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Hematopoietic stem cell transplantation in patients with gain-of-function signal transducer and activator of transcription 1 mutations

      Background

      Gain-of-function (GOF) mutations in signal transducer and activator of transcription 1 (STAT1) cause susceptibility to a range of infections, autoimmunity, immune dysregulation, and combined immunodeficiency. Disease manifestations can be mild or severe and life-threatening. Hematopoietic stem cell transplantation (HSCT) has been used in some patients with more severe symptoms to treat and cure the disorder. However, the outcome of HSCT for this disorder is not well established.

      Objective

      We sought to aggregate the worldwide experience of HSCT in patients with GOF-STAT1 mutations and to assess outcomes, including donor engraftment, overall survival, graft-versus-host disease, and transplant-related complications.

      Methods

      Data were collected from an international cohort of 15 patients with GOF-STAT1 mutations who had undergone HSCT using a variety of conditioning regimens and donor sources. Retrospective data collection allowed the outcome of transplantation to be assessed. In vitro functional testing was performed to confirm that each of the identified STAT1 variants was in fact a GOF mutation.

      Results

      Primary donor engraftment in this cohort of 15 patients with GOF-STAT1 mutations was 74%, and overall survival was only 40%. Secondary graft failure was common (50%), and posttransplantation event-free survival was poor (10% by 100 days). A subset of patients had hemophagocytic lymphohistiocytosis before transplant, contributing to their poor outcomes.

      Conclusion

      Our data indicate that HSCT for patients with GOF-STAT1 mutations is curative but has significant risk of secondary graft failure and death.

      Key words

      Abbreviations used:

      CID (Combined immunodeficiency), CMC (Chronic mucocutaneous candidiasis), EFS (Event-free survival), GAS (γ-Activated sequence), GOF (Gain of function), HLH (Hemophagocytic lymphohistiocytosis), HSCT (Hematopoietic stem cell transplantation), IPEX (Immune dysregulation–polyendocrinopathy–enteropathy–X-linked), JAK (Janus kinase), KREC (Kappa-deleting recombination excision circle), MMUD (Mismatched unrelated donor), MRD (Matched related donor), MUD (Matched unrelated donor), NK (Natural killer), OS (Overall survival), PBSC (Peripheral blood stem cell), RIC (Reduced-intensity conditioning), STAT (Signal transducer and activator of transcription), TREC (T-cell receptor excision circle), UCB (Unrelated umbilical cord blood), WT (Wild-type)
      Autosomal dominant gain-of-function (GOF) mutations in signal transducer and activator of transcription 1 (STAT1) cause susceptibility to a range of infections in human subjects, including severe viral and bacterial infections, nontuberculous mycobacterial disease, dimorphic fungal infections, and chronic mucocutaneous candidiasis (CMC), and are associated with development of autoimmunity.
      • Boisson-Dupuis S.
      • Kong X.F.
      • Okada S.
      • Cypowyj S.
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      Inborn errors of human STAT1: allelic heterogeneity governs the diversity of immunological and infectious phenotypes.
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      • Fuchs S.
      • Raabe J.
      • Frede N.
      • Glocker C.
      • Doffinger R.
      • et al.
      The extended clinical phenotype of 26 patients with chronic mucocutaneous candidiasis due to gain-of-function mutations in STAT1.
      • Liu L.
      • Okada S.
      • Kong X.F.
      • Kreins A.Y.
      • Cypowyj S.
      • Abhyankar A.
      • et al.
      Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.
      • Mizoguchi Y.
      • Tsumura M.
      • Okada S.
      • Hirata O.
      • Minegishi S.
      • Imai K.
      • et al.
      Simple diagnosis of STAT1 gain-of-function alleles in patients with chronic mucocutaneous candidiasis.
      • Romberg N.
      • Morbach H.
      • Lawrence M.G.
      • Kim S.
      • Kang I.
      • Holland S.M.
      • et al.
      Gain-of-function STAT1 mutations are associated with PD-L1 overexpression and a defect in B-cell survival.
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      • Hsu A.P.
      • Pechacek J.
      • Bax H.I.
      • Dias D.L.
      • Paulson M.L.
      • et al.
      Signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations and disseminated coccidioidomycosis and histoplasmosis.
      • Soltesz B.
      • Toth B.
      • Shabashova N.
      • Bondarenko A.
      • Okada S.
      • Cypowyj S.
      • et al.
      New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe.
      • Toubiana J.
      • Okada S.
      • Hiller J.
      • Oleastro M.
      • Lagos Gomez M.
      • Aldave Becerra J.C.
      • et al.
      Heterozygous STAT1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype.
      • van de Veerdonk F.L.
      • Plantinga T.S.
      • Hoischen A.
      • Smeekens S.P.
      • Joosten L.A.
      • Gilissen C.
      • et al.
      STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.
      Some patients with GOF-STAT1 mutations present with a syndrome that clinically resembles immune dysregulation–polyendocrinopathy–enteropathy–X-linked (IPEX) syndrome
      • Uzel G.
      • Sampaio E.P.
      • Lawrence M.G.
      • Hsu A.P.
      • Hackett M.
      • Dorsey M.J.
      • et al.
      Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.
      or with combined immunodeficiency (CID), with variable effects on immunoglobulin levels, antibody production, and lymphocyte development.
      • Sharfe N.
      • Nahum A.
      • Newell A.
      • Dadi H.
      • Ngan B.
      • Pereira S.L.
      • et al.
      Fatal combined immunodeficiency associated with heterozygous mutation in STAT1.
      However, the clinical phenotype can vary from mild to severe.
      • Boisson-Dupuis S.
      • Kong X.F.
      • Okada S.
      • Cypowyj S.
      • Puel A.
      • Abel L.
      • et al.
      Inborn errors of human STAT1: allelic heterogeneity governs the diversity of immunological and infectious phenotypes.
      • Depner M.
      • Fuchs S.
      • Raabe J.
      • Frede N.
      • Glocker C.
      • Doffinger R.
      • et al.
      The extended clinical phenotype of 26 patients with chronic mucocutaneous candidiasis due to gain-of-function mutations in STAT1.
      • Mizoguchi Y.
      • Tsumura M.
      • Okada S.
      • Hirata O.
      • Minegishi S.
      • Imai K.
      • et al.
      Simple diagnosis of STAT1 gain-of-function alleles in patients with chronic mucocutaneous candidiasis.
      • Soltesz B.
      • Toth B.
      • Shabashova N.
      • Bondarenko A.
      • Okada S.
      • Cypowyj S.
      • et al.
      New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe.
      • Toubiana J.
      • Okada S.
      • Hiller J.
      • Oleastro M.
      • Lagos Gomez M.
      • Aldave Becerra J.C.
      • et al.
      Heterozygous STAT1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype.
      • van de Veerdonk F.L.
      • Plantinga T.S.
      • Hoischen A.
      • Smeekens S.P.
      • Joosten L.A.
      • Gilissen C.
      • et al.
      STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.
      Other than treatment of the infectious manifestations with long-term antifungal, antibacterial, and antiviral therapies, the approach to management of patients with GOF-STAT1 mutations remains challenging and controversial. Immunosuppression to treat the autoimmune phenomena can cause exacerbation of infections. Ruxolitinib, a Janus kinase (JAK) family tyrosine kinase inhibitor targeting the JAK-STAT1 pathway, has been used to successfully treat CMC and alopecia areata
      • Higgins E.
      • Al Shehri T.
      • McAleer M.A.
      • Conlon N.
      • Feighery C.
      • Lilic D.
      • et al.
      Use of ruxolitinib to successfully treat chronic mucocutaneous candidiasis caused by gain-of-function signal transducer and activator of transcription 1 (STAT1) mutation.
      and autoimmune cytopenias and CMC
      • Weinacht K.G.
      • Charbonnier L.M.
      • Alroqi F.
      • Plant A.
      • Qiao Q.
      • Wu H.
      • et al.
      Ruxolitinib reverses dysregulated T helper cell responses and controls autoimmunity caused by a novel signal transducer and activator of transcription 1 (STAT1) gain-of-function mutation.
      in 2 patients with GOF-STAT1 mutations. Its mechanism is thought to include reduction of exaggerated cytokine-driven STAT1 phosphorylation,
      • Baris S.
      • Alroqi F.
      • Kiykim A.
      • Karakoc-Aydiner E.
      • Ogulur I.
      • Ozen A.
      • et al.
      Severe early-onset combined immunodeficiency due to heterozygous gain-of-function mutations in STAT1.
      reduction of follicular helper T-cell responses,
      • Weinacht K.G.
      • Charbonnier L.M.
      • Alroqi F.
      • Plant A.
      • Qiao Q.
      • Wu H.
      • et al.
      Ruxolitinib reverses dysregulated T helper cell responses and controls autoimmunity caused by a novel signal transducer and activator of transcription 1 (STAT1) gain-of-function mutation.
      improved TH17 cell differentiation,
      • Weinacht K.G.
      • Charbonnier L.M.
      • Alroqi F.
      • Plant A.
      • Qiao Q.
      • Wu H.
      • et al.
      Ruxolitinib reverses dysregulated T helper cell responses and controls autoimmunity caused by a novel signal transducer and activator of transcription 1 (STAT1) gain-of-function mutation.
      and induced IL-17 production in vitro.
      • Mossner R.
      • Diering N.
      • Bader O.
      • Forkel S.
      • Overbeck T.
      • Gross U.
      • et al.
      Ruxolitinib induces interleukin 17 and ameliorates chronic mucocutaneous candidiasis caused by STAT1 gain-of-function mutation.
      Granulocyte colony-stimulating factor has also been used to successfully treat CMC in 1 patient with a GOF-STAT1 mutation.
      • Wildbaum G.
      • Shahar E.
      • Katz R.
      • Karin N.
      • Etzioni A.
      • Pollack S.
      Continuous G-CSF therapy for isolated chronic mucocutaneous candidiasis: complete clinical remission with restoration of IL-17 secretion.
      In patients with severe recalcitrant disease, hematopoietic stem cell transplantation (HSCT) has been attempted with mixed results. Successful transplants were reported in 1 patient with CMC and a confirmed DNA-binding domain GOF-STAT1 mutation
      • Grunebaum E.
      • Kim V.H.
      • Somers G.R.
      • Shammas A.
      • Roifman C.M.
      Bone marrow transplantation for monoallelic signal transducer and activator of transcription 1 deficiency.
      and in 2 patients with severe CMC who were not evaluated for STAT1 mutations,
      • Deeg H.J.
      • Lum L.G.
      • Sanders J.
      • Levy G.J.
      • Sullivan K.M.
      • Beatty P.
      • et al.
      Severe aplastic anemia associated with chronic mucocutaneous candidiasis. Immunologic and hematologic reconstitution after allogeneic bone marrow transplantation.
      • Hoh M.C.
      • Lin H.P.
      • Chan L.L.
      • Lam S.K.
      Successful allogeneic bone marrow transplantation in severe chronic mucocutaneous candidiasis syndrome.
      whereas transplantations in 3 patients with confirmed GOF-STAT1 mutations were unsuccessful.
      • Aldave J.C.
      • Cachay E.
      • Nunez L.
      • Chunga A.
      • Murillo S.
      • Cypowyj S.
      • et al.
      A 1-year-old girl with a gain-of-function STAT1 mutation treated with hematopoietic stem cell transplantation.
      • Faitelson Y.B.A.
      • Shroff M.
      • Grunebaum E.
      • Roifman C.R.
      • Naqvi A.
      A mutation in the STAT1 DNA-binding domain associated with hemophagocytic lymphohistiocytosis.
      • Kilic S.S.
      • Puel A.
      • Casanova J.L.
      Orf infection in a patient with Stat1 gain-of-function.
      Because of the need for viable treatment options for patients with GOF-STAT1 mutations who have severe disease, we set out to obtain a more complete assessment of outcomes of HSCT and the challenges and complications encountered by using this approach.

