PNH Research and Support Foundation | Aplastic Anemia & MDS International Foundation Return to top.

PNH Research and Support Foundation

Sushant Kumar, PhD

Pubmed Author Name: 
Kumar S
Grant Year: 
2022
Original Research Center: 
University of Pennsylvania Perelman School of Medicine
Research Title: 
The mechanisms of immune evasion by PNH clones in aplastic anemia
Summary: 

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare blood disease in which blood cells are destroyed, leading to anemia, fatigue, and increased risk of blood clots. While several effective therapies can prevent the destruction of PNH red blood cells by blocking abnormal activation of a part of the immune system called “complement”, these treatments are life-long, extremely costly and do not lead to cure. In many places around the world, access to PNH treatments is limited. The main barrier to developing curative therapies for PNH has been a poor understanding of why PNH patients develop large outgrowths (“clones”) of PNH cells. Interestingly, most healthy individuals also harbor a few, very rare, isolated PNH cells, but these do not outgrow normal cells and do not turn into PNH clones, nor cause PNH disease The main risk factor for developing PNH is an autoimmune blood disease called aplastic anemia, in which nearly half of aplastic anemia patients develop PNH clones. The close association of aplastic anemia and PNH led to the current leading theory in the field that PNH cells outgrow normal cells because they escape autoimmune attack in aplastic anemia. However, how this occurs continues to be a matter of active debate in the field. In recent studies, we observed differences in how intracellular proteins in PNH cells compared to healthy cells are presented for immune surveillance. In the proposed studies, we will perform a comprehensive analysis of how intracellular proteins are processed and presented for immune recognition by PNH cells. We will also test for PNH cell immune evasion from T lymphocytes from aplastic anemia patients. The results of these studies will bring us closer to understanding why PNH disease develops. If successful, these studies will open the door to the future development of more effective, targeted therapies to prevent the onset of PNH and to eradicate PNH clones.

First Year Report: 
Final Report: 
Current Position/Title: 
Postdoctoral Researcher
Current Institution: 
University of Pennsylvania Perelman School of Medicine

Anastasios Karadimitris, MD, PhD

Pubmed Author Name: 
Karadimitris, A
Grant Year: 
2015
Original Research Center: 
Imperial College London
Research Title: 
CD1d-restricted, GPI-specific T cells in paroxysmal nocturnal hemoglobinuria
Summary: 

Our proposed research addresses one of the main and unresolved issues in the biology of paroxysmal nocturnal hemoglobinuria (PNH) and idiopathic aplastic anemia (IAA). Both disorders are considered to have an autoimmune mechanism involving auto-reactive T cells targeting hematopoietic stem cells (HSC) leading to their profound depletion. It is widely documented that the pathogenesis of IAA and PNH overlap to a great extent, with up to 50% of patients with IAA developing a PNH clone or bonafide PNH and a subgroup of patients with PNH evolving into IAA. The proposed work has the potential to unravel novel cellular and molecular mechanisms that underpin HSC depletion in PNH and IAA, with wider implications for both the hematology and immunology scientific communities.

First Year Report: 

Paroxysmal nocturnal Haemoglobinuria (PNH) is a bone marrow failure  disease in which there is a severe depletion of haematopoietic stem cells (HSC). This is compounded by exquisite sensitivity of red blood cells to the destructive effect of a complex molecular machine called complement, hence the anaemia and the propensity to developing blood clots. Eculizumab prevents the deleterious effects of complement, improves the anaemia and protects from blood clots.

However how depletion of HSC comes about is not known. We previously suggested that self-aggressive  T cells carrying a molecule called invariant T cell receptor (iTCR) can target a molecule called GPI and this interaction might be responsible for depletion of HSC. Towards addressing this idea we have employed advanced genetic tools to generate T cells that carry the candidate iTCR as well as haematopoietic cells in which presence of GPI and of CD1d, another molecule that the iTCR is expected to interact with on the target cells, has been altered.
 
