Dr. Apuri

1. A case of thrombosis caused by
paroxysmal nocturnal
hemoglobinuria (PNH)
Susmitha Apuri M.D
Fellow, Hematology/Oncology
USF/Moffitt Cancer Center

2. Case
27 yr. woman presented to the ED with syncopal episode in June 2003. She reports
four month history of fatigue, worsening shortness of breath on exertion and easy
bruising. Four months ago, she was evaluated at a different ED for dysuria and treated
for urinary tract infection. She denies fever, chills, night sweats, abnormal bleeding or
weight loss.
Labs in ED : CBC- WBC- 3.2, ANC 1500, hemoglobin- 9.9 g/dl, reticulocyte count -
5.8%, platelet count- 51,000, haptoglobin - < 30, ESR-110
CMP- BUN- 13, Cr- 0.8, LDH -883. U/A- 4+ blood, 8-10 RBC
She received two units of PRBC and was referred to MCC for management
Medical history: no history of chronic medical problems or childhood illness
Surgical history: wisdom teeth extraction
Medications : oral contraceptives

3. Case
Family History: negative
Social history: migrated from Puerto Rico at the age of 6. She works full time
as a customer service representative.
Physical examination:
VITAL SIGNS: Weight is 56.9 kg. Temperature is 38.1. Heart rate is 66 and
regular. Respirations are 20. Blood pressure is 124/56
GENERAL APPEARANCE: This is a well-nourished woman, in no acute distress
SKIN: There is ecchymosis on the right upper extremity. There is also a 4 x
6-cm oval area of erythema, without warmth, on the right forearm, which the
patient corresponds to an injection of a medication

4. LYMPH NODES: No cervical, supraclavicular, axillary or inguinal
lymphadenopathy.
LUNGS: Clear to auscultation and percussion anteriorly and posteriorly.
CARDIAC: Regular rate, with a very soft systolic murmur heard throughout
the precordium. No gallop, thrill or rub.
ABDOMEN: Bowel sounds are present, soft and non tender. No
hepatosplenomegaly, mass or midline abdominal bruit.
EXTREMITIES: No cyanosis or clubbing at the fingertips. No peripheral
edema.
NEUROLOGIC: Alert and oriented to person, place and time.
LABS: July 2003
CBC : WBC – 0.33, H/H- 8.8/25.4, MCV- 96.1, platelet count – 18,000
CMP: BUN-9. Creatinine – 0.9, Haptoglobin <30, LDH – 1655, total bilirubin -1.7
PT – 9.7, INR – 0.9, APTT – 24.7
Urine analysis : large amount of blood, RBC : 6-10, WBC : 8-12, bacteria : 1+
Case

5. Sugar water test –positive. Coombs test – negative
August 2003
Bone marrow biopsy –
Hypocellular marrow with trilineage hypoplasia, consistent with aplastic
anemia.
COMMENT:
Section of bone marrow and clot shows a hypocellular marrow with trilineage
hypoplasia. The marrow cellularity is less than 5%. Myeloid and erythroid
precursors are markedly reduced. Megakaryocytes are rare. M:E ratio is
approximately 3:1. These features are consistent with aplastic anemia most
likely associated with PNH.
Flow cytometry –
CD45 positive, CD14 negative, CD13 positive, CD11C positive, CD3 negative,
CD4 weak. In addition, granulocytes show lack of CD59.

6. She was evaluated by the BMT team at MCC and HLA typing showed one
matched sibling. She chose not to undergo allogeneic stem cell
transplantation.
September 2003
She was started on immunosuppressive therapy with ATG, cyclosporine and
prednisone. Hemoglobin at discharge- 8.6 g/dl, platelet count - 49,000.
(hemoglobin was 7.5 g/dl and PLT count was 45,000 on admission and she
required PRBC, platelet transfusions)
November 2003
Presented to ED with pleuritic chest pain, tingling of the right upper
extremity and right facial paresis. Imaging demonstrated multiple infarctions
in the left cerebral hemisphere. She was started on Lovenox and therapy was
switched to aspirin and Plavix at MCC. Hypercoagulation work up was
negative. Ultrasound Doppler of bilateral upper and lower extremities were
negative. She was also advised to discontinue hormonal therapy for birth
control.
Clinical Course