      Methods

       Patients

      Patients undergoing transplantation were identified from national and international sites by request to specific transplant centers and by announcements made through the European Society for Blood and Marrow Transplantation Inborn Errors Working Party and the Primary Immune Deficiency Treatment Consortium. To be included, patients were required to have a confirmed heterozygous mutation in STAT1 that confers GOF activity. Deidentified data for each case were collected by using a questionnaire/spreadsheet filled out by investigators at each institution contributing a patient. All studies involving human subjects were performed in accordance with site-specific Institutional Review Board–approved protocols, as well as the guidelines in the 1964 Declaration of Helsinki and its later amendments.
      Patients were categorized based on clinical and laboratory phenotype: IPEX-like, IPEX-like and CID, CID, and severe infections. Patients with an IPEX-like phenotype had evidence of polyendocrinopathy, enteropathy, and autoimmunity; patients with CID had low T-cell and/or B-cell quantities, abnormal lymphocyte proliferation to mitogens or antigens, or both. Patients categorized as those with only severe infections had no evidence of T- or B-cell defects, or immunologic assays had not been performed.

       DNA sequencing

      DNA was extracted, and full-length sequencing of STAT1 in genomic DNA was performed, as previously described.
      • Uzel G.
      • Sampaio E.P.
      • Lawrence M.G.
      • Hsu A.P.
      • Hackett M.
      • Dorsey M.J.
      • et al.
      Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.
      In some patients GOF-STAT1 mutations were identified by means of whole-exome sequencing and confirmed by using Sanger sequencing.
      • Liu L.
      • Okada S.
      • Kong X.F.
      • Kreins A.Y.
      • Cypowyj S.
      • Abhyankar A.
      • et al.
      Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.
      In one patient GOF-STAT1 mutation was identified by means of whole-genome sequencing.
      • Hu H.
      • Roach J.C.
      • Coon H.
      • Guthery S.L.
      • Voelkerding K.V.
      • Margraf R.L.
      • et al.
      A unified test of linkage analysis and rare-variant association for analysis of pedigree sequence data.

       Expression and phosphorylation of GOF-STAT1 mutants determined by means of immunoblotting

      Various STAT1 mutants were generated by using site-directed mutagenesis with a pcDNA3-V5–based wild-type (WT) STAT1 expression vector.
      • Kong X.F.
      • Ciancanelli M.
      • Al-Hajjar S.
      • Alsina L.
      • Zumwalt T.
      • Bustamante J.
      • et al.
      A novel form of human STAT1 deficiency impairing early but not late responses to interferons.
      Immunoblot analysis was performed, as previously described.
      • Mizoguchi Y.
      • Tsumura M.
      • Okada S.
      • Hirata O.
      • Minegishi S.
      • Imai K.
      • et al.
      Simple diagnosis of STAT1 gain-of-function alleles in patients with chronic mucocutaneous candidiasis.
      Briefly, WT or mutant STAT1-containing plasmids were transfected into a U3C STAT1-null fibrosarcoma cell line by using Lipofectamine LTX (Thermo Fisher Scientific, Waltham, Mass), according to the manufacturer's protocol. Twenty-four hours later, cells were stimulated with 1000 U/mL IFN-γ (R&D Systems, Minneapolis, Minn) for 20 minutes. Cells were then lysed and subjected to immunoblot analysis. Antibodies to detect Tyr701 phosphorylated STAT1 (D4A7; Cell Signaling, Danvers, Mass), STAT1 (BD Biosciences, San Jose, Calif), and β-actin (Sigma-Aldrich, St Louis, Mo) were used. Experiments were performed in triplicate to confirm reproducibility.

       Luciferase reporter assay to evaluate transcriptional activation by GOF-STAT1 mutants

      WT and the STAT1 mutants generated by means of site-directed mutagenesis (see above) were evaluated by using a Luciferase assay that measures luciferase activity of a reporter gene under the control of the γ-activated sequence (GAS) promoter, as previously described.
      • Liu L.
      • Okada S.
      • Kong X.F.
      • Kreins A.Y.
      • Cypowyj S.
      • Abhyankar A.
      • et al.
      Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.
      Reporter plasmids (Cignal GAS Reporter Assay Kit; SABiosciences, Frederick, Md) and WT or mutant STAT1-containing plasmids were transferred into U3C cells by means of lipofection. Twenty-four hours after transfection, the cells were stimulated with 10, 102, or 103 U/mL IFN-γ for 16 hours. The Dual-Glo Luciferase Assay System (Promega, Madison, Wis) was used to analyze firefly and Renilla luciferase activities. Experiments were performed in triplicate, and the data were expressed in relative luciferase units.

       Cytokine-induced STAT1 phosphorylation

      Intracellular staining for phosphorylated STAT1 (pSTAT1) was performed on thawed PBMCs (from patients 1 and 2) allowed to recover for 2 hours in complete medium. Cells were then stained with anti-human CD4 antibody (fluorescein isothiocyanate–conjugated mouse clone M-T441; Ancell Immunology Research Products, Bayport, Minn) in sodium azide–free buffer (0.1% BSA in 1× PBS), washed twice in complete medium, and seeded at 100,000 cells per well in 96-well plates at a volume of 100 μL. After 30 minutes of incubation at 37°C, cells were stimulated with either IL-27 (200 ng/mL) or IL-6 (50 ng/mL) for 7.5, 15, or 30 minutes and fixed for 10 minutes at 37°C with prewarmed paraformaldehyde (at a final concentration of 2%). Cells were then washed twice and permeabilized with prechilled (20°C) BD Phosflow Perm Buffer III (BD Biosciences). After 30 minutes on ice, cells were washed twice with staining buffer (2% FBS in DPBS) and stained for 40 minutes on ice with anti-human phosphorylated STAT1 antibody (Alexa Fluor 647–conjugated mouse clone 4a, BD Biosciences) directed against the phosphorylated tyrosine at position 701 (Y701). Data were collected with a BD LSR II (BD Biosciences). FlowJo software (version 7.2.5/version 10.0.7 (TreeStar, Ashland, Ore) was used for data analysis.