With these at hand we will be able to determine whether the iTCR-carrying T cells can target and harm haematopoietic cells that express CD1d and GPI but not  cells that don’t express either or both. The outcome of these experiments will be crucial for understanding how depletion of HSC in PNH comes about.

Chao-Yie Yang, MD, PhD

Pubmed Author Name: 
Yang Chao-Yi
Grant Year: 
2014
Original Research Center: 
University of Michigan
Research Title: 
Discovery of small molecule complement inhibitors as the treatment for PNH
Summary: 

Uncontrolled complement activation caused by a gene (PIG-A) mutation in hematopoietic stem cells has been characterized in PNH. Patients suffer from hemolysis, thrombosis and bone marrow failure. Current FDA-approved only treatment for PNH is eculizumab which is expensive, unable to eradicate PNH clone, not orally available, requires clinic-bound infusions via iv access and lifelong therapy. In this proposed work, we will discover and make rational designs to small molecules guided by protein structures to develop complement inhibitors. The promising small molecule inhibitors identified from this work will be evaluated and used to develop orally-available therapeutics to treat PNH patients.

First Year Report: 

Uncontrolled complement activation caused by a gene (PGI-A) mutation has been characterized in PNH. Current only FDA-approved treatment for PNH is eculizumab which is expensive, unable to eradicate PNH clone, not oral available and requires lifelong administration in clinics. Another molecule (AMY-101) based on the discovery of compstatin has received orphan status in Europe and US for treatment of PNH in 2014. AMY-101 inhibits C3, a key component of the complement activation system responsible for the cause of PNH, and the use of AMY-101 is a new therapeutic strategy to treat PNH. First in-human trial of AMY-101 will start in 2015. In preclinical study, AMY-101 was administered in monkey via intravenous injection. Currently, no oral drug is available to treat PNH. In this proposed work, we will discover and develop small molecules to inhibit complement activation with the guide of protein structures information. Promising small molecules identified from this work will be used to develop oral-available drugs for clinical development to treat PNH patients.

For the first part of the project, we first studied the structure of compstatin and generated a structural model recapitulating the important functional groups in compstatin. Using the structural model, we screened against 514957 compounds available from a commercial compound library to select a smaller set of 76800 compounds using a computer program. To determine which of the 76800 compounds fit well to C3c and capture the important interaction similarly to compstatin, we performed a second computational docking simulation which finds the best way each molecule fits into the C3c. The computer program also provides theoretical estimates of the potency of each compound to C3c. Finally, we selected 24 compounds for biochemical evaluations. Currently, we are waiting for the collection of patients’ blood samples to validate these compounds. We will use the same workflow to continue screening against millions of other small molecules libraries. This will be a cost-effective approach to discovery new candidate compounds. Once we confirmed that the compounds bind to C3 with good potencies, we will select the best candidates for additional inhibitor optimization.

A second therapeutic strategy proposed in this work is to target another protein, Factor D, that controls the complement activation via the alternative pathway. We first experimentally screened against the Factor D using 2670 fragment compounds to identify novel compounds that bind with Factor D. These fragment compounds are generally small in sizes but have great potentials to be chemically modified into potent molecules that can be made orally bioavailable. This screening allowed us to identified 11 promising candidates including several new classes of compounds that were not reported previously. Using the information of the new molecules identified from our screening experiment and the templates from Novartis’ inhibitors, we have designed new classes of Factor D inhibitors based on computer-aided drug design method. The computer-aided drug design method allows us to evaluate if our proposed chemical modifications of the small molecules will be compatible with the pocket in Factor D and set priority of which modifications should be tried first. With the assistance of the computer-aided design, we can make the drug design process more efficient as opposed to the traditional trial-and-error medicinal chemistry approach.

In the first year of this project, we have constructed critical computational models, established experimental biochemical tools to evaluate the selected compounds, discovered novel chemical molecules used for drug design, and devised a computer-aided drug design workflow. New compounds have been synthesized and will be evaluated using PNH patients’ blood samples. We anticipated that we will obtain encouraging data in the second year and publish the results to the research community to benefit the therapeutic development for treating this rare blood disorder.  

