria, as a matter of fact, probably hide different diseases raising the point of additional specific mechanisms that can be targeted by effective therapies.19
The aim of the present study is to compare the 2 groups ALPS-FAS/CASP10 versus ALPS-U to identify any possible surrogate markers that may direct the diagnosis toward more precise genetic categories.
PATIENTS AND METHODS
Patients
Patients were defined as ALPS definitive and probable according to the criteria established by the NIH in 2009.2 (Table 1) Demographic data, clinical findings, biochemical markers (vitamin B12, interleukin 10 and interleukin 18, sFASL, immunoglobulins serum level, lymphocyte subsets analysis including CD3+CD4-CD8- TCR alfabeta+, named DNT cells), autoimmune markers (antiplatelet and antineutrophil indirect antibodies, direct and indirect Coombs test, antinuclear antibodies [ANA], extractable nuclear antigen antibodies, antismooth muscle antibody [ASMA], anti double-stranded DNA [anti-dsDNA] antibodies, antiglyadin, antitransglutaminase, antiendomysium, and antithyroglobulin antibodies) were entered into an electronic national ALPS database (ALPS Italian Network) (Tables 4 and 5).
| Required |
|---|
| 1. Chronic nonmalignant lymphoproliferation (<6 mo lymphadenopathy and/or splenomegaly) |
| 2. Elevated peripheral blood DNTs |
| Accessory |
| Primary |
| 1. Defective in vitro Fas-mediated apoptosis (in 2 separate assays) |
| 2. Somatic or germline mutation in ALPS causative gene (FAS, FASL, CASP10) |
| Secondary |
| 1. Elevated biomarkers (any of the following): (a) Plasma sFASL > 200 pg/mL; (b) Plasma IL-10 > 20 pg/mL; (c) Plasma or serum vitamin B12 > 1500 ng/L; (d) Plasma IL-18 > 500 pg/mL |
| 2. Immunohistochemical findings consistent with ALPS as determined by experienced histopathologist |
| 3. Autoimmune cytopenias AND polyclonal hypergammaglobulinemia |
| 4. Family history of ALPS or nonmalignant lymphoproliferation |
| Definitive diagnosis: the presence of both required criteria plus one primary accessory criterion. |
| Probable diagnosis: both required criteria plus one secondary accessory criterion. |
| Of note, probable and definitive ALPS should be treated the same in the clinic. |
- ALPS = autoimmune lymphoproliferative syndrome; DNTs = double-negative T cells; NIH = National Institutes of Health; sFASL = soluble FAS-ligand.
| Pt n | Sex | Gene | Dg ALPS NIH criteria | DNT%>1,5% | Apoptosis | Lymphoproliferation | Cytopenia | Lymphocytopenia | IL-10 > 20 pg/mL | IL-18 > 500 pg/mL | VitB12 > 1500 mg/dL | CD19+CD27+<15% | TCR αβ B220+>60% | CD3CD25+/CD3HLADR+<1 | AUTOIMMUNE markers | Multiorgan Involvement |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | M | FAS | DEF | 2.4 | NP | Ly+S | Neg | Neg | NA | NA | NA | NA | NA | NA | Neg | Y |
| 2 | M | FAS | DEF | 5.8 | Pos | Ly+S | AIHA, ITP | Neg | 35 | 550 | 291 | 12 | 89.8 | 0.5 | DAT | Neg |
| 3 | M | FAS | DEF | 6.1 | Pos | S | AIHA | Neg | NA | NA | 467 | NA | NA | NA | Neg | Neg |
| 4 | F | FAS | DEF | 6.3 | Pos | Ly+S | ITP, L | Neg | 79 | 1175 | 2000 | 10.3 | 86 | 0.3 | Neg | Y |
| 5 | M | FAS | DEF | 13 | Pos | Ly+S | AIHA, ITP | Neg | NA | NA | 1581 | NA | NA | NA | DAT | Neg |
| 6 | F | FAS | DEF | 5.5 | Neg | S | L, AIHA, ITP | Y | 40 | 950 | 10233 | 2.7 | 68.7 | 0.4 | Neg | Neg |
| 7 | M | FAS | DEF | 3.7 | Pos | Ly+S | Neg | Neg | 0 | 1321 | 1747 | 2.2 | 69.4 | 0.2 | Neg | Neg |
| 8 | M | FAS | DEF | 4.8 | Pos | Ly+S | ITP | Neg | NA | NA | 879 | NA | NA | NA | Neg | Neg |
| 9 | M | FAS | DEF | 18 | Pos | Ly+S | ITP | Neg | NA | NA | 2000 | NA | NA | NA | Neg | Neg |
| 10 | M | FAS | DEF | 13 | Neg | Ly+S | ITP+AIHA | Neg | NA | NA | 1940 | NA | NA | NA | Neg | Neg |
| 11 | F | FAS | DEF | 3.7 | Pos | Ly | NA | NA | NA | NA | 376 | NA | NA | NA | Neg | Neg |
| 12 | F | FAS | DEF | 22 | Pos | Ly+S | ITP+AHIA+L | Neg | 115 | 1700 | 849 | 4.6 | 89 | 0.1 | Neg | Neg |
| 13 | M | CASP10 | DEF | 6.3 | Neg | Ly | NA | NA | 0 | 567 | NA | 25.7 | 66.4 | 1.2 | Neg | Neg |
| 14 | F | CASP10 | DEF | 1.9 | Pos | S | ITP | Neg | 0.9 | 250 | 547 | 49 | 41.7 | 1.9 | ANA | Neg |
| 15 | F | CASP10 | DEF | 2.3 | Neg | Ly+S | Neg | Neg | 0 | 1050 | 393 | 15 | 81 | 0.2 | DAT | Neg |
| 16 | F | CASP10 | DEF | 4.1 | Pos | Ly+S | L, AIHA | Neg | 90 | 5001 | 1492 | 3 | 62.8 | 0.3 | DAT, ASMA, anti N and PLT | Y |
| 17 | M | CASP10 | DEF | 2.6 | Pos | S | ITP | Neg | 7.2 | 230 | 585 | 13.6 | 10.4 | 0.3 | Neg | Neg |
| 18 | F | CASP10 | DEF | 3.1 | Pos | Ly+S | ITP | Neg | 3.1 | 1200 | 373 | 18 | 73.5 | 0.2 | ANA | Y |
- AIHA = autoimmune hemolytic anemia; AIN = autoimmune neutropenia; ALPS = autoimmune lymphoproliferative syndrome; ANA = antinuclear antibodies; antiN = antineutrophil antibodies; antiPLT = antiplatelets antibodies; ASMA = antismooth muscle antibodies; DAT = direct antibodies test; DEF = definitive; F = female; ITP = immune thrombocytopenia; L = leucopenia; Ly = lymphoproliferation; M = male; NA = not applicable; Neg = negative; NP = notproliferative; Pt = patient; Pos = pathologic; S = splenomegaly; Y = present.