       Statistical analysis

      Kaplan-Meier survival curves and significance of differences in overall survival (OS) and event-free survival (EFS) were generated and evaluated by using the Online Application for Survival Analysis (OASIS).
      • Yang J.S.
      • Nam H.J.
      • Seo M.
      • Han S.K.
      • Choi Y.
      • Nam H.G.
      • et al.
      OASIS: online application for the survival analysis of lifespan assays performed in aging research.
      EFS was determined by analysis of time to first transplant-related complication.

      Results

       Patients

      The clinical characteristics of the 15 patients with GOF-STAT1 mutations included in this retrospective study are presented in Table I. They were submitted by 12 participating centers from Canada, the United Kingdom, The Netherlands, Japan, Peru, Russia, Spain, Turkey, and the United States and met the criteria for study inclusion. The clinical course of 4 patients (P7, P11, P12, and P15) has been published previously,
      • Sharfe N.
      • Nahum A.
      • Newell A.
      • Dadi H.
      • Ngan B.
      • Pereira S.L.
      • et al.
      Fatal combined immunodeficiency associated with heterozygous mutation in STAT1.
      • Grunebaum E.
      • Kim V.H.
      • Somers G.R.
      • Shammas A.
      • Roifman C.M.
      Bone marrow transplantation for monoallelic signal transducer and activator of transcription 1 deficiency.
      • Aldave J.C.
      • Cachay E.
      • Nunez L.
      • Chunga A.
      • Murillo S.
      • Cypowyj S.
      • et al.
      A 1-year-old girl with a gain-of-function STAT1 mutation treated with hematopoietic stem cell transplantation.
      • Faitelson Y.B.A.
      • Shroff M.
      • Grunebaum E.
      • Roifman C.R.
      • Naqvi A.
      A mutation in the STAT1 DNA-binding domain associated with hemophagocytic lymphohistiocytosis.
      • Kilic S.S.
      • Puel A.
      • Casanova J.L.
      Orf infection in a patient with Stat1 gain-of-function.
      but those reports lack comprehensive data about HSCT, which have been included in this report. The cohort consists of 9 male and 6 female subjects who ranged in age from 13 months to 33 years at the time of transplantation (Table I). Of this cohort, 6 are alive and 9 died of complications related to HSCT (Table II and see Table E1 in this article's Online Repository at www.jacionline.org).
      Table IClinical phenotype of GOF-STAT1 mutations
      No. and country of originSexcDNA mutation protein change domainMutation analysis done before/after transplantationPhenotypePretransplantation complications
      InfectionsAutoimmunityGrowthOther
      1. United StatesMalec. 983 A>G

      p.H328R

      CC
      Pre-HSCT

      Sanger sequencing
      IPEX-likeNorovirus enteritis

      VRE and Pseudomonas abscess

      Clostridium difficile enteritis

      Mycobacterium fortuitum mediastinal lymphadenitis
      Enteropathy

      Type 1 diabetes

      Thyroiditis
      FTT
      2. RussiaMalec. 1154C>T

      p. T385M

      DB
      Pre-HSCT

      Sanger sequencing
      IPEX-likeCMC

      Pulmonary aspergillosis

      Recurrent pneumonia

      BCG lymphadenitis
      Enteropathy

      Autoimmune neutropenia

      Thyroiditis
      FTTEczema
      3. United StatesMalec. 1154C>T

      p. T385M

      DB
      Pre-HSCT

      Whole-genome sequencing
      IPEX-likeCMCEnteropathy

      Thyroiditis

      Growth hormone deficiency + anti-GAD antibody – hyperglycemia with concurrent steroid use
      FTT
      4. SpainMalec. 1154C>T

      p. T385M

      DB
      Pre-HSCT

      Sanger sequencing
      IPEX-like

      CID
      CMC

      Cryptococcal meningitis

      Pneumonia

      Pulmonary tuberculosis
      AIHA

      Type 1 diabetes

      IBD

      Glomerulonephritis

      Autoimmune hepatitis
      Normal
      5. CanadaFemalec. 1154C>T

      p. T385M

      DB
      Post-HSCT

      Sanger sequencing
      IPEX-like

      CID
      VZV

      Systemic CMV

      Pneumonia
      Type 1 diabetes

      IBD

      Thyroiditis
      FTT
      6. The NetherlandsMalec. 494A>G

      p.D165G

      CC
      Pre-HSCT

      Sanger sequencing
      CIDPulmonary aspergillosis

      MCV
      Autoimmune hepatitis

      Thyroiditis
      FTT
      7. TurkeyMalec.820G>A

      p.R274W

      CC
      Pre-HSCT

      Sanger sequencing
      CIDCMC

      Orf cutaneous infection

      Mycotic cerebral aneurysms

      Pulmonary CMV

      Pneumonia (Haemophilus influenzae, Pseudomonas aeruginosa, Streptococcus pneumoniae)
      AIHA

      Thyroiditis

      Autoimmune hepatitis

      Pernicious anemia

      Anti-phospholipid syndrome
      Normal
      8. JapanFemalec. 821 G>A

      p.R274Q

      CC
      Pre-HSCT

      Sanger sequencing
      CIDCMC

      Pulmonary aspergillosis

      VZV

      Pneumonia (H influenzae, S pneumoniae)
      NoneFTTSevere gastroesophageal reflux
      9. JapanMalec.876C>A

      p.D292E

      CC
      Pre-HSCT

      Sanger sequencing
      CID

      HLH
      CMC

      Parvovirus B19

      Recurrent sinusitis (H influenza, S pneumoniae)
      Vitiligo

      Pure red cell aplasia
      Pure red cell aplasia likely secondary to parvovirus.
      FTTGray hair depigmentation
      10. JapanFemalec.881T>C

      p.I294T

      CC
      Post-HSCT

      Whole-exome sequencing
      CIDCMC

      MCV

      VZV

      CMV

      Norovirus

      BK virus

      JC viremia

      Chronic sinusitis, otitis media, tonsillitis, bronchitis, pneumonia

      Bronchiectasis

      Cutaneous abscess
      AIHA

      Autoimmune neutropenia
      No anti-neutrophil antibody detected.


      Autoimmune thrombocytopenia

      Colitis
      NormalMalrotated kidney and pancreas divisum laryngeal edema chronic liver dysfunction
      11. CanadaMalec.1154C>T

      p.T385M

      DB
      Pre-HSCT

      Whole-exome sequencing
      CIDCMC

      Pneumonia

      Otitis media
      NoneShort stature
      12. CanadaFemalec.1189 A>G

      p.N397D

      DB
      Post-HSCT

      Sanger sequencing
      CID

      HLH
      CMC

      EBV with microabscessed spleen and kidneys

      Cutaneous HSV

      Pneumonia
      NoneNormal
      13. United KingdomFemalec.1398 C>G

      p.S466R

      DB
      Pre-HSCT

      Sanger sequencing
      CIDCMC

      Bronchiectasis

      EBV and Adenovirus pneumonia and colitis

      VZV

      HHV-6

      CMV

      Pyelonephritis

      Blepharitis

      Paronychia
      NoneFTT
      14. JapanMalec.1169 T>C

      p.M390T

      DB
      Post-HSCT

      Sanger sequencing
      Severe infectionsCMC

      HSV

      VZV

      Chronic EBV

      Recurrent otitis media (Staphylococcus aureus)

      Recurrent pneumonia (S aureus, S pneumoniae)

      Sepsis (S aureus)

      Urinary tract infection
      HypothyroidismFTT
      15. PeruFemalec.1189 A>G

      p.N397D

      DB
      Pre-HSCT

      Sanger sequencing
      Severe infectionsCMC

      CMV

      Severe diarrhea (Escherichia coli, Klebsiella pneumoniae)

      Sepsis
      NoneFTT
      AIHA, Autoimmune hemolytic anemia; CC, coiled-coil; CMV, cytomegalovirus; DB, DNA binding; FTT, failure to thrive; GAD, glutamic acid decarboxylase; HHV-6, human herpes virus 6; HSV, herpes simplex virus; IBD, inflammatory bowel disease; MCV, molluscum contagiosum virus; VRE, vancomycin-resistant Enterococcus; VZV, varicella zoster virus.
      Pure red cell aplasia likely secondary to parvovirus.
      No anti-neutrophil antibody detected.
      Table IITransplantation course and outcome of patients with GOF-STAT1 mutations
      Patient no.PhenotypeAge at transplantationDonor HLA matchCD34 doseConditioningGvHD prophylaxisEngraftmentTransplant-related complicationsGvHDOutcome of transplantation, current conditionSurvival
      1IPEX-like4 yMUD, 10/10 match8 × 106/kgFludarabine, 150 mg/m2, days −8 to −4