7. December 2003
Flow Cytometry (peripheral blood)-Cytologically and phenotypically shows
significant residual PNH affected neutrophils with quantitation equivalent
to 72% CD59 negative, CD13 + neutrophils.
BONE MARROW, LEFT POSTERIOR ILIAC CREST, ASPIRATE, TOUCH IMPRINT,
BIOPSY AND
CLOT:
Mildly hypo cellular bone marrow (40%) with marked erythroid
hyperplasia with M:E ratio of 0.6 with megaloblastoid maturation and
relatively decreased megakaryopoiesis and granulopoiesis.
August 2004
CBC: WBC 1.91, hemoglobin 9.3, hematocrit 29.4%, platelets
88,000, ANC 1110, eosinophils 10, monocytes 160, lymphocytes 630.
CMP: BUN 12, creatinine 0.7, total bilirubin 1.4, alkaline phosphatase 62,
AST 82, ALT 18, LDH 5220
Clinical course

8. Clinical course
February 2005
Reports worsening fatigue and dark urine. White blood cell count was 2.4,
hemoglobin 10.2 (which has decreased approximately 1 g in a 1-week time
interval), and platelets are 89,000. LDH was 5434. Required 4 PRBC
transfusions in past year. She was started on Darbepoetin 200 mcg every 2
weeks.
April 2005
White blood cell count 2.1,hemoglobin 11.5, hematocrit 34, MCV 95.6,
platelet count 95,000
CMP: bilirubin 1.4, alkaline phosphatase 61, AST 74, ALT 14, LDH 1869
Developed recurrent transient left cerebrovascular accident with right
hemiparesis and was diagnosed with progression of internal carotid artery
vaso occlusive disease. Transfusion threshold was increased to hemoglobin of
9 g/dl.

9. Clinical course
August 2011
WBC 4.41, hemoglobin 10.3, hematocrit 31.4, MCV 99.1, platelet count
107,000
BUN 11, creatinine 0.6, calcium 9.5,total protein 9.0, albumin 4.9, total
bilirubin 1.6, alkaline phosphatase 104, AST 111, ALT less than 12, and LDH
6957
Flow cytometry:
Supports the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), with
GPI-deficient erythrocytes, monocytes and granulocytes. 53.43% of red cells
are type II cells (decreased expression), while 5.34% are type III cells (absent
expression). 92% of monocytes lack CD14 (PNH monocytes).
She was started on Eculizumab q14 days and receiving treatment for past 5
years.

10. Paroxysmal Nocturnal Hemoglobinuria (PNH)
• Acquired, somatic mutation in the phosphatidylinositol
glycan class A (PIG-A) gene
• Clonal, non-malignant hematologic disease
• Hemolytic anemia resulting from the clonal expansion of one
or a few abnormal hematopoietic stem cells
• Rare disorder, associated with aplastic anemia and
myelodysplastic syndrome (MDS)
Triad
• Hemolytic anemia
• Bone marrow failure
• Thromboembolism
Devalet B et al. European Journal of Hematology 2015;95(3):190–8.

11. CD59 (Membrane inhibitor of reactive lysis)
•Defensive shield for red blood cells against
complement-mediated lysis
•Inhibits the assembly of membrane attack
cells
):1–11.
, et al. Biologics: targets & therapy 2008;2(2):205.
CD55 (Decay Acceleration Factor)
•Prevents formation of C3 convertase
•Augments instability of the C3
convertase
•Attenuates complement cascade
Sharma VR, et al. Clin Adv. Hematology Oncol. 2013;11(9):1–11.

12. Devalet B et al. . European Journal of Hematology 2015;95(3):190–8.

13. Diagnosis
PNH should be confirmed with peripheral blood flow cytometry to
detect the absence or severe deficiency of GPI anchored proteins-
(CD55&CD59) on ≥ 2 lineages (granulocytes and RBCs).
Sharma VR, et al. Clin Adv. Hematology Oncol. 2013;11(9):1–11.

14. Hypercoagulable
State
• Nitrous oxide depletion
• Complement-mediated
platelet activation
• Exposure of the
procoagulant interior of the
red cell membranes
•Thrombosis is the leading cause of death (occurs in 40% of
patients)
• Venous > arterial. Atypical sites of venous thromboses such as
splanchnic , portal, cerebral and hepatic veins.
•Accounts for 40% to 70% of the mortality from the disease
•History of even 1 thrombotic event is associated with a 5-fold
to 10-fold higher risk of death
Sharma VR, et al. Clin Adv. Hematol Oncol 2013;11(9):1–11.
Risitano AM, et al. Biologics: targets & therapy 2008;2(2):205.
Devalet, B. Et al. EJH. 95 (190-198)
Sharma VR, et al. Clin Adv Hematol Oncol 2013;11(9):1–11.
Risitano AM, et al. Biologics: targets & therapy
2008;2(2):205.
Devalet, B. Et al. EJH. 95 (190-198)