| Pt n | Sex | Gene | Dg ALPS NIH criteria | DNT%>1,5% | Apoptosis | Lymphoproliferation | Cytopenia | Lymphocytopenia | IL-10 > 20 pg/mL | IL-18 > 500 pg/mL | VitB12 > 1500 mg/dL | CD19+CD27+<15% | TCR αβ B220+>60% | CD3CD25+/CD3HLADR+<1 | Autoimmune Markers | Multiorgan Involvement |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 19 | M | ARPC1B c.64 + 1G>C | DEF | 3.6 | Pos | Ly | ITP | Y | NA | NA | NA | 10.6 | 64.7 | 0.8 | ANA, ASCA | Y |
| 20 | F | AIRE p.Arg9Trp AIRE p.Val484Met | DEF | 3.1 | Pos | S | ITP | Neg | 11.2 | 245 | 1016 | 9.4 | 29 | 1 | Neg | Neg |
| 21 | M | STAT3 p.Arg152Trp | PROB | 3.5 | ND | S | ITP+L | Y | 4 | 500 | NA | 14.7 | 48 | 0.8 | CD | Y |
| 22 | F | CTLA4 p.Cys58fs LRBA p.Asp2294Asn | DEF | 2.2 | Pos | Ly+S | AIHA+ITP+L | Y | 7 | 425 | 370 | 3.3 | 62.3 | 1.9 | Anti GP | Y |
| 23 | F | ADA2 p.Leu188Pro ADA2 p.Thr187Pro Somatic STAT3 p.Lys658Arg | DEF | 5.1 | Pos | Ly+S | AIHA+ITP | Neg | 14 | 5250 | 374 | 15.9 | 82.7 | 0 | DAT, ANA | Y |
| 24 | M | TNFRSF13C p.His159Tyr TNFRSF13C p.Pro21Arg | PROB | 4 | Neg | Ly+S | ITP | Y | 3 | 660 | 356 | 6.6 | 70.6 | 0.2 | DAT | Neg |
| 25 | F | NLRC4 p.Arg492Trp | DEF | 2.1 | Pos | Ly+S | Neg | Neg | 2.5 | 100 | 345 | 11 | 29.9 | 1.7 | Neg | Y |
| 26 | F | LRBA p.Gln2561Ter LRBA c.1359 + 1G>A | DEF | 2.6 | Pos | Ly+S | ITP+L | Neg | NA | NA | NA | NA | NA | NA | DAT,ASMA,ICA,CD | Y |
| 27 | M | LRBA p.Arg655Ter | DEF | 2.2 | Pos | Ly+S | Neg | Neg | 7.5 | 815 | 990 | 12.3 | 85 | 0.8 | ANCA, ASMA | Y |
| 28 | M | IKBKG p.Glu125Lys | DEF | 9 | Pos | Ly+S | ITP | Neg | 3 | 450 | 316 | 6.8 | 67.5 | 1 | ANA, anti N | Y |
| 29 | F | TNFRSF13B p.Arg202His | DEF | 5 | Pos | Ly+S | ITP+AIHA+L | Y | 0.9 | 1225 | 807 | 15 | 46.6 | 2.9 | ANA, DAT, CD | Y |
| 30 | M | GBA p.Asn370Ser GBA p.Gly202Arg | DEF | 2.8 | Pos | Ly+S | ITP+L | Y | 4 | 775 | NA | 9.4 | 56 | 1.5 | Neg | Neg |
| 31 | M | FOXP3 p.Leu260Gln | DEF | 3.7 | Neg | Ly | Neg | Neg | 4 | 600 | 1176 | 4.2 | 52 | 0.9 | Neg | Neg |
| 32 | F | NEG | PROB | 2.5 | Neg | S | L | Neg | 11 | 475 | 1029 | 22.3 | 62.5 | 1.3 | ANA, ASCA, anti N | Y |
| 33 | M | NEG | DEF | 2 | Pos | S | L | Neg | 0.9 | 736 | 819 | 14.4 | 61 | 3 | Neg | Y |
| 34 | M | NEG | PROB | 4.6 | Neg | Ly+S | ITP+L | Y | 2 | 600 | 274 | 6.2 | 70 | 0.9 | ENA | Y |
| 35 | M | NEG | PROB | 2.2 | Neg | Ly+S | ITP | Neg | 2.4 | 900 | 529 | 7.9 | 47.8 | 1.6 | Neg | Y |
| 36 | F | NEG | PROB | 2.4 | Neg | Ly+S | Neg | Neg | 0 | 700 | 1400 | 11.3 | 60 | 0.4 | Neg | Y |
| 37 | M | NEG | DEF | 1.8 | Pos | S | ITP+L | Y | 305 | 360 | 725 | 12.5 | 68.7 | 0.1 | ANA,ENA,ASMA,CD, anti N | Y |
| 38 | M | NEG | PROB | 2.5 | Neg | Ly+S | L | Neg | 0.9 | 925 | 567 | 3.8 | 47.7 | 1 | Neg | Y |
| 39 | M | NEG | DEF | 1.7 | Pos | S | ITP+L | Neg | 3.5 | 2150 | 430 | 33.6 | 78.7 | 0.6 | Anti N | Y |
| 40 | F | NEG | DEF | 1.9 | Pos | S | Neg | Neg | 0 | 375 | 698 | 26.6 | 83 | 0.7 | CD | Neg |
| 41 | F | NEG | DEF | 2.3 | Pos | S | AIHA+ITP | Y | NA | NA | NA | NA | NA | NA | DAT | Y |
| 42 | F | NEG | DEF | 2.4 | Pos | Ly+S | ITP | Neg | 12 | 1165 | 339 | 13.9 | 57.4 | 0.3 | Neg | Y |
| 43 | M | NEG | PROB | 3.4 | Neg | S | AIHA+L | Y | 0 | 275 | NA | 23 | 48.3 | 0.8 | ASMA, ASCA | Y |
| 44 | M | NEG | PROB | 3.5 | Neg | Ly+S | ITP+L | Neg | 13 | 600 | 641 | 12.9 | 43.9 | 0.8 | DAT | Neg |
| 45 | M | NEG | DEF | 14.8 | Pos | Ly | AIHA+ITP+L | Y | 0 | 550 | 684 | 34.2 | 69 | 1 | ANA,DAT | Neg |
| 46 | F | IKBKG p.Glu125Lys | DEF | 4.7 | Pos | S | AIHA+ITP+L | Y | 0.2 | 265 | 999 | 0 | 59 | 2.4 | ANA, CD, tyroid | Y |
- AIHA = autoimmune hemolytic anemia; AIN = autoimmune neutropenia; ALPS = autoimmune lymphoproliferative syndrome; ANA = antinuclear antibodies; antiGP = antigastric parietal cells; antiN = antineutrophil antibodies; antiPLT = antiplatelets antibodies; ASMA = antismooth muscle antibodies; CD = celiac disease; DAT = direct antibodies test; DEF = definitive; ICA = antipancreatic insula; F = female; ITP = immune thrombocytopenia; L = leucopenia; Ly = lymphoproliferation; M = male; NA = not applicable; Neg = negative; NP = not proliferative; Pos = pathologic ; Pt = patient; S = splenomegaly; Y = present.