      Melphalan, 140 mg/m2, day −3

      Almetuzumab, 10, 15, or 20, days −21 to −19
      Cyclosporine

      MMF
      D+12EBV reactivation

      catheter-associated venous thrombus

      Supraventricular tachycardia
      NoneSecondary graft loss within 30 d

      Mixed chimera with 2% donor myeloid cells and 39% donor lymphoid cells

      Recurrence of Clostridium difficile colitis

      Recurrent sinusitis and pneumonia (bacterial and viral)

      Abnormal pulmonary diffusion capacity

      Progressive lymphopenia

      Hypogammaglobulinemia

      On IVIG
      Alive
      2IPEX-like
      • (1)
        6 y
      • (2)
        6 y
      • (1)
        T cell α/β/CD19+ depleted matched unrelated PBSCs, 10/10 match
      • (2)
        T cell α/β/CD19+ depleted haploidentical PBSCs
      • (1)
        11.21 × 106/kg
      • (2)
        14.56 × 106/kg
      • (1)
        Treosulfan, 42 mg/m2, days −5 to −3
      • Fludarabine, 150 mg/m2, days −6 to −2
      • ATG, 5 mg/kg, days −5 to −4
      • Rituximab, 375 mg/m2, day −1
      • (2)
        TBI, 6 gray
      • Fludarabine, 30 mg/m2, days −6 to −2
      • ATG, 2.5 mg/kg, days −5 and −4
      • Cyclophosphamide, 60 mg/kg, days −3 and −2
      • Melphalan, 140 mg/m2, day −1
      • Rituximab, 375 mg/m2, day −1
      • (1)
        Tacrolimus
      • (2)
        Tacrolimus
      • (1) Day +26 (lymphocytes)
      • (2) Day +35 (lymphocytes)
      • (1) Streptococcus parasanguis bacteremia
      • CMV viremia
      • (2) Drug-associated nephropathy
      • (1) None
      • (2) Grade I skin
      • (1) Secondary graft loss in first 30 d
      • (2) Full immune reconstitution
      • Alive
      • 100% donor
      • Enteropathy resolved
      • Improved growth
      Alive
      3IPEX-like12 yMUD, 10/10 match4.58 × 106/kgFludarabine, 150 mg/m2, days −8 to −4

      Melphalan, 140 mg/m2, day −3

      Almetuzumab, 10, 15, 20 mg; days −21 to −19
      Tacrolimus

      MMF

      Methotrexate
      D+16NoneGrade III skinFull immune reconstitution

      Alive

      100% donor

      Enteropathy resolved

      Improved growth
      Alive
      4IPEX-like

      CID
      29 yMUD PBSCs, 10/10 match3.33 × 106/kgFludarabine, 50 mg/d, days −6 to −3

      Melphalan, 140 mg/m2/d, day −3 to −2

      Alemtuzumab, 12 mg/d, days −10 to −6
      Sirolimus

      Tacrolimus
      D+23Severe thrombocytopenia

      Reaction to almetuzumab

      Pneumonia
      Grade I skinSecondary graft loss over 2 mo

      Continued lymphopenia

      Continued hypogammaglobulinemia

      Continued infections
      Dead

      2 y after pneumonia and sepsis
      5IPEX-like

      CID
      7 yMUD, 6/6 match6.5 × 106/kgCyclophosphamide, 50 mg/kg/d, days −5 to −2

      Busulfan, 4 mg/kg/d, days −9 to −6
      Prednisone

      Cyclosporine
      No engraftmentCMVGrade I skinDied D+12Died

      Multiorgan failure
      6CID
      • (1)
        17 y
      • (2)
        17 y
      • (1)
        UCB, 5/6 matched
      • (2)
        UCB, 6/6 matched
      • (1)
        0.14 × 106/kg
      • (2)
        0.14 × 106/kg
      • (1)
        Busulfan (myeloablative TDM AUC 90 mg*h*I)
      • Fludarabine, 160 mg/m2
      • ATG, 10 mg/kg
      • (2)
        Treosulfan, 42 g/m2
      • Fludarabine, 160 mg/m2
      • TBI, 2 × 2 Gy
        (1)
      • Cyclosporine
      • Prednisone
      • (2)
        Cyclosporine
      • MMF
      • (1) Primary graft failure
      • (2) D+15
      • (1) Fungal Pneumonia
      • (2) BK virus reactivation
      • Adenovirus reactivation
      • Cryptosporidium gut
      • Pneumonia
      • (2) Grade III gut
      • (1) Primary graft failure
      • (2) Full immune reconstitution
      Died

      4 mo after pneumonia
      7CID33 yMRD, 6/6 matched3.84 × 106/kgNoneNoneD+25Severe thrombocytopeniaNonePrimary engraftment but not immune reconstitutedDied 3 mo after bleeding from mycotic aneurysms
      8CID18 yMRD, 8/8 matched8.14 × 106/kgBusulfan, 4 mg/kg/d, days −9 to −6

      Cyclophosphamide, 50 mg/kg/d, days −5 to −2
      Tacrolimus

      Methotrexate
      No engraftmentCardiomyopathy and heart failure secondary to cyclophosphamideNoneDied

      D+3 from heart failure
      9CID

      HLH
      • (1)
        8 y
      • (2)
        8 y
      • (3)
        8 y
      • (1)
        MMUD, 6/8 matched
      • (2)
        UCB, 7/8 matched
      • (3)
        Haploidentical marrow, 5/8 matched
      • (1)
        2.3 × 106/kg
      • (2)
        0.77 × 105/kg
      • (3)
        2.09 × 106/kg
      • (1)
        Fludarabine, 180 mg/m2, days −9 to −6
      • Busulfan, 16 mg/kg, days −5 to −2
      • rATG, 8 mg/kg, days −9 to −6
      • (2)
        Fludarabine, 90 mg/m2, days −4 to −2
      • Cyclophosphamide, 1 g/m2, day −2
      • Etoposide, 100 mg/m2, day −3
      • TBI, 4 Gy; day −5
      • (3)
        No conditioning
      • (1)
        Tacrolimus
      • Short methotrexate
      • (2)
        Tacrolimus
      • Short methotrexate
      • (3)
        Cyclosporine
      • Dexamethasone
      • (1) D+19
      • (2) None
      • (3) None
      Refractory HLH

      Streptococcus mitis sepsis

      Cardiac effusion

      Ascites

      Pancreatitis

      Adenovirus viremia and cystitis
      None
      • (1) Secondary graft loss in first 30 d
      • (2) Refractory HLH
      • (3) Refractory HLH
      Died

      D+109 from multiorgan failure
      10CID12 yUCB, 5/8 matched0.126 × 106/kgFludarabine, 180 mg/m2, days −7 to −2

      Busulfan, 7.6 mg/kg, days −3 to −2

      ATG, 8 mg/kg, days −9 to −6
      Tacrolimus

      Methotrexate

      Prednisolone
      D+33Acute pulmonary edema

      TMA

      Recurrent pancreatitis
      NoneFull immune reconstitution

      100% Donor
      Alive
      11CID7 yMRD, HLA identical5.7 × 108/kg nucleated cellsBusulfan, 16 mg/kg

      Cyclophosphamide, 200 mg/kg
      Cyclosporine

      Methylprednisolone
      D+12Hemorrhagic ulcers with massive GI bleeding

      Hemorrhagic cystitis

      Pulmonary aspergillosis
      Acute GvHD skin and GI tractFull immune reconstitution

      95% Donor in myeloid and lymphoid lines
      Alive
      12CID

      HLH
      10 yMUD, 10/10 matched6.32 × 106/kgBusulfan, 3.6 mg/kg, days −8 to −5

      Etoposide, 30 mg/kg/dose, day −4

      Cyclophosphamide, 60 mg/kg, days −3 to −2
      Prednisone

      Cyclosporine

      Methotrexate

      Almetuzumab used as salvage therapy after HSCT
      D+16Refractory HLH

      GI bleeding

      Pulmonary hemorrhage

      Toxic epidermal necrosis

      Renal failure
      Grade II skinRefractory HLHDied

      D+42 from multiorgan failure
      13CID7.5 yMUD PBSCs31.2 × 106/kgTreosulfan, 14 mg/m2/d, days −7 to −5

      Fludarabine, 30 mg/m2/d, days −6 to −2

      Alemtuzumab, 0.2 mg/kg/d, days −8 to −4

      Ruxolitinib, 2-wk course (5 mg/d); stopped day −9
      Cyclosporine

      MMF
      D+16NoneGrade II skinFull immune reconstitution

      100% Donor
      Alive
      14Severe infections29 yMUD, 6/6 matched3 × 108/kg nucleated cellsCyclophosphamide, 200 mg/kg, days −10 to −7

