Severe reduced level of memory B cells – is it diagnostic tool for CVID?

1. Belarusian Center for Primary Immunodeficiencies Belarusian Research Center for Pediatric Oncology and Hematology Severe reduced level of memory B cells – is it diagnostic tool for CVID? ESID – Prague May 2009 Svetlana SharapovaImmunology Department Sasha MigasMolecular Biology Department Galina KachanClinical Department Mihael BelevtsevImmunology Department

2. Common Variable Immunodeficiency Commonvariableimmunodeficiency(CVID)isaheterogeneousdisorderthatisassociatedwithlowserumimmunoglobulinconcentrations,defectivespecific-antibodyproductionandanincreasedsusceptibilitytobacterialinfectionsoftherespiratoryandgastrointestinaltracts.(Ochs H. et alPrimary Immunodeficincy Disease: Oxford University Press 2007)CVID has two picks of manifestation: from 6 to 10 and from 26 to 40 years.Gastrointestinal complications are fairly common inCVID—up to 50% of patients with CVID havechronic diarrhoea withmalabsorption.Othergastrointestinal diagnoses in patients with CVID includeCrohn’s disease, intestinal granulomatous disease,intestinal parasitic bacterial or viral infections, coeliacsprue, and intestinal lymphangiectasia.Autoimmune thrombocytopenic purpura and autoimmune haemolytic anaemia are the most common autoimmune consequences, occurring in 5–8% of all patients with CVID. (Lancet, 2008) PatientswithCVIDoftenhavedefectsinpost-antigenicB-celldifferentiation: -- areductionofCD27+ memoryBcells; -- impairedclassswitching; -- expansion of CD21low immatureBcells; -- poordiferentiationtoplasmacells. (Warnatz etal.)

3. Date Clinical case History Male was born to healthy unrelated parents. Delivery at term. Body weight: 3 530g. Early neonatal period was without features. At his first year hegrown up and developed according to his age, get oversomerespiratory infections (not severe disease course). (4 years 9 months) Pt had an infectious mononucleosis (diagnose was made on clinical data: fever, intoxication, lymphadenopathy, hepatosplenomegaly, in blood there were foundatypical mononuclear cells – 28%).On the background of which appears:pneumoniain left S5,thrombocytopenia 12,000 per liter,agranulocytosis 100* 10(9)/L. That was the reason for admittance to our hospital. 1999 December2003

4. Date Clinical case Lab Evaluation Thr-penia: 12,000/L Leu-penia: 3,3*10(9)/LAgran-cytosis: 100* 10(9)/L,Anemia: Hbg 92 g/L NORMAL: Urine analysis, electrolyte levels, lymphocyte subsets, Ig concetration, blood coagulation, Liver enzymes. In our hospital pneumonia progressed(despite antibacterial therapy); It was supposed to be tuberculosis; The patient was sent for clinical examination and treatment at the Institute of Tuberculosis, where specific treatment was not effective. The effect was reached at single dosingIV Ig (0,8g/kg) + Zienam (Imipenem), prednisolone 2mg/kg. Tuberculosis was excluded; Consequently appeared hemolysis on the basis of antituberculous therapy. February 2004 March 2004

5. Date Patient’s data Autoimmune Manifestation Lab Evaluation From December 2003 to April 2004 BM cytology: a lot of megakaryocyte, absent functioning it’s Immune thrombocytopenia (ITP)is mediated by platelet autoantibodies that accelerate platelet destruction and inhibit their production. Was stopped by prednisolone, IV Ig Autoimmune haemolytic anaemia(AIHA)is an immune disorder is an immune disorder caused by antibodies directed against unmodified autologous red cells.Most AIHA are caused by warm antibodies, whereas cold antibodies are less commonly detected. Wien Klin Wochenschr. 2008;120(5-6):136-51. Repeated hemolytic crises (at the attempt to reduce the dose of corticosteroids) Reticulocytosis:140 ‰, norma 5-10‰;Haptoglobin:<13g/L norma > 30 Anemia: Hbg: 40 g/L; Coombs test:direct positive 1:8 From April 2004 to December 2004

6. Date Intractable hemolysis Treatment IV Ig 0,2 g/kg Prednisolone 2mg/kg December 2004 Severe hemolytic crisis was the reason for admittance to hospital (intensive care unit) Our patient with severe autoimmune hemolysis with both coldand warm antibodies waspreviously refractory to conventional treatments was treated with weekly infusions of Rituximab. The only treatment with steroids, was allowed during the period of rituximab administration. Rituximab reacts specifically with the CD20 antigen and induces B-cell depletion. This could interfere with the production of autoantibodies in some immune diseases. Rituximab 375mg/m2, for 2 weeks