All the information were retrieved from the medical records of patients after informed consent according to the Helsinki declaration of principles. Patients were referred at the ALPS Italian network through the AIEOP (Italian Pediatric Hemato-Oncology Association) centers.
Response to therapy was evaluated after at least 4–6 weeks of drug administration for each of the following clinical symptoms: cytopenia, lymphoproliferation, fever, or other autoimmune symptoms and scored as complete (CR), partial (PR), or nonresponse (NR) (See for further detail the footnotes of Table 6). In keeping with the response criteria from our previous publication,19 a patient was considered as complete responder if all the aforementioned clinical symptoms disappeared/normalized 4–6 weeks after treatment start. A patient was considered as partial responder if at least one of the aforementioned clinical symptom improved/normalized 4–6 weeks after treatment start. A patient was considered as nonresponder if none of the above symptoms improved/normalized 4–6 weeks after treatment start.
| ALPS-FAS/CASP + | ALPS-U | P | |
|---|---|---|---|
| Cytopenia (overall) | 13/16 (81%) | 23/28 (82%) | 0.94 |
| Thrombocytopenia (ITP) | 11/13 (85%) | 19/23 (83%) | 0.87 |
| Hemolytic anemia (AIHA) | 7/13 (54%) | 7/23 (30%) | 0.16 |
| Autoimmune neutropenia (AIN) | 4/13 (31%) | 15/23 (65%) | 0.04 |
| Lymphocytopenia | 1/16 (6%) | 16/28 (57%) | 0.01 |
| Isolated ITP | 5/13 (38%) | 6/23 (26%) | 0.43 |
| Isolated AIHA | 1/13 (14%) | 0/23 (0%) | 0.17 |
| Isolated leucopenia | 0/13 (0%) | 3/23 (13%) | 0.17 |
| Isolated AIN | 0/13 (0%) | 3/23 (13%) | 0.17 |
| ITP + AIHA | 3/13 (23%) | 2/23 (9%) | 0.23 |
| ITP+ leucopenia | 1/13 (8%) | 7/23 (30%) | 0.11 |
| AIHA + leucopenia | 1/13 (8%) | 1/23 (4%) | 0.67 |
| AIHA + leucopenia + ITP | 2/13 (15%) | 4/23 (17%) | 0.87 |
| Isolated lymphoproliferation | 2/18 (11%) | 3/28 (10%) | 0.96 |
| Isolated splenomegaly | 4/18 (22%) | 10/28 (36%) | 0.33 |
| Lymphoproliferation + splenomegaly | 12/18 (67%) | 15/28 (54%) | 0.37 |
| Vitamin B12 > 1500 ng/L | 6/16 (37%) | 0/22 (4%) | 0.001 |
| IL-10 > 20 pg/mL | 5/11 (45%) | 1/25 (4%) | 0.002 |
| IL-18 > 500 pg/mL | 9/11 (82%) | 16/25 (64%) | 0.28 |
| Autoimmunity markers | 6/18 (33%) | 19/28 (68%) | 0.02 |
| FAS-mediated apoptosis | 13/18 (72%) | 18/27 (67%) | 0.69 |
| Multiorgan involvement | 4/18 (22%) | 21/28 (75%) | 0.001 |
| No therapy | 2/18 (11%) | 1/28 (4%) | 0.31 |
| Response to MMF or rapamycina | 12/12 (100%) | 7/21 (33%) | <0.001 |
| Associated therapies | 1/16 (6%) | 17/27 (63%) | <0.001 |
| Number of drugs | 2/16 (12%) | 14/27 (52%) | 0.009 |
- CR for cytopenia: Hb 80–100 g/L (NCI-CTCAE grade 2 anemia) and/or platelet count >100 × 109/L and/or neutrophil count >1.5 × 109/L.
- PR: red cell transfusion independence and/or platelet count 30–100 × 109/L and/or neutrophil count >1.5 × 109/L.
- NR for cytopenia: failure to achieve partial/complete response objectives.
- CR for lymphoproliferation: lymphoid organs return to normal size.
- PR for lymphoproliferation: lymphoid organs reduce their Volume.
- NR for lymphoproliferation: failure to achieve partial/complete response objectives.
- CR for fever and other symptoms: disappearance.
- PR for fever and other symptoms: attenuation.
- NR for fever and other symptoms: failure to achieve partial/complete response objectives.
- Degree of cytopenia was defined according to NCI-CTCAE v.4. Available from: https://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf.
- a Response to therapy was evaluated after at least 4–6 weeks of drug administration for each of the following clinical symptoms: cytopenia, lymphoproliferation, fever, or other autoimmune symptoms and scored as CR, PR, or NR.
- AIHA = autoimmune hemolytic anemia; AIN = autoimmune neutropenia; ALPS = autoimmune lymphoproliferative syndrome; CR = complete response; DNTs = double-negative T cells; ITP = immune thrombocytopenia; MMF = mychopenolate-mophetil; NR = nonresponse; PR = partial response.
First-line treatment was steroids or intravenous immunoglobulins, whereas the second-line was MMF or rapamycin. Further lines of treatment included: Rituximab plus MMF or rapamycin, anakinra, cyclosporine A, eltrombopag or Nplate (whenever cytopenia was the dominating sign) either alone or in combination with MMF or rapamycin.
Lymphocyte immunophenotypes
A flow cytometry panel including 4 parameters suggestive of ALPS (CD3CD4-CD8+TCRαβ+>1.5% of total lymphocytes, CD3CD25+/CD3HLADR+<1%, TCR αβ+ B220+>60% of DNT, and CD19+CD27+<15% of B cells)7, 11 was used as immunological screening. Peripheral lymphocyte subsets were evaluated from whole blood using an 8-color immunostaining panel (lyse and wash procedure), a FACS Canto II flow cytometer (BD) equipped with three lasers (blue, red, violet), FACSDiva software (BD), and a large panel of RUO mAbs and fluorochromes variously combined (all BD).
Functional tests
FAS-mediated apoptosis test
Fas-induced cell death was evaluated on T-cell lines obtained by activating peripheral blood mononuclear cells (PBMCs) with phytohemagglutinin (PHA) at days 0 (1 μg/mL) and 12–15 (0.1 μg/ml) and cultured in RPMI 1640 + 10% FCS + rIL-2 (2 U/mL). Fas function was assessed 6 days after the second stimulation (day 21). Cells were incubated with control medium or anti-Fas mAb (CH11, IgM isotype) (1 μg/mL) in the presence of rIL-2 (1 U/mL) to minimize spontaneous cell death. Cell survival was evaluated after 18 hours by counting live cells in each well by the trypan blue exclusion test and by flow cytometry of cells excluding propidium iodide. Cells from 2 normal donors were included in experiment as positive controls. Results were expressed as specific cell survival % calculated as follows: (total live cell count in the assay well/total live cell count in the control well) × 100. Fas function was defined as defective when cell survival was >78% (the 95th percentile of data obtained from 200 normal controls).20
Not proliferative (NP) indicates that tested lymphocyte are normally culture for 18 days and occasionally at time of testing cells may not proliferate in sufficient number. The cells are kept in culture for 18 days to select the population of T-lymphocytes on which to perform the test. In some cases it may happen that at the time of setting up the test the number of cells is insufficient, and in this case, we indicate insufficient cell proliferation in the report.