      Fludarabine, 125 mg/m2, days −6 to −2

      ATG, 25 mg/kg, days −6 to −2

      TBI, 3 Gy, day −7
      Tacrolimus

      Methotrexate
      D+17CMV

      Candidiasis

      Sepsis
      NoneSecondary graft loss in first 90 dDied

      D+410 from sepsis
      15Severe infections13 moMRD 6/6, matched6.56 × 106/kgFludarabine, 30 mg/m2/d, days −8 to −3

      Melphalan, 140 mg/m2/d, day −3

      ATG, 5 mg/kg/d, days −3 to −2
      Cyclosporine

      Methotrexate
      D+15Severe thrombocytopenia

      Increased transaminase levels
      NoneSecondary graft loss within first 100 dDied

      10 mo after from fulminant lung infection
      ATG, Antithymocyte globulin; AUC, area under the blood concentration time curve; CMV, cytomegalovirus; GvHD, graft-versus-host disease; MMF, mycophenolate mofetil; IVIG, intravenous immunoglobulin; rATG, rabbit anti-thymocyte globulin; TMA, thrombotic microangiopathy; GI, gastrointestinal; TBI, total body irradiation; TDM, therapeutic drug monitoring; UCB, unrelated cord blood.
      All 15 patients had heterozygous missense mutations in either the coiled-coil or DNA-binding domain of STAT1 (Table I). Five patients (P2-P5 and P11) from 4 different countries shared the same c.1154C>T, p.T385M missense mutation, and 2 patients (P12 and P14) from Canada and Peru shared the same c.1189A>G, p.N397D missense mutation, both of which were located in the DNA-binding domain. The remaining mutations were identified in only a single patient. All 10 unique mutations conferred GOF activity with enhanced STAT1 phosphorylation in response to IFN-γ (Fig 1) and showed increased GAS-dependent reporter gene transcriptional activity after stimulation with IFN-γ (Fig 2): P1, who was studied before HSCT, demonstrated enhanced and prolonged STAT1 phosphorylation after stimulation with IL-6 or IL-27 (Fig 3, A).
      Figure thumbnail gr1
      Fig 1Evaluation of expression and phosphorylation of GOF-STAT1 mutants by using immunoblotting. GOF-STAT1 mutations led to enhanced phosphorylated STAT1 (pSTAT1) expression after stimulation with IFN-γ. U3C cells were transfected with STAT1 mutants or WT and stimulated with IFN-γ for 20 minutes. Western blotting was performed with anti–phosphorylated STAT1, anti-STAT1, and anti–β-actin antibodies. Mutations affecting Y701 prevent STAT1 phosphorylation.
      Figure thumbnail gr2
      Fig 2Luciferase reporter assay to evaluate transcriptional activation by GOF-STAT1 mutants. STAT1 mutants led to enhanced luciferase GAS-induced activity. Transcriptional responses to increasing concentrations of IFN-γ (white bar, nonstimulated; light gray bar, 10 U/mL; dark gray bar, 100 U/mL; black bar, 1000 U/mL) were added to U3C cells transfected with STAT1 mutants or WT STAT1 and cultured for 16 hours before GAS-induced activity was measured. Experiments were performed in triplicate, and data are expressed in relative luciferase units (RLU). Individual reporter assays were performed 3 times to confirm reproducibility. Y701 mutation eliminated transcriptional activity of STAT1.
      Figure thumbnail gr3
      Fig 3Cytokine-induced STAT1 phosphorylation. A, Pre-HSCT PBMCs from patient 1 (H328R) led to hyperphosphorylation and delayed dephosphorylation after IL-6 and IL-27 stimulation. B, Post-HSCT PBMCs from patient 2 (T385M) show normal phosphorylation kinetics compared with healthy control subjects (HC).

       Clinical phenotype and pre-HSCT complications

      Infections occurred in all 15 patients before HSCT, with fungal infections being the most common. Twelve patients had CMC affecting the nails, skin, oral mucosa, and intestinal tract. Five had invasive fungal infections, including pulmonary aspergillosis in 3 patients, candidemia in 1 patient, and cryptococcal meningitis in 1 patient. Bacterial infections were also frequent, most commonly affecting the upper and lower respiratory tract, but also causing sepsis in 2 patients and gastroenteritis in 2 patients. When culture results were available, encapsulated organisms were predominant (Haemophilus influenzae, n = 3; Streptococcus pneumoniae, n = 3), followed by Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, and Clostridium difficile. Recurrent pneumonia led to bronchiectasis in 2 patients, one of whom has recurrent Pseudomonas species pneumonia. Varicella zoster virus and cytomegalovirus were the most common viral infections among this cohort (n = 5 each), but multiple other cutaneous and invasive viral infections occurred (EBV in 3 patients; molluscum contagiosum virus, norovirus, and herpes simplex virus each in 2 patients; and parvovirus, BK virus, JC virus, adenovirus, and human herpes virus 6 each in 1 patient). Disseminated EBV led to hemophagocytic lymphohistiocytosis (HLH) in 1 patient (P12), and parvovirus B19 induced pure red cell aplasia in 1 patient (P9). BK and JC viremia occurred in the context of immunosuppression for treatment of chronic neutropenia and thrombocytopenia in another (P10). Nontuberculous mycobacterial infections were observed in patients 1 and 2, both with lymphadenitis. One patient (P4) had pulmonary tuberculosis.
      Autoimmune disorders were observed in 10 patients, 5 of whom had an IPEX-like syndrome (P1-P5). Enteropathy, the hallmark feature of those with IPEX-like syndrome, was often associated with other autoantibody/T cell–mediated manifestations, including type 1 diabetes mellitus, thyroiditis, autoimmune neutropenia, hemolytic anemia, and growth hormone deficiency. Four of the IPEX-like patients had the same missense mutation affecting the DNA binding domain (c.1154C>T p.T385M). In 6 of the 10 patients without a typical IPEX-like syndrome, a variety of autoimmune symptoms occurred, including vitiligo, hypothyroidism, autoimmune cytopenias, antiphospholipid syndrome, pernicious anemia, and autoimmune hepatitis.
      Two patients in this cohort had features consistent with HLH that were refractory to medical therapy, contributing to the decision to initiate HSCT (see Fig E1 in this article's Online Repository at www.jacionline.org). Patient 9 had HLH associated with pneumonia at age 7 years. He was successfully treated with prednisolone and high-dose intravenous immunoglobulin, but HLH relapsed a few months later, leading to agranulocytosis unresponsive to granulocyte colony-stimulating factor, prednisone, intravenous immunoglobulin, and cyclosporine. Despite 3 attempted HSCTs, HLH never cleared, and the patient died of multiorgan failure 3 months after the third transplantation and secondary graft failure from the first transplantation.
      Patient 12 had HLH at age 10 years in association with EBV viremia while also having microabscesses of the spleen and kidneys and cutaneous herpes zoster infection. She did not respond to dexamethasone, and HLH progressed to involve the central nervous system. Treatment was escalated to include etoposide, cyclosporine, and intrathecal methotrexate but unfortunately failed to induce remission, and the patient died of multiorgan failure within 1 week of transplantation.
      Both patients had classic presentations of HLH with very increased inflammatory markers and ferritin and hemophagocytosis visualized in the bone marrow. Assessment of natural killer (NK) cell cytotoxicity was not performed in patient 9 and was normal in patient 12.
      Antibody deficiency was present in 9 patients, prompting treatment with immunoglobulin supplementation (P4, P6-P11, P13, and P14). Nine patients (P4, P5, and P7-13) had T-cell lymphopenia, and 3 had abnormal T-cell function when tested (P5, P6, and P11). Eight patients with T-cell defects also had concurrent B-cell and/or NK cell lymphopenia (P4, P6, and P8-13). T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) were quantified in 3 patients (P8-P10) and were less than the limit of detection in all 3. Defects in cellular and humoral immunity occurred independent of the presence of autoimmunity in some and coincided with autoimmunity in others.

