Wiskott-Aldrich Syndrome

Jaichat Mekaroonkamol, MD

CASECASE

• A 3 year 5 month old Thai boy• Referred to KCMH since 2 month old because

Pneumonia with anemia and thrombocytopenia

Refractory CMV infection

• FTT(3.22 kg: < P3)• Microcephaly(HC 33 cm: < P3)• Thrombocytopenia

Initial CMV viral load 52800Total Gancyclovia iv 80 days

Persistent thrombocytopenia

CMV-induced thrombocytopenia

• Fully recovered a month after initial diagnosis• Acute CMV-induced thrombocytopenia VS ITP

secondary to a recent infection

British Journal of Haematology,2010. 149, 451–462

BMA: normocellular marrow adjust from age, increase megakaryocyte, normal maturation

of myeloid and erythroid series, increase eosinophil

Difficult to treat eczemaDifficult to treat eczema

• Age 7 day old: Diaper rash• Age 2 month old: systemic cadidiasis• Age 4 month old: Eczema

Wiskott Aldrich Wiskott Aldrich syndromesyndrome

• Severe viral infection with Impaired T cell function

• Thrombocytopenia• Eczema• Hyper IgE

Gene analysis: Gene analysis: WAS gene mutationWAS gene mutation

Recurrent infections

• 6 month old: Rota diarrhea, Salmonella gr C diarrhea• 8 month old: Viral pneumonia• 9 month old and 1 year 3 month old: PCP pneumonia• 1 year 6 month old: Klebsiella pneumonia• 1 year 7 month old: Multiple brain abscess• 1 year 10 month old: Pulmonary aspergillosis• 2 year 5 month old: Salmonella enteritis (gr D)

Persistent Persistent thrombocytopeniathrombocytopenia

Atopic Atopic diseasedisease

Multiple food Multiple food allergyallergy

Eczema with Eczema with recurrent recurrent

bacterial and bacterial and fungal skin fungal skin infectionsinfections

Reactive airway Reactive airway diseasedisease

OutlinesOutlines

• Pathogenesis & Etiology• Clinical manifestations• Management• Morbidity and Mortality • Genetic Counseling

History

• Alfred Wiskott, 1937– Diagnosed 3 brothers• Thrombocytopenia• Bloody diarrhea• Eczema• Recurrent ear infections

– Proposed hereditary thrombocytopenia

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

History

• Robert Aldrich, 1954– reported a similar clinical

phenotype• Observed six

generations.• 16 of 40 males• no females

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

History

• 1950’s & 1960’s – Classified as a

primary immunodeficiency disease

– involving T lymphs, B lymphs, and platelets

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

History

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

WAS protein (WASP), is a key regulator of actin polymerization in hematopoietic cells

Pathophysiology

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

Wiskott-Aldrich Syndrome Protein (WASP)

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

Wiskott-Aldrich Syndrome Protein (WASP)

Target Organs

Michael P. Blundella, Austen Wortha,b, Gerben Boumaa and Adrian J. Thrashera. The Wiskott-Aldrich syndrome: The actin cytoskeleton and immune cell function. Disease Markers 29 (2010) 157–17

5

Thrombocytopenia

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

• Most common symptom and occurs irrespective of the mutation severity in WASP

• Platelets are also often small in size• Main cause – Membrane/structural defects cause the spleen to

overdo it in disposing of platelets– Antiplatelet antibodies– Normal to increased megakaryocytes

Tcell Defects

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

Tcell Defects

• Cytoskeleton reorganization is involved in the binding of T lymphocytes to antigen-presenting cells through CD3 crosslinking

• Without actin reorganization, CD3 is not properly presented at the cells surface and the T cell is not activated

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

Regulatory T cell Defects

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

• Treg cell development is not impaired in WAS patients

• Decreased suppression of effector T cell proliferation and IFN-gramma production in vitro

• Allergies, autoimmunity

B cell Defects

• Thymus dependent B lymphocytes need T cells for activation and differentiation.

• Deletion of WASp lead to production of autoabtibodies and development of autoimmunity

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome:

Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

Other cell Defects

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

• Natural killer (NK) cells– normal to elevated levels of NK cells in peripheral

blood– cytotoxic activity of WAS NK cells is severely

decreased• DCs– express normal surface protein markers.– F-actin is abnormally distributed – fail to make sustained ruffles, lamellopodia, and

filopodia

Other cell Defects

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

• Monocytes/marcophages– Fewer filopodia, have a severe defect in

podosome formation and cell polarization– Fail to migrate to a chemokine gradient despite

having normal expression of chemokine receptors

• Neutrophils– Fail to polymerize actin and migrate normally– Reduced activation of respiratory burst