ADA2 functional test
Two previously unreported variants of gene ADA2 were found through next-generation sequencing (NGS) analysis, and a pathogenic role was demonstrated through a functional study.
A functional analysis on peripheral monocytes was performed to test their effect on ADA2 activity.
These cells were isolated by adherence, after PBMC Ficoll–Paque separation and were then cultured in phosphate-buffered saline with exogenous adenosine (Sigma Aldrich) with or without ADA1 inhibitorerythro-9-(2-hydroxy-3-nonyl) adenine (Sigma Aldrich) for4 h at 37°C with 5% of CO2. The supernatants were collected, and the activity enzyme was indirectly evaluated in high-performance liquid chromatography through the measurement of the adenosine-derived products (inosine and hypoxanthine) as a surrogate marker of enzyme activity.21
CASP10 functional test
In CASP10 variants, functional studies were performed after inducing apoptosis by FAS-ligand/TRIAL stimulation and analyzing cell death and the function of CASP10, CASP8 and PARP proteins. Human lymphoblast cell lines, obtained from patients’ samples after informed consent, were grown in RPMI medium with glutamine and antibiotics to 37°C and 5% CO2. Recombinant FAS-ligand (FASL) was from Enzo Life Science (Farmingdale, NY) and recombinant super killer TRAIL was from Alexis Biochemicals (Farmingdale). Both primary cells and lymphoblasts were treated with FASL(10 ng/mL) or TRAIL (100 ng/mL) for 24 hours to induce apoptosis and cell death, which were measured by the cytofluorimeter. For Western blotting, the cells were incubated for 4 hours, then lysed with ice-cold radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors. Protein quantification in cell lysates was done with the DC Protein assay Kit (BioRad, Hercules, CA). An equal amount of protein (20 μg/lane) were loaded on precast 8% gel (GenScript, Piscataway, NJ) and electrotransferred to polyvinylidene difluoride membranes (GE Healthcare, Chicago, IL). After blocking, membranes were probed over night at 4°C with the following antihuman primary antibodies diluted according to the manufacturer's instructions: mouse monoclonal anti-Caspase-8 (1C12), rabbit polyclonal anti-PARP (Cell Signaling, Danvers, MA) and rabbit monoclonal anti-Caspase-10 (Abcam, Cambridge, United Kingdom). After washing, the membranes were incubated for 1 hours at room temperature with the relevant horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling) and proteins were detected by chemiluminescent HRP substrate (Immobilon Western, Millipore, Burlington, MA). Anti-actin HRP-conjugated (Cell Signaling) was used as loading control. To better investigate the causes of this resistance to apoptosis, we assessed cell extracts after treatment with FAS-L and TRAIL by Western blotting.
Interleukin 18, Interleukin 10, and FasL
Serum interleukin 18, interleukin 10, and FasL assays were performed using commercially available enzyme-linked immuno sorbent assay kits (MBL, Woburn, MA; Invitrogen, Waltham, MA; and Abnova, Taipei, Taiwan, respectively).
Genetic analysis
Forthysix ALPS-FAS/CASP10 and ALPS-U patients underwent genetic analysis. Molecular variants were detected by Sanger sequencing in the less recent diagnoses and specifically 10 of 18 subjects of the ALPS-FAS/CASP10 group and 3 of 28 of the ALPS-U group or by NGS with Sanger confirmation (the most recent diagnoses; 8/18 patients of the ALPS-FAS/CASP10 group and 25/28 of the ALPS-U group).
Analysis were carried out on peripheral blood and not on sorted DNT cells.
We used the Sanger PCR technique to search for mutations in the pre-NGS period. We studied with NGS new patients who had not yet undergone any molecular investigation. To confirm the presence of the variants thus selected, the Sanger PCR protocol was set up for each variant to confirm genetic mutations detected by NGS.
The HaloPlex Target Enrichment System was used for the NGS panel (Version C1, December 2016; Version E1, July 2015) and included 315 genes involved in hematological disorders, immunodeficiencies, immune dysregulation, inflammatory, and bone marrow failure syndromes (Table 2).
| Target ID | Coverage | Target ID | Coverage | Target ID | Coverage | Target ID | Coverage | Target ID | Coverage |
|---|---|---|---|---|---|---|---|---|---|
| A20 | 99.49 | CENPS | 100 | HAX1 | 100 | NHEJ1 | 100 | SERPING1 | 100 |
| ACP5 | 100 | CENPX | 100 | HOIP | 100 | NLRC4 | 100 | SH2D1A | 100 |
| ACT1 | 100 | CFB | 100 | ICOS | 100 | NLRP12 | 100 | SH3BP2 | 100 |
| ACTB | 61.77 | CFD | 100 | IFIH1 | 100 | NLRP3 | 100 | SLC29A3 | 100 |
| ADA2 | 100 | CFH | 100 | IFNGR1 | 100 | NLRP7 | 99.77 | SLC37A4 | 100 |
| ADAR1 | 100 | CFHR1 | 95.24 | IFNGR2 | 100 | NOD2 | 100 | SLC46A1 | 100 |
| AICDA | 100 | CFHR3 | 88.54 | IGLL1 | 100 | NOLA2 | 100 | SLC7A7 | 100 |
| AIRE | 100 | CFI | 100 | IKAROS | 100 | NOLA3 | 100 | SLX4 | 100 |
| AK2 | 100 | CFP | 100 | IKBA | 100 | NRAS | 100 | SMARCAL1 | 100 |
| AP1S3 | 100 | CHD7 | 100 | IKBKB | 100 | ORAI1 | 100 | SP110 | 100 |
| AP3B1 | 100 | CIITA | 100 | IKBKG | 46.03 | OTULIN | 100 | SPINK5 | 100 |
| APOL1 | 100 | COLEC11 | 100 | IKZF1 | 100 | OX40 | 100 | STAT1 | 100 |