       Transplantation course and complications

      Nineteen HSCTs were performed in this cohort of 15 patients (Fig 4 and Table II). One patient (P9) received 3 transplants, and 2 (P2 and P6) received 2 transplants. Indications for HSCT were severe clinical manifestations, including recurrent infections, autoimmunity, IPEX-like symptoms refractory to medical therapy, HLH, and CID (see Fig E1). Of those patients who survived HSCT, the mean age at HSCT was 8.5 years (range, 4-12 years); of those who died, the mean age was 16.5 years (range, 13 months to 33 years) at the time of transplantation. A variety of graft sources were used, including matched related donors (MRDs; 4 HSCTs), matched unrelated donors (MUDs; 5 HSCTs), mismatched unrelated donors (MMUDs; 1 HSCT), a haploidentical donor (1 HSCT), partially matched unrelated umbilical cord blood (UCB; 3 HSCTs), and fully matched UCB (1 HSCT). Peripheral blood stem cells (PBSCs) were used in 4 transplants. Four of six survivors received unrelated grafts (2 MUD-marrow, 1 MUD-PBSCs, and 1 partially matched UCB); the fifth and sixth survivors received haploidentical PBSCs and matched related bone marrow, respectively.
      Figure thumbnail gr4
      Fig 4Outcome of patients with GOF-STAT1 mutations after HSCT. Fifteen patients with GOF-STAT1 mutations underwent HSCT. Primary engraftment occurred in 12. Six had secondary graft loss. Six are alive, and 5 have full immune reconstitution and reversal of disease manifestations.
      Reduced-intensity conditioning (RIC) regimens were used most frequently (P1, P2 [first HSCT], P3, P4, P6 [second HSCT], P9 [second HSCT], P10, P13, P14, and P15) and were associated with higher OS (P = .11; Fig 5, E) and EFS (P = .07; see Fig E2, E, in this article's Online Repository at www.jacionline.org). The decision to use RIC regimens was institution specific. Patients 1, 3, and 4, 2 of whom survived, received the same RIC regimen consisting of fludarabine, melphalan, and alemtuzumab based on its previous success in patients with IPEX syndrome.
      • Rao A.
      • Kamani N.
      • Filipovich A.
      • Lee S.M.
      • Davies S.M.
      • Dalal J.
      • et al.
      Successful bone marrow transplantation for IPEX syndrome after reduced-intensity conditioning.
      Myeloablative regimens were used in 7 HSCTs (haploidentical PBSCs in P2, MMUD in P9, MRD in P8 and P11, partially matched UCB in P6, and MUD in P5 and P12). Five of 7 patients (P5, P6, P8, P9, and P12) who received myeloablative regimens died; however, death was complicated by chronic HLH in 2 patients (P9 and P12). Patients 5 and 8 died before primary engraftment, the latter of which was directly related to cyclophosphamide toxicity. Patient 6 had secondary graft failure and did not survive a second transplantation. Patient 2 received myeloablative conditioning before a second HSCT after secondary graft loss from the first transplantation with RIC. Patient 11 had full immune reconstitution and resolution of disease symptoms and is alive 2 years after HSCT.
      Figure thumbnail gr5
      Fig 5Overall posttransplantation survival and survival analysis. A, Overall posttransplantation survival in patients with GOF-STAT1 mutations. B, Survival analysis comparing patients with an IPEX-like presentation versus patients with CID, significant infections, or both. C, Survival analysis comparing patients with T385M amino acid substitution versus patients with other GOF-STAT1 mutations. D, Survival analysis comparing patients younger or older than 12 years of age. E, Survival analysis comparing myeloablative and nonmyeloablative protocols. In , B-E, numbers in brackets represent the number of patients in each group. In patients undergoing more than 1 HSCT, survival is calculated since the last transplantation performed.
      A total of 80 posttransplantation events occurred (see Table E1), with infections consisting of viral reactivation and sepsis being the most common. Median event-free time from a posttransplantation complication was 31.5 days. There were also a high number of vascular and cardiac-related transplant complications: catheter-associated venous thrombus formation and supraventricular tachycardia in the same patient and cardiac effusion, thrombotic microangiopathy, and cyclophosphamide-induced cardiac toxicity each in 1 patient. The association of vascular aneurysms and complications in patients with GOF-STAT1 mutations
      • Toubiana J.
      • Okada S.
      • Hiller J.
      • Oleastro M.
      • Lagos Gomez M.
      • Aldave Becerra J.C.
      • et al.
      Heterozygous STAT1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype.
      • Uzel G.
      • Sampaio E.P.
      • Lawrence M.G.
      • Hsu A.P.
      • Hackett M.
      • Dorsey M.J.
      • et al.
      Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.
      • Kataoka S.
      • Muramatsu H.
      • Okuno Y.
      • Hayashi Y.
      • Mizoguchi Y.
      • Tsumura M.
      • et al.
      Extrapulmonary tuberculosis mimicking Mendelian susceptibility to mycobacterial disease in a patient with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutation.
      could be a risk factor for cardiac- and vascular-related events after HSCT. Other noninfectious complications of HSCT included pancreatitis, acute pulmonary edema, gastrointestinal bleeding, and hepatitis. Overall EFS was low (10% by day +100 after HSCT) and was not affected by age at transplantation, phenotype, genotype, or conditioning regimen (see Fig E2, B-E).

       Immune reconstitution

      Primary engraftment, which was defined as an absolute neutrophil count of greater than 500 cells/μL for 3 consecutive days, occurred at a median of day +18 in 14 (74%) of 19 HSCTs and in 13 (80%) of 15 patients with their first HSCT. With the exception of patients 5 and 8, who died soon after transplantation and before engraftment, 12 of 13 patients engrafted with their first transplantation. Primary graft failure occurred in patient 6.
      Secondary graft failure occurred in 6 patients (within the first 4 months in 5 patients) after HSCT that had primary engraftment and survived the first 100 days (Fig 4 and Table II and see Fig E3, A, in this article's Online Repository at www.jacionline.org). No isolated risk factors for secondary graft loss, including phenotype, genotype, age at transplantation, conditioning regimen, or donor source, were identified (see Fig E3, B). Of the 6 patients with secondary graft failure: 2 (P2 and P9) underwent subsequent transplant, with 1 (P2) surviving with full immune reconstitution. The other survivor with secondary graft failure (P1) is a mixed chimera, has experienced return of symptoms, and has not yet undergone retransplantation (Fig 4 and Table II). All 5 survivors (P2, P3, P10, P11, and P13) with 95% to 100% donor chimerism established full immune reconstitution and complete reversal of immunodeficiency and resolution of infectious and autoimmune manifestations.
      Poor thrombocyte recovery was observed in 3 patients (P4, P7, and P15), resulting in severe transfusion-dependent thrombocytopenia. Patient 7, who received an MRD transplant, engrafted without conditioning but died 3 months after HSCT from bleeding at sites of mycotic brain aneurysms; no bleeding episodes occurred in patients 4 or 15.
      Graft-versus-host disease (GvHD) prophylaxis was used in all but 1 patient, P7, who was pancytopenic and received an MRD transplant. Acute GvHD was generally mild or well controlled by immunosuppression. Acute GvHD occurred after 8 of 18 evaluated HSCTs in 8 (57%) patients (P2 [second HSCT], P3, P4, P5, P6 [second HSCT], P11, P12, and P13; Table II). Seven patients had acute GvHD of the skin (grades 1-3), and in all cases there was good response to topical and systemic corticosteroids. Two patients (P6 and P11) had acute GvHD of the gastrointestinal tract.

       Outcome

      Overall, 6 (40%) patients survived and are presently more than 1 year after transplantation (Fig 4 and Table II). Nine patients died, 7 less than 1 year after HSCT (Fig 4 and Table II and Table E1). Of the surviving patients, 5 of 6 had full immune reconstitution, and 1 is a mixed chimera. One survivor had complete secondary graft loss (P2), and 1 (P1) has split donor chimerism with 2% donor myeloid cells and 39% donor lymphoid cells. Patient 2 underwent a second HSCT and had full immune reconstitution and reversal of in vitro hyperphosphorylation of STAT1 (Fig 3, B). Graft loss was gradual in patient 1 and associated with return of infections, enteropathy, continued failure to thrive, and development of lymphopenia and hypogammaglobulinemia. Symptoms remain milder than those in his pre-HSCT condition.
      Those patients with IPEX-like syndrome who engrafted and have 95% to 100% immune reconstitution (P2 and P3) resolved enteropathy within the first 100 days, and immunosuppression could be discontinued within 1 year. Patient 3 was able to discontinue parenteral nutrition by day +38. All surviving patients with 95% to 100% donor engraftment (P2, P3, P10, P11, and P13) have complete resolution of autoimmunity and infections and underwent catch-up growth.
      Two patients with CID had HLH several months before HSCT (P9 and P12). In both patients HLH was lethal when the patients underwent HSCT in the presence of active disease. HLH-associated death accounted for 22% of the lethal outcomes of HSCT in this cohort. A CID phenotype was associated with a higher frequency of posttransplantation infections (12/22 posttransplantation infections, see Table E1) and negatively affected OS (P = .24; Fig 5, B) and EFS (P = .14; see Fig E2, B), but neither reached statistical significance.
      Death occurred in all patients by the end of this study who did not have some degree of donor engraftment and immune reconstitution. Only patients with 95% to 100% immune reconstitution had resolution of infections and autoimmunity (P2, P3, P10, P11, and P13). Younger age (P = .05; Fig 5, D), a mutation leading to T385M (P = .24; Fig 5, C), lack of CID (P = .24; Fig 5, B), and use of RIC (P = .11; Fig 5, E) were associated with increased OS, but only age at transplantation reached statistical significance. IPEX-like disease and mutation leading to T385M were favorable factors; however, patients with these characteristics underwent transplantation at a younger age. Four of 5 patients with IPEX-like disease and 3 of 4 with mutations leading to T385M were 12 years or younger at the time of HSCT, suggesting that age and not phenotype or genotype most strongly predict OS and EFS.

