Reversible Secondary Myelofibrosis or Clonal Myeloproliferative Disorder

Pulmonary arterial hypertension: Pulmonary arterial hypertension (PAH) is a disease
primarily affecting the small precapillary pulmonary vessels. It is characterized by
sustained elevation of the pulmonary vascular resistance (PVR). Without therapy, right heart
failure and death eventually occur. PAH occurs in an idiopathic form, primary pulmonary
hypertension (PPH), and in association with other disorders such as connective tissue
diseases or congenital heart disease (CHD). PAH remains a disease of unknown etiology. Until
recently, prognosis for PAH was poor, with a median survival of less than 3 years.

Prostacyclin (PGI2) is the main product of arachidonic acid in all vascular endothelium. It
is a potent vasodilator in all vascular beds. In addition, it is a potent endogenous
inhibitor of platelet aggregation and smooth muscle growth. The prostacyclin receptor (IP)
is located on a variety of cell types, enabling prostacyclin to exert a range of biologic
actions by means of raising intracellular levels of cAMP through activation of adenylate
cyclase. The stable, freeze-dried salt preparation of prostacyclin is known as epoprostenol
and is available for IV administration under the brand name Flolan (Glaxo Wellcome, Research
Triangle Park, N.C.). The first randomized clinical trial in PPH showed that epoprostenol
improved quality of life, hemodynamics, exercise tolerance, and survival over a 12-week
period. Epoprostenol has become the standard of care for patients with advanced PPH.
Recently, chronic intravenous epoprostenol has been shown to be an effective therapy to
improve long-term quality of life and survival in patients with PPH. Epoprostenol also
improves hemodynamics, exercise capacity, and quality of life (but not survival) in PAH
associated with CHD and connective tissue disease.

Side effects of epoprostenol infusion include rash, headache, jaw pain, leg pain, diarrhea,
nausea, catheter infections, chest pain, anxiety dizziness, bradycardia, dyspnea, abdominal
pain, musculoskeletal pain, tachycardia, flu-like symptoms, and anxiety/nervousness.
Thrombocytopenia is commonly seen in patients with PPH treated with PG12. Occasionally, a
decrease in other blood cell lines occurs leading to anemia or pancytopenia. Splenomegaly
and hypersplenism are observed at times. While thrombocytopenia has been attributed to the
antiplatelet effects of prostacyclin, the pathophysiology of pancytopenia is poorly
understood. There are no studies that address thrombocytopenia and other hemopoietic
abnormalities in people with pulmonary hypertension receiving epoprostenol. The possibility
of myelofibrosis causing these hematologic abnormalities has not been investigated.

Primary myelofibrosis is a myeloproliferative disorder, characterized typically by
pancytopenia, splenomegaly, a leukoerythroblastic blood smear, fibrosis on the bone marrow
examination, and myeloid metaplasia. Like all myeloproliferative disorders, it is due to
somatic mutation and clonal proliferation of hematopoietic progenitors. Fibrosis occurs
secondary to cytokines secreted by malignant cells. These patients are symptomatic because
of cytopenias. Bone marrow failure and transformation to acute myeloid leukemia is the major
reason for mortality in these patients. Elevated blood levels of hematopoietic progenitors
(identified by positivity for the CD34 antigen) are well documented and correlates with
prognosis. Therefore two major identifying features of primary myelofibrosis as opposed to
secondary fibrosis of the marrow are: 1) clonal hematopoiesis and 2) high CD 34 positive
hematopoietic precursors.

We therefore hypothesize these two will be normal in patients with secondary myelofibrosis
and help us distinguish the two entities.

Clonality Studies: Clonality is based on the principal of X-chromosome inactivation and
thus, for clonality studies only females can be used as subjects. It is based on the
principal that while female cells have two X-chromosomes, one is randomly inactivated during
early embryonic development and thus female tissue is a mixture of cells expressing either
paternal or maternal inherited x-chromosome genes. In the first step, DNA analysis will
study 5 exonic polymorphisms for the X-chromosome genes which have previously been shown to
be useful for these purposes. If any of the five loci is heterozygous then the subject is
said to be informative for clonality. Studies which will proceed as follows: The fresh blood
platelets and granulocytes will be isolated and the RNA isolated and used later for reverse
transcription to cDNA which will then be analyzed for X-chromosome allelic usage ratio by
single-stranded conformational polymorphisms (SSCP). If only one allele is expressed in
platelets or granulocytes the patient is said to be clonal and favors a diagnosis of
myeloproliferative disorder (such as AMM), while polyclonal blood (both X-chromosome alleles
expressed in platelets and granulocytes) is compatible with secondary myelofibrosis.

A three-color direct immunofluorescent staining method will be used in the evaluation of
CD34+ cells. In our preliminary experience we observed four Flolan treated patients with PPH
who had splenomegaly, anemia and/or thrombocytopenia. All of these patients had severe
marrow fibrosis. The purpose of this study is to determine whether the incidence of the bone
marrow fibrosis in this patient population is primary or secondary, and whether a secondary
effect is a fibrosis that would be expected to be transient and resolve after
discontinuation of Flolan.

Participating subjects will Patients will undergo the following studies:

1. History and Physical examination

2. Peripheral blood smear

3. Bone marrow examination to determine the presence and severity of fibrosis. The bone
marrow specimen taken for diagnostic purposes will be examined by three independent
hematologists/hematopathologists in a masked fashion for the presence of fibrosis using
routine methods.

4. Blood: 30 cc of blood will be collected for

1. Clonality studies (female subjects only),

2. CD34 quantitation.

3. Further studies will be done to monitor the aberrant trafficking of hemopoietic
stem cells, the adhesion molecules, and any disease related genes, such as
JAK2V617F and cMPL, if indicated.

4. The above studies will be repeated in patients undergoing lung transplants at 3
months, 6 months and 1 year after transplantation.

Inclusion Criteria:

1. > 18 years of age

2. Signed Informed Consent

3. Subjects diagnosed with Primary Pulmonary Hypertension or Myelofibrosis (primary or
secondary)

Exclusion Criteria:

1. Anyone not meeting the above inclusion criteria

2. Pregnant women