| ARPC1B | 100 | COPA | 100 | IL-10 | 100 | PALB2 | 100 | STAT2 | 100 |
| ATM | 100 | CORO1A | 95.62 | IL10RA | 100 | PAX5 | 100 | STAT3 | 100 |
| BCL10 | 100 | CSF2RA | 22.43 | IL10RB | 100 | PGM3 | 100 | STAT5B | 90.23 |
| BLM | 100 | CSF3R | 100 | IL12B | 100 | PI3K | 99.63 | STIM1 | 100 |
| BLNK | 99.46 | CTC1 | 100 | IL12RB1 | 100 | PIK3CD | 100 | STK4 | 100 |
| BLOC1S6 | 100 | CTLA4 | 100 | IL17F | 100 | PIK3R1 | 100 | STN1 | 100 |
| BOD1L1 | 100 | CTPS1 | 100 | IL17RA | 100 | PLCG2 | 100 | STX11 | 100 |
| BRCA1 | 100 | CTSC | 100 | IL1RN | 100 | PMS2 | 74.9 | STXBP2 | 98.89 |
| BRCA2 | 99.97 | CXCR4 | 100 | IL21 | 100 | PNP | 100 | TAP1 | 100 |
| BRIP1 | 100 | CYBA | 100 | IL21R | 100 | POLE1 | 99.89 | TAP2 | 99.85 |
| BTK | 100 | CYBB | 100 | IL2RA | 100 | PRF1 | 100 | TAPBP | 100 |
| C1NH | 100 | DCLRE1C | 100 | IL2RG | 100 | PRKCD | 100 | TAZ | 100 |
| C1QA | 100 | DKC1 | 100 | IL36RN | 100 | PSMA3 | 100 | TBK1 | 100 |
| C1QB | 100 | DNASE1 | 100 | IL7R | 100 | PSMB4 | 100 | TBX1 | 100 |
| C1QC | 100 | DNASE1L3 | 100 | IRAK4 | 100 | PSMB8 | 100 | TCF3 | 100 |
| C1R | 100 | DNASE2 | 100 | IRF8 | 100 | PSMB9 | 100 | TCN2 | 100 |
| C1S | 100 | DNMT3B | 100 | ISG15 | 100 | PSTPIP1 | 99.53 | TERC | 100 |
| C2 | 99.06 | DOCK2 | 100 | ITCH | 100 | PTPRC | 97.54 | TERT | 100 |
| C3 | 100 | DOCK8 | 100 | ITGB2 | 100 | RAB27A | 100 | THBD | 100 |
| C4A | 21.47 | ELANE | 100 | ITK | 100 | RAC2 | 100 | TINF2 | 100 |
| C4B | 27.75 | ERCC4 | 100 | JAGN1 | 100 | RAD51 | 100 | TLR3 | 100 |
| C5 | 100 | EVER1 | 100 | JAK1 | 100 | RAD51C | 100 | TMEM173 | 100 |
| C6 | 100 | EVER2 | 100 | JAK3 | 100 | RAG1 | 100 | TNFAIP3 | 100 |
| C7 | 100 | EXTL3 | 100 | KIND3 | 100 | RAG2 | 100 | TNFRSF11A | 100 |
| C8A | 100 | FAAP100 | 100 | KRAS | 100 | RASGRP1 | 100 | TNFRSF13B | 100 |
| C8B | 100 | FAAP20 | 100 | LACC1 | 100 | RBCK1 | 100 | TNFRSF13C | 100 |
| C8G | 100 | FAAP24 | 100 | LAMTOR2 | 100 | RFX5 | 100 | TNFRSF1A | 98.34 |
| C9 | 96.03 | FADD | 100 | LCK | 100 | RFXANK | 93.94 | TPP2 | 100 |
| CARD11 | 100 | FAN1 | 100 | LIG4 | 100 | RFXAP | 100 | TRAF3 | 100 |
| CARD14 | 100 | FANCA | 97.66 | LPIN2 | 100 | RHOH | 100 | TREX1 | 100 |
| CARD9 | 100 | FANCB | 100 | LRBA | 99.94 | RMRP | 100 | TRIF | 100 |
| CASP10 | 100 | FANCC | 100 | LYST | 99.99 | RNASEH2A | 100 | TTC7A | 100 |
| CASP8 | 100 | FANCD2 | 99.42 | MAGT1 | 99.4 | RNASEH2B | 100 | TWEAK | 100 |
| CD19 | 100 | FANCE | 100 | MALT1 | 100 | RNASEH2C | 100 | TYK2 | 100 |
| CD20 | 99.57 | FANCF | 100 | MAP3K14 | 100 | RNF168 | 100 | UAF1 | 100 |
| CD21 | 100 | FANCG | 100 | MASP1 | 100 | RPL11 | 100 | UBE2T | 100 |
| CD27 | 100 | FANCI | 100 | MASP2 | 100 | RPL26 | 100 | UNC119 | 100 |
| CD3D | 100 | FANCL | 100 | MCM4 | 100 | RPL35A | 100 | UNC13D | 100 |
| CD3E | 100 | FANCM | 100 | MDA5 | 100 | RPL5 | 100 | UNC93B1 | 95.43 |
| CD3G | 99.65 | FAS | 100 | MEFV | 100 | RPS10 | 100 | UNG | 100 |
| CD3Z | 100 | FASLG | 100 | MPL | 100 | RPS17 | 15.97 | USB1 | 100 |
| CD40 | 100 | FCN3 | 100 | MRE11 | 100 | RPS19 | 100 | USP1 | 100 |
| CD40LG | 100 | FOXN1 | 100 | MTHFD1 | 100 | RPS24 | 100 | VPS13B | 100 |
| CD46 | 100 | FOXP3 | 100 | MVK | 100 | RPS26 | 100 | VPS45 | 100 |
| CD59 | 100 | FPR1 | 100 | MYD88 | 100 | RPS7 | 100 | WAS | 100 |
| CD70 | 99.52 | FUCT1 | 100 | NBN | 100 | RPSA | 100 | WDR1 | 100 |
| CD79A | 100 | G6PC | 100 | NCF1 | 42.96 | RTEL1 | 100 | WIPF1 | 100 |
| CD79B | 100 | G6PC3 | 100 | NCF2 | 100 | RUNX1 | 99.94 | WRAP53 | 100 |
| CD81 | 100 | GATA2 | 100 | NCF4 | 100 | SAMHD1 | 100 | XIAP | 100 |
| CD8A | 100 | GFI1 | 100 | NFKB2 | 100 | SBDS | 92.88 | ZAP70 | 100 |
| CEBPE | 100 | GIMAP5 | 100 | NFKBID | 100 | SEMA3E | 100 | ZBTB24 | 100 |
Raw data were analyzed and variants assessed by the Ion Reporter Software 5.0 (https://ionreporter.thermofisher.com/ir/). To confirm the presence of the variants thus selected, the Sanger PCR protocol was set up for each variant to confirm genetic mutations detected by NGS. Functional studies were performed to assess defects in the proteins encoded by the detected variants. Apoptosis induced by FAS-ligand and TRAIL stimulation was assessed on patient Epstein-Barr virus-immortalized B cells. Apoptosis pathway function was evaluated by Western blot analysis of CASP10, CASP8, and PARP proteins using rabbit monoclonal anti-Caspase-10 antibodies (Abcam), mouse monoclonal anti-Caspase-8 (1C12), and rabbit polyclonal anti-PARP (Cell Signaling). In one case, quantitation of the protein (ADA2) encoded by the defective gene was performed on the fresh blood.
Statistical analysis
Descriptive statistics were reported in terms of absolute frequencies and percentages. Distribution of data regarding continuous variables was described in terms of a median value and range. Comparison of frequency distribution was analyzed by the Chi-square test. The Fisher exact test was used in the case of at least one expected frequency <5. Comparison regarding demographic characteristics and double-negative values was analyzed by the Mann–Whitney test as nonparametric test. All test were two-tailed and P value <0.05 was considered statistically significant.
STATA statistical software (release 7.0, StataCorp 2001, College Station, TX) was used.