Clinical manifestations

Michael H. Alberta, Luigi D. Notarangelob and Hans D. Ochsc.Clinical spectrum, pathophysiology and treatment of the Wiskott–Aldrich syndrome Current Opinion in Hematology 2011, 18:42–48

Clinical manifestations

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

• WAS is classified by a triad of symptoms:– Microthromobocytopenia– Eczema– Immunodeficiency

• Classic triad appears only in 1/3 of patients

Microthromobocytopenia

HemorrhageHemorrhage

Microthromobocytopenia

• A clear correlation between platelet counts and risk for severe bleeding has never been established

• Recent retrospective analysis in a large cohort of XLT patients (173) found no such association

Albert MH, Bittner TC, Nonoyama S, et al. X-linked thrombocytopenia (XLT) due to WAS mutations: clinical characteristics, long-term outcome, and treatment options. Blood 2010

Eczema

Michel J. Massaad, Narayanaswamy Ramesh, and Raif S. Geha.Wiskott-Aldrich syndrome: Acomprehensive review. Ann. N.Y. Acad. Sci. 2013.

• Not usually manifest exclusively• Frequently associated with one

or more of symptoms• Incidence is higher in patients

with complete absence of WASp

• Imbalance of Th1/Th2 cytokine Imbalance of Th1/Th2 cytokine productionproduction

• Chemotaxis dysfunction of DC and LCChemotaxis dysfunction of DC and LC

Immunodeficiency

Autoimmune manifestations

• Most common– Autoimmune hemolytic anemia– Autoimmune neutropenia– Cutaneous vasculitis– Arthritis– Ig A Nephropathy

• Less Common– Inflammatory bowl syndrome– Idiopathic thrombocytopenic purpura

Tumor

• Mainly lymphoreticular malignancies with lymphoma esp. EBV-positive B cell lymphoma– Decrease cytotoxic T cell killing of cancer– Decreased NK cell function– Genomic instability

TreatmentTreatment

• Treatment of Manifestations• Prevention and Surveillance• Specific treatment

Treatment of Treatment of ManifestationsManifestations

• Infection: antibiotic, antifungal +/- IVIG• Eczema: Topical steroids +/- antibiotics

: Tacrolimus/ pimecrolimus???• Food allergy: avoidance• Thrombocytopenia with severe bleeding

– Platelet transfusions(must be irradiated and CMV free)

• Autoimmune disease– Hight dose corticosteroid, IVIG– Immunosuppressantsviral reactivation, risk for fungal infection

• Neutropenia: G-CSF and appropriate antibiotics.

PreventionPrevention

• Infection – Antibiotic prophylaxis: PCP in first 2-4 year– Intravenous immune globulin: every 3 to 4 weeks– Routine childhood immunizations (killed vaccine)

• Bleeding– Splenectomy:

• significantly increases the risk of life-threatening infections [Albert et al 2010]

• Caution in who subsequently undergo hematopoietic cell transplantation (HCT) [Ozsahin et al 2008]

– Avoid ASA

Specific treatmentSpecific treatment

• Only cure is hematopoeitic stem cell transplant– Matched sibling most successful– Unrelated donor much more risky – Greatest success when done <5yrs of age

• Gene therapy– If no matched dornor

Hematopoietic stem cell Hematopoietic stem cell transplantationtransplantation

• Retrospective study at 4 Israeli centers from 1996 to 2011: Fourteen WAS– Five children were transplanted from matched related

donors (4/5 siblings, 1/5 fully matched uncle)– Six patients were conditioned with full dose

busulfan/cyclophosphamide (Bu/Cy) – Thirteen of 14 patients (92.8%) are alive with a median

follow-up of 3.4 yr– Nine patients (64.3%) survive with complete clinical,

immunologic, and hematologic recovery– Children conditioned with full dose Bu/Cy had a 100% DFS

J Pediatr Hematol Oncol. 2013

Successful depend on……Successful depend on……

• HLA matched donor• Low clinical score at the time of treatment

Pai SY, Notarangelo LD. Hematopoietic cell transplantation for Wiskott– Aldrich syndrome: advances in biology and future directions for treatment. Immunol Allergy Clin North Am 2010.