      Discussion

      This multinational cohort of 15 patients is the largest aggregation of patients with GOF-STAT1 mutations who have undergone HSCT yet collected. In 6 patients GOF-STAT1 mutations were identified retrospectively after HSCT and postmortem in 3. In none of the patients was HSCT elective but rather intended to be lifesaving to reverse severe infections, HLH, or autoimmunity. For this reason, survival rates might have been more dismal and would likely be better in patients undergoing transplantation earlier before the development of severe manifestations.
      Data from this cohort suggest that HSCT is a viable and curative treatment option for patients with GOF-STAT1 mutations; however, disease-related complications are common and can strongly affect outcomes. Numerous questions surrounding appropriate patient selection, timing, donor, and conditioning regimen for HSCT in patients with GOF-STAT1 mutations still exist. However, our data suggest that HSCT can be considered curative, particularly if performed early in patients with GOF-STAT1 mutations with severe phenotypes, including those with IPEX-like symptoms, CID, serious life-threatening infections, and severe autoimmunity but not active HLH. Younger age was the strongest positive indicator of OS, suggesting a negative effect of disease-related morbidity on EFS and a higher rate of success when undergoing early transplantation. Because of the effect of age on OS and EFS, early recognition and diagnosis of GOF-STAT1 is imperative. OS and EFS were not affected by genotype, phenotype, or conditioning regimen. When assessed, abnormal TREC and KREC numbers coincided with immunodeficiency and predisposition to infections. The role of TREC and KREC analysis in early diagnosis and prognosis of patients with GOF-STAT1 mutations needs more investigation.
      Five of 6 patients in this series with IPEX-like symptoms had the common DNA binding domain mutation c.1154C>T, p.T385M, which is known to be associated with IPEX-like disease.
      • Uzel G.
      • Sampaio E.P.
      • Lawrence M.G.
      • Hsu A.P.
      • Hackett M.
      • Dorsey M.J.
      • et al.
      Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.
      Ten patients in this series had mutations (c.1154C>T, p.T385M in P2-P5 and P11; c.494A>G, p.D165G in P6; c.820G>A; R274W in P7, c.821G>A, p.R274Q in P8; and c.1189A>G, p.N397D in P12 and P15) that were previously described in other patients as associated with severe clinical manifestations, including severe infections and autoimmune disease.
      • Sharfe N.
      • Nahum A.
      • Newell A.
      • Dadi H.
      • Ngan B.
      • Pereira S.L.
      • et al.
      Fatal combined immunodeficiency associated with heterozygous mutation in STAT1.
      In agreement with this, these patients also had severe infections, autoimmunity, and immunodeficiency. With the exception of the common DNA binding domain mutation c.1154C>T, p.T385M, which was associated with a higher incidence of IPEX-like symptoms and overall better outcome (Fig 5, C), there was no specific genotype-phenotype outcome correlation. Within this cohort, patients with GOF-STAT1 mutations were preferentially observed in the DNA-binding domain (10/15), suggesting that mutations in this region are associated with worse infections, autoimmunity, and immunodeficiency, and patients with these mutations might benefit from early consideration of transplantation.
      Although HLH has not been described as a major problem in patients with GOF-STAT1 mutations,
      • Boisson-Dupuis S.
      • Kong X.F.
      • Okada S.
      • Cypowyj S.
      • Puel A.
      • Abel L.
      • et al.
      Inborn errors of human STAT1: allelic heterogeneity governs the diversity of immunological and infectious phenotypes.
      • Depner M.
      • Fuchs S.
      • Raabe J.
      • Frede N.
      • Glocker C.
      • Doffinger R.
      • et al.
      The extended clinical phenotype of 26 patients with chronic mucocutaneous candidiasis due to gain-of-function mutations in STAT1.
      • Liu L.
      • Okada S.
      • Kong X.F.
      • Kreins A.Y.
      • Cypowyj S.
      • Abhyankar A.
      • et al.
      Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.
      • Mizoguchi Y.
      • Tsumura M.
      • Okada S.
      • Hirata O.
      • Minegishi S.
      • Imai K.
      • et al.
      Simple diagnosis of STAT1 gain-of-function alleles in patients with chronic mucocutaneous candidiasis.
      • Romberg N.
      • Morbach H.
      • Lawrence M.G.
      • Kim S.
      • Kang I.
      • Holland S.M.
      • et al.
      Gain-of-function STAT1 mutations are associated with PD-L1 overexpression and a defect in B-cell survival.
      • Sampaio E.P.
      • Hsu A.P.
      • Pechacek J.
      • Bax H.I.
      • Dias D.L.
      • Paulson M.L.
      • et al.
      Signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations and disseminated coccidioidomycosis and histoplasmosis.
      • Soltesz B.
      • Toth B.
      • Shabashova N.
      • Bondarenko A.
      • Okada S.
      • Cypowyj S.
      • et al.
      New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe.
      • Toubiana J.
      • Okada S.
      • Hiller J.
      • Oleastro M.
      • Lagos Gomez M.
      • Aldave Becerra J.C.
      • et al.
      Heterozygous STAT1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype.
      • van de Veerdonk F.L.
      • Plantinga T.S.
      • Hoischen A.
      • Smeekens S.P.
      • Joosten L.A.
      • Gilissen C.
      • et al.
      STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.
      • Uzel G.
      • Sampaio E.P.
      • Lawrence M.G.
      • Hsu A.P.
      • Hackett M.
      • Dorsey M.J.
      • et al.
      Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.
      the 2 patients reported here had severe HLH without entering remission and died shortly after HSCT during active disease. The mechanism of HLH in patients with GOF-STAT1 mutations is not well elucidated, but given the proposed role played by IFN-γ in the pathogenesis of HLH,
      • Tang Y.
      • Xu X.
      • Song H.
      • Yang S.
      • Shi S.
      • Wei J.
      • et al.
      Early diagnostic and prognostic significance of a specific Th1/Th2 cytokine pattern in children with haemophagocytic syndrome.
      development of HLH associated with GOF-STAT1 mutations might be related to hyperactivation of IFN-γ–dependent STAT signaling pathways. Defects in NK cell cytotoxicity in patients with GOF-STAT1 mutations have recently been reported,
      • Tabellini G.
      • Vairo D.
      • Scomodon O.
      • Tamassia N.
      • Ferraro R.M.
      • Patrizi O.
      • et al.
      Impaired NK cell functions in patients with STAT1 gain-of-function mutations.
      • Vargas-Hernandez A.
      • Mace E.M.
      • Freeman A.F.
      • Rosenzweig S.
      • Chinn I.K.
      • Holland S.M.
      • et al.
      LASID Meeting 2015.
      and this might also contribute to an increased risk of HLH in these patients, particularly in the context of viral infections.
      IPEX-like symptoms are conventionally treated with long-term immunosuppression, including corticosteroids, calcineurin inhibitors, and rituximab. In the 3 patient with GOF-STAT1 mutations associated IPEX-like symptoms without CID (P1-P3), various immunosuppressive agents were ineffective in correcting the autoimmune features. HSCT was well tolerated in those with isolated IPEX-like disease. There were no deaths, and two thirds had full immune reconstitution with complete reversal of clinical manifestations, suggesting that HSCT should be considered in patients with GOF-STAT1 mutations with IPEX-like symptoms, especially if the symptoms are refractory to medical therapy.
      Secondary graft loss occurred frequently (6/12 with primary engraftment) and did not discriminate between phenotype, genotype, conditioning regimen, age, or donor source. The reasons behind the high rate of secondary graft loss are unclear, including what role heightened IFN-γ–induced STAT1 signaling plays in patients with GOF-STAT1 mutations.
      IFN-γ receptor deficiency and primary and secondary HLH are examples of primary immunodeficiency diseases in which heightened IFN-γ expression correlates with poor engraftment rates and worse outcome after transplantation.
      • Ouachee-Chardin M.
      • Elie C.
      • de Saint Basile G.
      • Le Deist F.
      • Mahlaoui N.
      • Picard C.
      • et al.
      Hematopoietic stem cell transplantation in hemophagocytic lymphohistiocytosis: a single-center report of 48 patients.
      • Roesler J.
      • Horwitz M.E.
      • Picard C.
      • Bordigoni P.
      • Davies G.
      • Koscielniak E.
      • et al.
      Hematopoietic stem cell transplantation for complete IFN-gamma receptor 1 deficiency: a multi-institutional survey.
      Recently, the JAK inhibitor ruxolitinib
      • Das R.
      • Guan P.
      • Sprague L.
      • Verbist K.
      • Tedrick P.
      • An Q.A.
      • et al.
      Janus kinase inhibition lessens inflammation and ameliorates disease in murine models of hemophagocytic lymphohistiocytosis.
      and anti–IFN-γ mAbs
      • Jordan M.
      • Locatelli F.
      • Allen C.
      • DeBendetti F.
      • Grom A.A.
      • Ballabio M.
      • et al.
      2015 annual meeting abstracts.
      have been effective at suppressing IFN-γ–induced inflammation in murine and human HLH, respectively. In vitro exposure of T lymphocytes from patients with GOF-STAT1 mutations to ruxolitinib resulted in profound suppression of IFN-α and IFN-β STAT1 phosphorylation,
      • Higgins E.
      • Al Shehri T.
      • McAleer M.A.
      • Conlon N.
      • Feighery C.
      • Lilic D.
      • et al.
      Use of ruxolitinib to successfully treat chronic mucocutaneous candidiasis caused by gain-of-function signal transducer and activator of transcription 1 (STAT1) mutation.
      • Baris S.
      • Alroqi F.
      • Kiykim A.
      • Karakoc-Aydiner E.
      • Ogulur I.
      • Ozen A.
      • et al.
      Severe early-onset combined immunodeficiency due to heterozygous gain-of-function mutations in STAT1.
      and treatment of a patients with a GOF-STAT1 mutation resulted in improvement of their individual symptoms.
      • Higgins E.
      • Al Shehri T.
      • McAleer M.A.
      • Conlon N.
      • Feighery C.
      • Lilic D.
      • et al.
      Use of ruxolitinib to successfully treat chronic mucocutaneous candidiasis caused by gain-of-function signal transducer and activator of transcription 1 (STAT1) mutation.
      • Weinacht K.G.
      • Charbonnier L.M.
      • Alroqi F.
      • Plant A.
      • Qiao Q.
      • Wu H.
      • et al.
      Ruxolitinib reverses dysregulated T helper cell responses and controls autoimmunity caused by a novel signal transducer and activator of transcription 1 (STAT1) gain-of-function mutation.
      Ruxolitinib was used as immunosuppressive therapy in 1 patient in this series (P13) for 2 weeks before transplantation. This patient was one of the 6 survivors and had full immune reconstitution with no secondary graft loss. With the exception of acute GvHD, he has no other posttransplantation complications. The success of this specific case might suggest that use of JAK inhibitors, as well as anti–IFN- γ therapies, might be an effective strategy in controlling IFN-γ–induced inflammation in patients with GOF-STAT1 mutations in the setting of transplantation and could perhaps improve engraftment rate and overall outcome.
      Our retrospective data demonstrate that HSCT can be curative for patients with GOF-STAT1 mutations. With full immune reconstitution, disease manifestations are reversed quickly and permanently. Because those with a severe phenotype have a less favorable outcome, patients should be considered for transplantation earlier to prevent serious complications and improve OS. Without novel strategies to control HLH, HSCT in patients with active disease is likely to fail.
      Clinical implications
      HSCT in patients with GOF-STAT1 mutations can be curative but might be associated with complications that can lead to secondary graft failure and decreased survival.