RESULTS
Characteristic of the whole cohort
Forty-six patients (20 females, 43%) diagnosed with ALPS referred at the ALPS Italian network through the AIEOP centers from 2002 to 2019 were considered eligible for the present study. In the ALPS-FAS/CASP10 group, all patients were affected with definitive ALPS; in the ALPS-U group, 19 of 28 were definitive.
Thirty-seven of 46 (81%) were affected with definitive ALPS and 9 of 46 (20%) with probable ALPS according the 2009 NIH criteria.2
The median age at clinical onset was 6 years (range 0–23 years) and median age at diagnosis was 12 years (range 1–39 years). Median duration of follow-up of the whole cohort was 3.3 years (range 0–14 years). Patients were genetically tested to differentiate classical ALPS (FAS/CASP 10 mutated) from ALPS-U (those negative for FAS/CASP10 variants). Then, ALPS-FAS/CASP10 and ALPS-U subjects were compared by several clinical, biochemical, and flow cytometry markers and treatment response.
Genetic testing
Genetic testing applied to the whole cohort of 46 subjects showed that 18 patients (39%) carried the classical FAS/CASP10 gene mutations 12 with FAS and 6 with CASP10), while 28 patients (61%) were negative for these mutations and were identified as ALPS-U (undetermined genetic defects).
In the ALPS-FAS/CASP10 group, 11 variants were known as pathogenic/likely pathogenic (Table 3).
| Pt | Gene | Inheritance | Protein | Zigosity | Segregation | GnomAD | Varsom | Type Variant |
|---|---|---|---|---|---|---|---|---|
| 1 | FAS | AD | p.Glu218Lys | HET | na | – | LP | Missense |
| 2 | FAS | AD | p.Glu218Lys | HET | na | – | LP | Missense |
| 3 | FAS | AD | p.Glu194Lys/p.Ser225Thr | HET | na | 1.41E-03 | VUS/VUS | Missense/Missense |
| 4 | FAS | AD | p.Cys129Arg | HET | Mother | 3.19E-5 | LP | Missense |
| 5 | FAS | AD | p.Gly286Ter | HET | na | – | P | Nonsense |
| 6 | FAS | AD | p.Gln273His | HET | na | – | LP | Missense |
| 7 | FAS | AD | p.Gln273His | HET | na | – | LP | Missense |
| 8 | FAS | AD | p.Val190Met | HET | na | – | LP | Missense |
| 9 | FAS | AD | c.650_651 + 3delCTGTAinsAGTG | HET | na | LP | Frameshift | |
| 10 | FAS | AD | p.Gly66Cys | HET | na | P | Missense | |
| 11 | FAS | AD | p.Val190Met | HET | na | LP | Missense | |
| 12 | FAS | AD | c.650_651 + 3delCTGTAinsAGTG | HET | na | LP | Frameshift | |
| 13 | CASP10 | AD | p.Ile406Leu | HET | na | 4.53E-02 | LB | Missense |
| 14 | CASP10 | AD | p.Ile406Leu | HET | Father | 4.53E-02 | LB | Missense |
| 15 | CASP10 | AD | p.Tyr446Cys | HET | Mother | 2.28E-02 | B | Missense |
| 16 | CASP10 | AD | p.Val410Ile | HET | Mother | 4.19E-02 | B | Missense |
| 17 | CASP10 | AD | p.Pro501Leu | HET | Mother | 9.07E-04 | B | Missense |
| 18 | CASP10 | AD | p.Val410Ile | HET | na | 4.19E-02 | B | Missense |
| 19 | ARPC1B | AR | HOMO | na | 4.22E-06 | P | Missense | |
| 20 | AIRE | AR/AD | p.Arg9Trp/p.Val484Met | HET | na | – | LP/VUS | Missense/Missense |
| 21 | STAT3 | AD | p.Arg152Trp | HET | na | 9.22E-05 | LP | Missense |
| 22 | CTLA4/LRBA | AD/AR | p.Cys58Serfs*13/p.Asp2294Asn | HET | Mother/mother | – | LP/VUS | Frameshift/Missense |
| 23 | ADA2 STAT3 | AR/AD | p.Leu188Pro-p.Thr187Pro7/p.Lys658Arg | HET | Father/somatic | – | LP-LP/LP | Missense-Missense/Missense |
| 24 | TNFRSF13C | AR | p.His159Tyr/p.Pro21Arg | HET | Father | 7.97E-06 | B/B | Missense/Missense |
| 25 | NLRC4 | AD | p.Arg492Trp | HET | na | 1.97E-05 | VUS | Missense |
| 26 | LRBA | AR | p.Gln2561Ter | HET | na | 3.99E-06 | P | Nonsense |
| 27 | LRBA | AR | p.Arg655Ter | HET | Mother/father | – | P | Missense |
| 28 | IKBKG | XLR | p.Glu125Lys | HET | Mother | 5.81E-03 | LP | Nonsense |
| 29 | TNFRSF13B | AD | p.Arg202His | HET | Father | 5.85E-02 | VUS | Missense |
| 30 | GBA | AR | p.Asn370Ser/p.Gly202Arg | HET | na | P | Missense | |
| 31 | FOXP3 | AD | p.Leu260Gln | HEMI | na | LP | Missense | |
| 32 | NEG | |||||||
| 33 | NEG | |||||||
| 34 | NEG | |||||||
| 35 | NEG | |||||||
| 36 | NEG | |||||||
| 37 | NEG | |||||||
| 38 | NEG | |||||||
| 39 | NEG | |||||||
| 40 | NEG | |||||||
| 41 | NEG | |||||||
| 42 | NEG | |||||||
| 43 | NEG | |||||||
| 44 | NEG | |||||||
| 45 | NEG | |||||||
| 46 | IKBKG | XLR | p.Glu125Lys | HET | mother | 1.50E-03 | LP | Missense |
- AD = autosomal dominant; AR = autosomal recessive; B = benign; HEMI = hemizygous; HET = heterozygous; HOMO = homozygous; LB = likely benign; LP = likely pathogenic; na = not available; NEG = negative; P = pathogenic; VUS = variant of unknown significance; XLR = X-linked-related.
Patient 1 and patient 2 (Table 3) are brothers. Both have lymphoproliferation, increased DNTs and one of them pathological FAS assay, while in the other the test failed. Moreover, one of them needs therapy for immune thrombocytopenia. The Varsome data base considers their variants as likely pathogenic. Putting together the biochemical and the immunological data, we reckon that these patients can be considered as having ALPS disease.
In patient 3 (Table 3), the variant is uncertain, but it was associated to a pathological FAS test and to clinical findings very suggestive of ALPS including the presence of lymphoproliferation, increased DNTs, and autoimmune cytopenia (AIHA).
According to Varsome, the variant p.Cys129Arg is likely pathogenic. Indeed, in patient 4 (Table 3), there are other elements supporting the pathogenic role of this variant including lymphoproliferation increase of DNTs and pathological FAS test.
Patients 6 and 7 (Table 3) with p.Gln273His variant are related (father and daughter).
According to Varsome, this variant is likely pathogenic. These patients have lymphoproliferation, increase of DNTs, pathological FAS test (only patient 7) AIHA, ITP, and leucopenia (only patient 7).