Final Report: 

Uncontrolled complement activation caused by a gene (PGI-A) mutation found in Paroxysmal nocturnal hemoglobinuria (PNH) is a main cause for intravascular hemolysis. Though effectively reducing hemolysis in the majority of PNH patients, the treatment cost of eculizumab (an antibody to C5 which is a downstream component of the complement system) remains a significant financial burden. Another molecule (AMY-101) based on the discovery of compstatin has been approved in Europe and US to treat complement related orphan diseases. Specifically, AMY-101 inhibits C3, a key upstream component of the complement activation, and is approved to treat C3 glomerulopathy in 2015. Its clinical use to treat PNH is still under investigation. Emerging and vibrant drug development activities in complement-targeted therapy in the past few years are encouraging and welcoming news for PNH patients awaiting affordable and effective treatments. Administration of eculizumab and AMY-101 to patients are via intravenous injection performed by healthcare professionals at the hospitals. No oral drug is currently available to treat PNH patients who require a long-term therapy in clinics. Our goal in this project is to discover small molecules that inhibit two upstream component proteins regulating the complement activation. Unlike eculizumab and AMY-101, these molecules can potentially be advanced into oral drug development to treat PNH.

In the first aim of the project, we have searched 514957 compounds available from a commercial compound library to select a smaller set of 76800 compounds that match with some chemical groups of compstatin using a computer program. The compounds were then subject to computational docking calculations which find the best way each molecule fits into the C3c protein. The computer program also estimates how strong each compound interacts with C3c in theory. Using compstatin (a known C3c inhibitor) as a reference compound, we have selected 24 compounds for biochemical evaluations. This is a cost-effective approach to discovery new candidate compounds for biological evaluation from large of amount of molecules available but difficult to be tested in a reasonable time. We plan to adopt this approach to continue searching for best candidates for follow-up inhibitor development.

In the second aim, we developed compounds to inhibit Factor D that controls an important pathway to affect the rate of complement activation. Similar to Aim 1, we screened commercially available compounds using computer programs and discovered two new molecules confirmed by our experimental validation. We have also successfully obtained crystal structures that provide atomic details of how three other compounds inhibit Factor D. The structural information provides critical guidance when we designed and modeled four novel different series of compounds. We are currently evaluating their activities that will provide feedback to our drug design pipeline. In addition, we have determined that our compounds have favorable stability profiles in mouse liver enzymes. This indicates that good amount of the compounds can enter the blood stream after intestine absorption through oral administration in mouse. Follow-up evaluation in human liver enzymes will be performed. We have also validated that these compounds inhibit complement activation in the rabbit blood cells induced by human serum. Our data showed that our compounds effectively inhibit Factor D and its associated activation of the complement system. Evaluation of these inhibitors in the PNH patients’ blood sample is planned and will be conducted in the near future.

We have established a drug design platform involving a strong team of scientists through the support from AA&MDSIF. We have made a significant progress especially in the Factor D inhibitor development and have presented our findings in the 2016 Bone Marrow Failure Disease Scientific Symposium hosted by AA&MDSIF. We received positive feedbacks from physician scientists working to treat PNH. We also submitted a manuscript documenting our results to benefit the research community. Additional research supports from our institution have been applied based on these data. We will continue to work on these projects initiated by the support from AA&MDSIF support and contribute to the research community dedicated to the therapeutic development for treating this rare blood disorder.

Patrizia Ricci, PhD

Pubmed Author Name: 
Ricci P
Grant Year: 
2014
Original Research Center: 
University of Naples, Italy
Research Title: 
Small peptide C3-inhibitors for the treatment of paroxysmal nocturnal hemoglobinuria: moving towards the bedside
Summary: 

PNH is a rare hematological disease characterized by spontaneous destruction of red blood cells (intravascular hemolysis), poor functioning of bone marrow, and thrombosis. Recently, the availability of the complement inhibitor eculizumab has dramatically improved the treatment of PNH. Nevertheless, about half of the patients treated with eculizumab shows a persistence of clinical signs of the disease. We have recently described the reasons underlying the limited efficacy of current anti-complement treatment for PNH. With this proposal we aim to complete the pre-clinical development of a novel complement inhibitor which targets early events in complement activation, the component-3 of the complement cascade.