15. Thrombosis in PNH
• Acute thrombotic events-
- Full dose anticoagulation with heparin therapy (aiming for anti-Xa
levels between 0.5 and 1.0 for those treated with low-molecular-
weight heparin) and the commencement of the monoclonal antibody
therapy with Eculizumab.
- Continuing anticoagulation with the vitamin K antagonists is generally
recommended in the long term if there are no contraindications
• No data on the use of antiplatelet therapy
Hill et al, Blood. June 20, 2013; 121 (25)

16. • Patients, especially those with a small PNH clone
associated with hematologic malignancies or those
with only mild symptoms should not be treated unless
thrombosis supervenes or symptoms are severe
• Treatment is reserved for transfusion-dependent
patients with severe hemolytic anemia and disabling
symptoms such as fatigue, thromboses, frequent
paroxysms of pain and/or end-organ damage.
Treatment

17. Devalet B et al . European Journal of Hematology 2015;95(3):190–8.
Treatment

18. • Open-label pilot study (n=11)
• Evaluate the effectiveness of Eculizumab in reducing the incidence of
intravascular hemolysis, hemoglobinuria and transfusion requirements in
patients with PNH
RESULTS:
• Eculizumab if safe and well tolerated in patients with PNH. It reduces hemolysis,
hemoglobinuria, transfusions and improves quality of life (QoL)

19. • Phase 3 double- blind RCT of Eculizumab vs. placebo in patients with PNH (n=87)
• Inclusion criteria:
- Men and women >18 year old with a diagnosis of PNH
- PNH Type III erythrocyte proportion of 10% or more, platelets ≥ 100 K and LDH ≥
1.5 x upper limit of normal (ULN)
- Received at least 4 RBC transfusions in the preceding 12 months
• Patients where stratified based on the number of pRBC transfusions during the past
year
• Primary endpoints: stabilization of Hemoglobin level and number of pRBCs
transfused during the study period (26 weeks)

20. Hillmen P et al. N Engl J Med 2006;355:1233-1243
Stabilization of Hemoglobin levels and
number of pRBC (units)transfused
Median time to first transfusion between
patients treated with Eculizumab and
patients treated with placebo.

21. Change of fatigue score from baseline
to week 26 between the two group.
Hillmen P et al. N Engl J Med 2006;355:1233-1243
Eculizumab in patients with PNH:
• Reduces or eliminates the need for transfusions
• Improves anemia, fatigue and improves QoL
• Effectively reduces intravascular hemolysis

22. SHEPHERD TRIAL- Blood 2008;111(4)1840-7
• Phase III non randomized ,open label clinical trial to evaluate the long term safety and efficacy
of Eculizumab in patients with PNH (n=97)
• Inclusion criteria:
- 18 years and above with a diagnosis of PNH for more than 6 months.
- At least 1 RBC transfusion in the past 2 years
- PNH Type III erythrocyte proportion of 10% or more, platelets ≥ 30,000 and LDH ≥ 1.5 x ULN
• Exclusion criteria:
- Patients with ANC < 500, active bacterial infection, known complement deficiency,
prior transplant, history of meningococcal disease or received a previous investigational
drug within 30 days of screening.
• Safety endpoints : Adverse events (labs, ECG and vital signs data).
• Primary endpoints: Hemolysis assess by LDH area under the curve (AUC)
• Secondary endpoints: fatigue and LDH change from baseline, QoL and thrombosis.

23. SHEPPERD Trial
• 97 patients were enrolled as the intent-to-treat population
• Median age: 41
• Median duration of PNH: 4.9 years
• Follow up : 52 weeks
• Transfusion requirements: 8 (0-66) units, LDH: 2051 (537-5245) U/L,
platelet count : 136 (23 – 355,000), hemoglobin : 93 (48-131) g/L,
history of thrombosis :42%
• Transfusion independence in 49 patients, 87% reduction in
hemolysis measured by LDH
• Most common adverse events (10% of patients): upper respiratory
infections, headache, nasopharyngitis, pyrexia
• Overall well tolerated

24. • Eculizumab increases risk of Neisseria sepsis serotype B
• Need to vaccinate against serotype A, C, W and Y . Type B vaccine is
approved in 2015
– 14 days prior to first dose
– Revaccinate every 3-5 years
• May consider vaccination for pneumococcal and influenza
Eculizumab

25. Stem cell transplantation
Indications
• Severe aplastic anemia with PNH clone in patients who
are young and have a suitable donor
• MDS
• PNH complications and:
- Severe symptoms with no access to Eculizumab.
- Resistance to Eculizumab (heterozygous c.2654G->A
mutation in C5) or intravascular hemolysis that persists
due to ongoing inflammation

26. Thank You