The 2 CASP10 p.Tyr446Cys and CASP10 p.Val410Ile variants found in patients 15, 16, and 18, respectively, known as benign were found to be associated with impaired apoptosis driven by FAS-ligand and TRAIL stimulation22 (Table 1). The remaining 4 variants are considered variant of unknown significance (VUS) or likely benign or benign.
Within the ALPS-U group, we found in 14 of 28 (50%) patients 19 variants; of these, 4 of 19 (21%) were known as pathogenic, 8 of 19 (42%) as likely pathogenic and 4 of 19 (21%) as VUS. In 1 patient (ALPS-U Pt 23), we found 2 ADA2 gene variants, reported as VUS/likely pathogenic, which was associated to reduced expression (assessed in Western blot) of the corresponding ADA2 protein (Table 3).
We outline that on the clinical point of view, some VUS may be of relevance if associated to a clinical phenotype that is clearly consistent with the diagnosis. In the specific case of patient 24 (Table 3), we reckoned that these 2 VUS in the context of the clinical phenotype might have been somehow contributory.23
Patient 23 (Table 3) showed 3 different causative variants. One rare STAT3 variant was a somatic mosaicism, being present in the DNA extracted from peripheral blood but not in the DNA extracted from a different source. Two other rare variants affected the 2 different alleles of the ADA2 gene: indeed, the parents turned out to carry one variant each (p.Leu188Pro for the mother and p.Thr187Pro for the father).24-26
In patient 27, genetic analysis detected a pathogenic heterozygous variant of LRBA. This patient has a composite clinical phenotype including symptoms fully compatible with a diagnosis of ALPS but also others not consistent with this diagnosis (diarrhea, recurrent upper respiratory tract infections).27, 28
Patients 28 carried pathogenic variants in the X-linked IKBKG gene, showing a clinical overlap between inborn errors of immunity and bone marrow failure, in line with what was recently demonstrated.25
Clinical, biochemical, and flow cytometry markers
The phenotypes of the 46 ALPS-FAS/CASP10 and ALPS-U patients were compared by a large number of clinical and biological parameters (Table 6).
As expected, chronic (<6 months) nonmalignant lymphoproliferation expressed by lymphadenopathy and/or splenomegaly14, 29 was present in all patients in both groups. Lymphoadenopathy and splenomegaly was contemporarily present in 67% of the ALPS-FAS/CASP10 group and in 54% of the ALPS-U group (P = 0.37).
Cytopenia affecting one or more hematopoietic lineages without difference in distribution between ALPS-FAS/CASP10 and ALPS-U (81% versus 82%; P = ns) groups.
Thrombocytopenia was the most frequent cytopenia in both groups (85% versus 83%; P = ns), whereas lymphocytopenia and autoimmune neutropenia were most commonly seen in ALPS-U (12/28 and 15/23; 43% and 65%) than that in the ALPS-FAS/CASP10 group (1/16 and 4/13; 6% and 31%) (P = 0.01 and P = 0.04).
A flow cytometry panel identifying 4 specific lymphocyte subpopulations (CD3CD4-CD8-+TCRαβ+, CD3+CD25+/CD3HLADR+, TCR αβ+ B220+, and CD19+CD27+) and proposed as a diagnostic tool for screening ALPS30 was compared in ALPS-FAS/CASP10 versus ALPS-U. When taken individually, each of these lymphocyte subpopulation was not significantly different in the 2 groups. However, when taken together, the whole pathologic panel was significantly associated (P = 0.01) with ALPS-FAS/CAS10, thus suggesting that the contemporary positivity of these parameters might be a hallmark for ALPS-FAS/CASP10 and useful for differentiating from ALPS-U (Table 7).
| ALPS-FAS CASP + | ALPS-U | P | |
|---|---|---|---|
| A) CD3CD4-CD8-+TCRαβ+ (DN) (>1.5%) | 18/18 (100%) | 28/28 (100%) | |
| B) CD19+CD27+ (<15%) | 8/11 (73%) | 19/26 (73%) | 0.98 |
| C) TCR αβ+ B220+ (>60%) | 9/11 (82%) | 14/26 (50%) | 0.10 |
| D) CD3CD25+/CD3HLADR+ (<1) | 9/11 (82%) | 18/26 (69%) | 0.43 |
| Concomitant pathological values A+B+C+D | 7/11 (64%) | 7/26 (27%) | 0.01 |
- ALPS = autoimmune lymphoproliferative syndrome.
Increased vitamin B12 serum levels (>1500 mg/dL) were significantly more frequent in ALPS-FAS/CASP10 than in ALPS-U (P = 0.001) and so was IL-10 (> 20pg/mL) (P = 0.002) (Table 6).
Not surprisingly, DNTs, the primary hallmark of ALPS, were not significant different in ALPS-FAS/CAS10 population (median value of 3.9% [range 1.9%–13%]) as compared to ALPS-U (median value of 2.6% [range 1.7%–14.8%]; P = 0.11).
Somatic Fas mutations were not detected in our patient. This may reflect a lack of sensitivity of the sequencing assay because genetic analysis was not performed on sorted DNT cells.
FAS-mediated apoptosis test has been done in all subjects of ALPS-FAS/CASP10 (1 failed) and in 27 of 28 of the ALPS-U group. The comparison was not statistically significant.
Overall, markers of autoimmunity were positive in 6 of 18 (33%) subjects of the ALPS-FAS/CASP10 group and in 19 of 28 (68%) ALPS-U subjects (P = 0.02). In terms of frequency, direct antibodies test (DAT+) was the most common positive marker in the ALPS-FAS/CASP10 group (4/14; 28%), while ANAs were so in the ALPS-U group (7/28; 25%).
Signs and symptoms of autoimmune diseases (arthritis, celiac disease, primary sclerosing cholangitis, thyroiditis, autoimmune hepatitis, oral ulcers, chronic inflammatory GI diseases, type 1 diabetes, nephrotic syndrome, and eczema) were significantly more frequent in ALPS-U (21/28, 75%) than in ALPS-FAS/CASP10 patients (4/18, 22%) (P = 0.001).
Only the ALPS-U group comprised both definitive ALPS and probable ALPS and the only statistical differences between them was in FAS-mediated apoptosis (P < 0.05).
Treatment
Three of 46 (6%) patients did not require any treatment, while the remaining 43 (93%) received one or more lines or drug combinations. First-line treatment (steroid or intravenous immunoglobulins) was effective in only 13 of 38 subjects (30%), 6of 13 (46%) in ALPS-FAS/CASP10, and 7 of 22 (32%) in the ALPS-U group (P = 0.39).
The response rate to second-line (MMF or rapamycin) was 100% (12/12) in ALPS-FAS/CASP10 and 33% (7/21) in the ALPS-U group (P < 0.001). In the ALPS-U patients not responding to second-line therapy, further lines of treatment were needed. Abatacept (a recombinant fusion protein-drug that mimics the action of CTLA4), was successfully administered in 2 LRBA-mutated patients. In the remaining ALPS-U subjects, various combination treatments were adopted (Rituximab, Anakinra, Eltrombopag or Nplate, either alone or in combination with MMF or rapamycin). In the ALPS-U group, a CR was achieved in 12 of 27 (44%) and a PR in 10 of 27 (37%).