Gene TherapyGene Therapy

Anne Galya and Adrian J. Thrasher. Gene therapy for the Wiskott–Aldrich syndrome. Allergy and ClinicalImmunology 2011

Boztug, Germany. 2010Boztug, Germany. 2010

• The first gene therapy for ten WAS patients• Gammaretroviral vector (GV) derived from a

Moloney murine leukaemia virus expressing WASp as a transgene

• A report on two of the patients demonstrated stable engraftment

• One patient was reported to have developed acute T cell leukemia

Boztug, Germany. 2010Boztug, Germany. 2010

After gene therapy, WASP-positive cells in variousleukocyte subgroups were detected on flow cytometry

The level of WASP-positive NK cellsranged from 25 to 90% over time

Boztug, Germany. 2010Boztug, Germany. 2010

An increase inthe percentage of

WASP-positive CD4+ and CD8+

T cells was seen 6 to 12 months after

gene therapyand has remained stable ever since

Boztug, Germany. 2010Boztug, Germany. 2010

An increase in platelet counts was noted, starting6 to 9 months after gene therapy

Boztug, Germany. 2010Boztug, Germany. 2010

WASP expression in platelet-rich plasma fromboth patients

was confirmed on Western

blot andflow cytometric

analysis

Boztug, Germany. 2010Boztug, Germany. 2010

Boztug, Germany. 2010Boztug, Germany. 2010

Boztug, Germany. 2010Boztug, Germany. 2010

• After gene therapy 2.5 years, WASP+CD19+ cells of – 57% in Patient 1– 69% in Patient 2

• Abnormal immunoglobulin levels normalized after gene therapy– IgG levels have not remained at a protective level in

Patient 1, who had undergone earlier splenectomy– Both patients were immunized with hexavalent vaccine 12

months after gene therapy and successfully produced specific antibodies to tetanus, diphtheria, and Haemophilus influenzae

Boztug, Germany. 2010Boztug, Germany. 2010

Boztug, Germany. 2010Boztug, Germany. 2010

• Infection – Patient 1 had an episode of pneumococcal

meningitis– his previous splenectomy or insufficient B-cell

reconstitution

• Autoimmunity– Signs and symptoms of autoimmunity disappeared

in both patients within the first year after gene therapy

Boztug, Germany. 2010Boztug, Germany. 2010

• GV shows preferential insertion at transcription start sites, promoters, and enhancer regions of active genes and at conserved noncoding DNA, resulting in a high rate of transformations

• The genotoxic risk of GV can be reduced by replacing the strong promoter/enhancer long terminal repeat (LTR) of the virus to produce a self inactivating (SIN) vector

Boztug, Germany. 2010Boztug, Germany. 2010

WAS-LVWAS-LV

• Recombinant lentiviral vector (LV) derived from the HIV-1 and carrying the WAS cDNA under the control of 1.6 kb derived from the endogenous WAS promoter (WAS-LV) was developed for treatment of WAS

WAS-LVWAS-LV

• Stable and broad tropism• Randomly integrate into DNA with no bias to a

specific area in the genome– lower rates of insertional mutagenesis

• Elimination of the LTR– its own promoter decreases the risk of insertional

mediated oncogene activation

WAS-LVWAS-LV

• Several clinical studies are currently open in Europe and the United States– Pre-clinical studies demonstrated high transduction

efficiency and survival advantage of human PBMCs and HSCs

– correction of Factin polarization, T cell proliferation and cytokine production, enhancedmigration of B cells and DCs, and increased podosome formation and turnover in DCs

– B cell immunoglobulin production against polysaccharide Ags, and decreased autoantibody production

WAS-LVWAS-LV

• The WAS promoter is not very efficient in sustaining adequate levels of WASp expression in human and murine HSCs

• Development of autoimmune manifestations

Lower incidence of transformation

induced by SIN-LV compared to SIN-GV

Modlich, U. et al. 2009.

Morbidity and Mortality Morbidity and Mortality

• The reported median survival of children with Wiskott-Aldrich syndrome who do not undergo successful allogeneic BMT is between eight and 14.5 years– infection (44% of individuals)– malignancy (26%)– bleeding (23%)

• 30% of males prior to diagnosis• Particularly when the intrinsic thrombocytopenia is complicated by

superadded autoimmunity

Dupuis-Girod et al 2003

Genetic CounselingGenetic Counseling

Genetic CounselingGenetic Counseling

• A de novo mutation: – 1/3 of affected individuals with no Fx

• The risk to sibs depends on the carrier status of the mother– If the mother is a carrier:

• 50% Males will be affected in each pregnancy • females who inherit the mutation will be carriers and may

occasionally have mild thrombocytopenia

• Males will pass the disease-causing mutation to all of their daughters and none of their sons

[Parolini et al 1998, Inoue et al 2002, Lutskiy et al 2002, Andreu et al 2003].

Autosomal dominantAutosomal dominant

Blood Journal hematology 1996

Prenatal TestingPrenatal Testing

• Pregnancies of women who are carriers of a WAS mutation. – Fetal sex by chromosome analysis of fetal cells – If the karyotype is 46,XY DNA analyzed

• Preimplantation genetic diagnosis (PGD)

SummarySummary

• WAS is caused by a mutation in the WASP gene, inhibits actin reorganization.

• Clinical phenotypes that include classic WAS, XLT or XLN

• Patients with severe WAS) without HLA-compatible donors are candidates for treatment by gene therapy

• GV vs LV gene therapy