Dr Tiu

Paroxysmal Nocturnal Hemoglobinuria Sharing Hope NORD/ PNH Regional Meeting

Ramon V. Tiu, MD Assistant Professor of Molecular Medicine Cleveland Clinic Taussig Cancer Institute March 1, 2014 San Antonio, Texas

Objectives 1. Overview of PNH • Historical Background, Epidemiology of PNH, Pathogenesis of PNH, Clinical Manifestations in PNH, Classification of PNH

2. 3. 4. 5. 6. 7.

Diagnosis of PNH Thrombosis in PNH Treatment of PNH Other Long term complications of PNH Updates in PNH Other Issues in PNH

Overview of PNH

Historical Background (selected timelines)

• 1866 – Dr. William Gull published important observations on the clinical feature of PNH but did not distinguish it from paroxysmal cold hemoglobinuria • 1882 – paper of Dr. Paul Strubing that clearly described PNH and distinguished it from paroxysmal cold hemoglobinuria and mrch hemoglobinuria • 1939 – Dr. Thomas Hale Ham reported evidence that complement mediates abnormal lysis of PNH RBCs in acidified serum • 1950s – Louis Pillemir discovered the alternative pathway of complement • 1993 – Kinoshita and colleagues found that PNH is a consequence of mutant PIG-A gene Parker CJ. Hematol Am Soc Hematol Educ Program.2008

Overview of PNH

Epidemiology of PNH

• A type of bone marrow failure syndrome • A rare disease (1-2 per million annual rates)

• However mortality rates in this disease is high

Overview of PNH

Genetic defect in PNH and effect on disease phenotype

• Somatic mutation in the PIGA gene, rarely mutation in PIG-T gene • This leads to loss in GPI link proteins which anchors important proteins to the surface of cells • This results in absence of CD55 and CD59 which makes the RBC prone to hemolysis

Krawitz Blood 2013. Luzzatto L Blood 2013;122:1099-1100

Overview of PNH

The Complement Pathway

Jozsi M. Book Chapter. An Update on Glomerulopathies.

Overview of PNH

Classification of PNH

3 groups 1. Classic PNH 2. PNH in the setting of a BM disorder (MDS, AA, MF) 3. Subclinical PNH – very small GPI negative clones with no obvious clinical significance (In this context, using the term PNH may be misleading since none of the clinicl manifestions of this condition are present. Some have proposed the term “acquired GPI-AP deficiency.”

Parker CJ et al. Blood. 2005; Risitano A & Rotoli B. Biologics: targets & Therapy. 2008

Overview of PNH • • • • • • • • •

Clinical Manifestations of Patients with PNH

Thrombosis Fatigue Dysphagia Abdominal Pain Erectile dysfunction Pulmonary HTN Pain symptoms Other symptoms related to Anemi Other symptoms related to thrombocytopenia or neutropenia if these abnormalities are present

Standard Diagnostic Test for PNH  Flow cytometry performed on peripheral blood

 Granulocytes and at least one additional cell line should be evaluated – Red blood cells (RBCs) – Monocytes  Quantitative results – Optimal-High sensitivity analysis: ≥0.01% – Routine analysis: ≥1%  Easy to understand PNH reports  Use more than one reagent against GPI-anchored proteins Borowitz MJ et al. for International Clinical Cytometry Society. Part B Clin Cytometry. 2010;78B:211-230.

9

Testing for PNH in Red Blood Cells Normal RBC’s with normal CD59 expression (Type I cells)

PNH clone with complete CD59 deficiency (Type III cells)

Gating on GPA+ RBC’s

PNH clone with complete CD59 deficiency (Type III cells) and partial CD59 deficiency (Type II cells)

GPA = glycophorin A. Data Source - Dahl-Chase Diagnostic Services.

10

Why Look Beyond RBCs for PNH?  Granulocytes provide more accurate representation of PNH clone size  Percentages of PNH RBCs may be affected by – Hemolysis – Blood transfusion PNH reports should provide quantitative results expressing clone size on both granulocytes and red blood cells

Borowitz MJ et al. for International Clinical Cytometry Society. Part B Clin Cytometry. 2010;78B:211-230.

11

PNH Patient With an 80% WBC Clone Size and 31% RBC Clone Size Indicating Hemolysis RBC

CD24- Granulocytes

WBC

FLAER- GPI Anchor Binding Marker

80.1 % of Granulocytes lack GPI proteins

Data Source - Dahl-Chase Diagnostic Services.

CD59 – GPI Anchored Protein

31.4% RBCs are Type III PNH cells

12

Important to Monitor Granulocytes and RBCs Over Time

FLAER-GPI Anchor Marker

Mar 09

FLAER-GPI Anchor Marker

May 09

CD24-Granulocytes

CD24-Granulocytes

CD14-Granulocytes

Dec 08

CD24-Granulocytes

CD24-Granulocytes

Sept 08

FLAER-GPI Anchor Marker

FLAER-GPI Anchor Marker

Gran clone: 3.8%

Gran clone: 7.6%

Gran clone: 14.2%

Gran clone: 23.3%

RBC clone: 0.8%

RBC clone: 1.6%

RBC clone: 1.8%

RBC clone: 2.4%

CD59 –GPI Anchor Protein

CD59 –GPI Anchor Protein

3 Months

CD59 –GPI Anchor Protein

CD59 –GPI Anchor Protein

6 Months 9 Months

PNH Clone Expanded in