Reversible Secondary Myelofibrosis or Clonal Myeloproliferative Disorder
Reversible Secondary Myelofibrosis or Clonal Myeloproliferative Disorder
To determine the prevalence of myelofibrosis in patients with primary pulmonary hypertension,
 and to discover if the fibrosis in these patients is primary (AMM) or secondary.
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