Overall, ALPS-U received a significantly higher number of combination treatments (no 17/27, 63%) over ALPS-FAS/CAS10 (no 1/16.6%) (P = 0.01) and a significantly greater number of drugs (14/27 [52%] versus 2/16 [12%] of ALPS-FAS/CASP10) to achieve a response (P = 0.009) (Table 6).
DISCUSSION
In this study, we report on the largest comparison of classical ALPS (FAS/CASP10) with ALPS-U patients, diagnosed according to the NIH 2009 criteria2 (Table 1). The analysis shows that a consistent number of ALPS-U patients display a distinct clinical and biological phenotype and also have a different genetic background when compared to ALPS-FAS/CASP10 subjects.
Indeed, the clinical phenotype of ALPS-U patients appeared to be characterized by an increased frequency of lymphocytopenia, of associated autoimmune makers/symptoms and consequently of multiple organ involvement and by a greater difficulty in achieving response as expressed by the need for more lineages of treatments and drug combinations with a far inferior response to MMF and rapamycin (the second-line treatment) versus ALPS-FAS/CASP10 subjects. Incidentally, although the numerosity of the sample size is relatively limited, it has to be noted the higher response rate achieved in classical ALPS patients with second-line treatment MMF or rapamycin compared to first-line therapies steroids or immunoglobulins.
This highlights the option for MMF or rapamycin to shift to upfront therapies in documented classical ALPS.31
Also biological phenotype was different from ALPS-FAS/CASP10 since ALPS-U patients displayed a significantly lower association with elevated Vitamin B 12 and IL-10 serum levels.
Another important difference is related to the flow cytometry panel proposed in past years as a screening for ALPS diagnosis.30 Indeed, in our cohort, this panel was significantly associated to ALPS-FAS/CASP10 when all the 4 parameters were contemporarily positive. Although this may somehow orient clinicians toward ALPS-FAS/CAPS10 versus ALPS-U, it has to be outlined that the final diagnosis can only rely on genetic analysis.
The percentage of 64% of sharing the quadruple positivity of immunophenotype elects in the ALPS-FAS/CAPS10 group, probably delineates a peculiar characteristic partially represents in ALPS-U patients surely including a portion of overlap.
In classical studies,2, 14, 17 ALPS patients were divided into 2 groups based on the genetic background: those in whom a pathogenic mutations in FAS, FASL, and CASP10 genes were found, and those, named ALPS-U and accounting to up to one-third of the total, in whom no pathogenic genetic lesion was detectable.12, 13 In our study, we found that ALPS-U subjects represented the majority (61%) of the whole cohort. The reason for this difference is not obvious and might possibly be related to selection bias inherent in registry studies. However, thanks to NGS resources, genetic variants were found in about half (14/28) of ALPS-U patients (Table 3). The pathogenic role of the detected mutations was known only for a minority of them (4/14). In one additional patient (ALPS-U patient 23), we found an ADA2 gene variant (p.Leu188Pro; p.Thr187Pro) reported as likely pathogenic. Indeed, we proved that this variant caused the reduced expression of the corresponding protein ADA2, thus supporting the idea of its pathogenic role for this variant. In all ALPS CASP10 subjects carrying benign/likely benign variants of CASP10, an altered apoptosis induced by FAS-ligand and TRAIL stimulation was shown. In addition to this, we also proved a reduced cleavage of CASP8 and PARP likely reflecting a reduced upstreamed CASP10 activity; thus, indirectly supporting a potential pathogenic role for this variants.22 The variants of CASP10 have always been reported in the literature with conflicting interpretation; nevertheless, some of them have been recently reported to impaired apoptosis and suggest that could play a role in predisposition of immune dysregulation.22
The remaining patients either carried variants considered as probably pathogenic or as VUS.
VUS have a probable effect on the phenotype. Although taking VUS variants into consideration for diagnostic purpose may be questionable, clinicians, who found VUS in patients showing symptoms and laboratory alterations similar to ALPS-FAS/CASP10 patients, may have an impact.32
Still with the bias of having adopted a NGS panel largely composed of immune genes virtually, all of detected variants are related to immunodeficiencies/immune dysregulation diseases. Interestingly, the same was shown in a recent study on a large cohort of pediatric Evans syndrome33 patients where an association was found with high frequency of potentially damaging variants in immune genes.
The availability of the new genetic techniques such as NGS and Whole Genome Sequencing will probably modify the “classical genotype/phenotype associations” widening the clinical variability referred to a single-gene variant as recently described for LRBA- and CTLA4-deficient patients.33, 34 Nevertheless, still this with standing the need remains for functional validation analyses aimed to prove the real pathogenic role of likely pathogenic variants or VUS.
Overall, our study suggests that the ALPS-FAS/CASP10 represents a well-defined disease with specific genetic background, rather definite clinical signs and immunological markers and a rather predictable response to treatment that appears to be optimal to MMF or rapamycin.
On the contrary, ALPS-U differs from ALPS-FAS/CASP10 because it has a clearer nonhematological autoimmune signature, it is more associated to lymphopenia, to lower response to treatment in general and to drugs classically effective in ALPS-FAS/CASP10 such a MMF and rapamycin, and appears to be a more heterogeneous group often generated by pathogenic variants of immunodeficiency or immune dysregulation genes.
Although some genetic diagnosis that we detected correspond to well-defined disorders different form ALPS (eg, CTLA4 and LRBA) we think that this study does not diminish the role of genetic analysis, but rather outlines the need to revise ALPS diagnostic criteria. On the contrary, this study envisages the importance to apply enlarged genetic panels to ALPS-U patients including several immunodeficiencies and immune dysregulation genes and to recur to WES analysis when a pathogenic gene is not found and to always look at genetic finings in the context of the clinical phenotype.
ACKNOWLEDGMENTS
The authors would like to thanks ERG S.p.A., Rimorchiatori Riuniti (Genoa), Cambiaso Risso Marine (Genoa), Saar Depositi Oleari Portuali (Genoa) and ONLUS Nicola Ferrari are acknowledged for supporting the activity of the Hematology Unit of IRCCS Istituto Giannina Gaslini, Genoa for supporting the activity of the Hematology Unit of IRCCS Istituto Giannina Gaslini, Genoa.
AUTHOR CONTRIBUTIONS
All authors contributed to the study conception and design. EP designed the research, contributed essential data, cared for the patients and wrote the manuscript. AG, IC, ML performed the genetic analysis. ML performed the IL-18, IL-10, and FAS-ligand tests. PT performed the study of lymphocyte immune phenotype. FT performed functional tests. NC performed the FAS-mediated aptosis test. EM, MC, FP, CM, RM, DG, EF, AM, EM, PC, GR, MP, PF, SC, AB, and UR contributed essential data, cared for the patients. JB discussed the results and critically reviewed the manuscript. MM, CD, and FF designed the study protocol, contributed essential data, cared for the patients, revised the manuscript.
DISCLOSURES
The authors have no conflicts of interest to disclose.