7. Date Patient Data Treatment Result April 2005 Complete hematologic response (+90) Hemolysis was stopped, Hb became normal(+10). The hematologic improvement was prompt, appearing by the second infusion of rituximab. (8-12 times ) watery stool with negative bacterial seeding. Skin: hyperemic spotswith desquamation(atopy) possible reaction to medicines. Despite intensive antibiotic therapy, IVIg the patient is constantly ill (sinusitis, ethmoiditis) Secondary malabsorbtion syndrome with secondary lactase deficiency was revealed; Clinically important electrolytic disturbances;Physicaldevelopment delay, Osteoporosis (as manifestation of secondary hyperparathyroidism) Hbg: 112 g/L Coombs test: negative June 2005 Hypo-electrolytemia (K, Ca, Ph, Mg,)

8. Patient’s Lab Data Recovery humoral immunity IV Ig replacement 0,2g/kg 2004 2005 2007 2009

9. Patient’s Lab Data Recovery humoral immunity 2004 2005 2007 2009

10. CVID Diagnosis Humoral immunity has not recovered during 4 years and 4 months after Rituximab treatment!! • After absence of recovery of humoral immunity was suggested CVID as diagnosis • The well-accepted definition of CVID includes • three key features: • the presence of hypogammaglobulinaemia Pt has decreasing of • of two or more immunoglobulin isotypes three isotypes • (low IgG, IgA, or IgM), • 2)recurrent sinopulmonary infections, Pt has constant sinusitis, • ethmoiditis • 3) impaired functional antibody responsesPt (I-0): • α-antibodies - very low • β-antibodies - absent • In addition to these date, there can be other clinical findings • including autoimmunity, granulomatous disease, and • neoplasia. Pt – Evans syndrome Common variable immunodefi ciency: a new look at an old disease, Lancet, 2008

11. Date Diagnostics procedures Patient’s Bone Marrow Investigation Healthy Donor April 2008 +52months after treatment Pro-B cells 4,7% B-cell precursors 4,9% Pre-B cells 55,9% Patient Pro-B cells 0.09% B-cell precursors 0% 0% Pre-B cells 0,05% CD19 CD34 CD34 Gated on CD45+CD14- HANDBOOK OF DIAGNOSTIC HEMATOPATHOLOGY TESTS, 2001

12. Date Diagnostics procedures Patient’s Bone Marrow Investigation Healthy Donor Pro-B cells 7,5% February 2009 +62months after treatment 17,1% Pre-B1 cell 75,4% Patient Pro-B cells 3% 28,2% 2,3% Pre-B1 cell 69,1% CD19 CD34

13. Patient data Extended Immunological Investigations Memory B cells (CD19+CD27+IgD-) detection Healthy Donor 57,3% 11,1% 95,9% 4,02% 18,6% Patient 97,3% 1,9% 51,4% 48,6% 0% IgD CD21

14. Patient data Mutational analysis Resequencing of 1-5 exons of TNFRSF13B gene has not revealed any mutations; The only nucleotide substitution was synonymous homogenic SNP in exon 2 : rs8072293 C/T;

15. Patient data Extended Immunological Investigations Phenotyping of naïve and memory CD4 T cells (gated on CD3+) CD4+CD45RA+ CD4+CD45RO+ Patient 14,5% 35,9% CD45RA CD45RO The immune dysregulation in our patient was extended to a reduction of naive CD4+CD45RA+ T cells

16. Summary At present moment our Pt’s intestinal problems (severe malabsorbtion) occupy the first place. Since 2005 Pt has had 8-12 times stool in a day. Most days of his life he spends in our Hospital.The Pt moves in hospital room with great difficulties (severe osteoporosis).He has stopped to grow since 2005 and has poor quality of life.-- According to literature data our patient may have CVID (severe phenotype) but usually such patient’s respond to IV Ig replacement therapy, treatment with steroids etc.-- Severe clinical data is a full manifestation of CVID in first age-specific period or complication of rituximab administration?-- We observed B-cells populations disturbance in BM of patien’t with CVID before but have never seen absolute absence of CD19+ in BM ?-- We do not know if it is possible to regarded that synonymous homogenic SNP in exon 2 : rs8072293 C/T formed such severe phenotype?-- It is possible to consider BMT as curable therapy?

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