      Appendix

      Figure thumbnail fx1
      Fig E1Indication for HSCT in patients with GOF-STAT1 mutations. All patients in this cohort had severe infections (n = 15). Indications for HSCT were IPEX-like symptoms (n = 3), CID (n = 6), CID with HLH (n = 2), IPEX-like symptoms and CID (n = 2), or only severe infections (n = 2).
      Figure thumbnail fx2
      Fig E2Overall posttransplantation event-free survival (EFS). A, Overall posttransplantation EFS in patients with GOF-STAT1 mutations. B, EFS analysis comparing patients with IPEX-like presentation versus patients with CID, significant infections, or both. C, EFS analysis comparing patients with T385M amino acid substitution versus patients with other GOF-STAT1 mutations. D, EFS analysis comparing patients younger or older than 12 years. E, EFS analysis comparing patients receiving myeloablative versus nonmyeloablative conditioning regimen. In , B and D, numbers in brackets represent the number of patients in each group.
      Figure thumbnail fx3
      Fig E3Secondary graft failure data. A total of 19 HSCTs were performed in 15 patients with GOF-STAT1 mutations, including 3 patients who underwent 2 HSCTs and 1 patient who underwent 3 HSCTs. A total of 6 events of secondary graft failure were reported, 2 of which occurred in the same patient (patient no. 9). A, Survival curve showing the timing of each of the 6 events. B, Incidence of different variants in the graft failure group compared with their incidence in all HSCTs performed does not show any statistical significance. Each of the following variants was analyzed: presence of T385M amino acid substitution, clinical phenotype of IPEX-like disease, age less than or equal to 12 years at transplantation, use of a myeloablative conditioning protocol, and use of any stem cell donor other than an MRD (T385M mutation: 33.3% vs 31.58%, P = .94; IPEX-like: 50% vs 31.58%, P = . 43; age < 12 years: 66.67% vs 73.68%, P = .7512; myeloablative conditioning: 16.66% vs 31.58%, P = .49; non-MRD: 100% vs 78.95%, P = .24).
      Table E1Posttransplantation complications in patients with GOF-STAT1 mutations
      Day after transplantationReversibleFatal
      Patient 1
       Infection
      EBV reactivation+159YesNo
      Recurrent pneumonia+1464YesNo
       Bleeding (coagulopathy associated with colitis)+2765YesNo
       CV
      Catheter-associated thrombus+159YesNo
      SVT+159YesNo
       GI
      Recurrence of colitis+2756YesNo
       Heme
      Lymphopenia+2249NoNo
      Hypogammaglobulinemia+1611NoNo
       Secondary graft loss+30NoNo
      Patient 2 (first transplantation)
       Infection
      S parasanguis bacteremia−2YesNo
      CMV viremia+19YesNo
       Secondary graft loss+33NoNo
      Patient 2 (second transplantation)
       Renal/GU
      Drug-associated nephropathy+28YesNo
       GvHD+30YesNo
      Patient 3
       GvHD+16YesNo
      Patient 4
       Heme
      Severe thrombocytopenia+5Yes (+490)No
       Lymphopenia+30Nono
       Hypogammaglobulinemia+304NoNo
       Secondary graft loss+120NoNo
       Death (sepsis, pneumonia)+779NoYes
      Patient 5
       Infection
      CMVBefore transplantationNo+7
      GvHD+10YesNo
       Death+12
      Patient 6 (first transplantation)
       Infection
      Fungal pneumonia+24YesNo
      BK virus reactivation+24NoNo
      Patient 6 (second transplantation)
       Infection
      Adenovirus reactivation+2YesNo
      Cryptosporidium gut+20YesNo
      Fungal pneumonia+74NoYes
       GvHD+17YesNo
       Death+4 moNoYes
      Patient 7: Turkey
       Infection
      Mycotic aneurysmBefore transplantationNoYes
       Bleeding+88NoYes
       Death+3 mo
      Patient 8
       CV
      Cardiomyopathy+1NoYes
       Death+3
      Patient 9 (first transplantation)
       Infection
      S mitis sepsis+22YesNo
       Hemophagocytosis+25 (first)NoNo
       Secondary graft loss+45NoYes
      Patient 9 (second transplantation)
       Infection+14NoYes
      Adenovirus viremia+35
       CV
      Cardiac/pleural effusion+21NoYes
       GI
      Ascites+21NoYes
       Renal/GU
      Adenovirus cystitis+29NoNo
       Secondary graft loss+32NoYes
      Patient 9 (third transplantation)
       Bleeding+17NoYes
       GI
      Pancreatitis+13YesNo
       Death+109NoYes
      Patient 10: Japan
       CV
      TMA+20YesNo
       Lung
      Acute pulmonary edema+4YesNo
       GI
      Recurrent pancreatitis−1, +32, +42, +53, +58, +63YesNo
      Patient 11
       Infection
      Pulmonary aspergillosis+150YesNo
       GI
      Hemorrhagic bleeding+30YesNo
       Renal/GU
      Hemorrhagic cystitis+18, +120YesNo
       Heme
      Lymphopenia+50YesNo
      Hypogammaglobulinemia+50
       GvHD
      Skin+7YesNo
      GI+17YesNo
      Patient 12: Canada
       Lung
      Pulmonary hemorrhage+20NoYes
       GI
      GI bleeding+13YesNo
       Renal/GU
      Renal failure+21NoYes
       Other
      TEN+16NoYes
       GvHD+12NoNo
       Death+42NoYes
      Patient 13: United Kingdom
       GvHD+50YesNo
      Patient 14
       Infection
      CMV+31YesNo
      Candidiasis+72YesNo
      Sepsis+214YesNo
       Secondary graft loss+28, +90NoNo
       Death (sepsis)+410NoYes
      Patient 15
       Infection
      Pneumonia+52, +300Yes, noNo, yes
       GI
      Increased transaminase levels+150NoNo
       Heme
      Severe thrombocytopenia+150NoNo
       Secondary graft loss+130NoNo
       Death+10 moNoYes
      CV, Cardiovascular; CVM, cytomegalovirus; GI, gastrointestinal; GU, genitourinary; SVT, supraventricular tachycardia; TEN, toxic epidermal necrolysis; TMA, thrombotic microangiopathy.

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