First Year Report: 

PNH is a rare hematological disease caused by a defective expression of regulatory proteins on the surface of blood cells, leaving them vulnerable to complement attack. This can lead to premature death of the red blood cells, a process called hemolysis, which results in severe anemia and contributes to a high risk of clotting. The availability of the anti-C5 complement inhibitor Eculizumab has dramatically improved the treatment of PNH; however, since a substantial proportion of patients continue to show sign and symptoms of the disease, there is a room for improving the current anti-complement treatment of PNH.

To date, clinical translation plans have been started with the fusion protein TT30 only. However, pre-clinical data with other agents seem robust enough to support clinical translation with other second-generation complement inhibitors that, by blocking early phases of complement activation, should result in a better control of both intravascular and extravascular hemolysis in PNH patients. This is the case of compstatin analogs, and in particular of the derivative AMY-101, which has shown in vitro a complete abrogation of intravascular hemolysis of PNH erythrocytes, as well as a full prevention of their C3 opsonization. Combined with the excellent bio-availability and the safety profile demonstrated in non-human primates, these data pave the way for forthcoming human studies.

In our proposal, we aimed to complete the pre-clinical investigation of a novel class of peptides which target the complement system at the level of C3, the key component of the complement. 

The inhibitors tested are all derivatives of compstatin, an agent that prevents cleavage and activation of the complement protein C3; in particular, our data support a clinical translation with the second generation compstatin analog Cp40 (AMY-101). 

In the first year of our research, we have definitely confirmed the efficacy of Cp40 in an in vitro model of PNH, providing strong evidence that the compound prevents the destruction of red blood cells obtained from PNH patients. We also demonstrated that pharmacological levels of Cp40 were able to prevent the modification of PNH red blood cells (e.g., the decoration with the complement component 3) which usually impair the efficacy of Soliris in a substantial proportion of PNH patients. 

These in vitro data were accompanied by extensive investigations in non-human primates, which ruled out possible side effects, and provided detailed information about the pharmacokinetics and the pharmacodynamics of Cp40 in vivo. Indeed, a single intravenous injection of PEG Cp40 resulted in

a prolonged elimination half-life of >5 days but may potentially affect the plasma levels of C3. Despite faster elimination kinetics, saturating inhibitor concentration could be reached with unmodified Cp40 through repetitive subcutaneous administration. It was important to demonstrate that sustained plasma inhibitor levels could be achieved even with unmodified Cp 40 when ijected subcutaneosly into non-humane primate in a multi-dose regimen (at 12h-intervals). These findings suggest that Cp-40 may prove a valuable option for long-term systemic treatment of PNH patients potentially even allowing for self-administration by the patient via a subcutaneous formulation.

According to our data, the inhibition of the complement cascade using small inhibitory molecules like AMY-101 would be a better strategy to prevent hemolysis and immune cell recognition while being potentially more cost-effective. Our pre-clinical data are almost complete to lead to the design of clinical trials for human subjects, including PNH patients. In the meantime, based on the potential benefit of this treatment over current standard therapies for PNH, together with Amyndas (a small biotech Company based in Europe) we have applied for the status of Orphan Drug to competent authorities. Indeed, without further discussion AMY-101 has been granted AMY-101 with the Orphan Drug Designation for the treatment of Paroxysmal Nocturnal Hemoglobinuria (PNH) in July 2014 by the European Medicines Agency (EMA), and more recently by the U.S. Food and Drug Administration (FDA).

This achievement may be useful to speed the clinical translation of AMY-101, eventually leading to first-in-human studies before the end of 2015; in addition, the Orphan Drug Designation may expedite the clinical development, and possibly a future marketing authorization for PNH patients.

Final Report: 

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare hematological disease caused by a defective expression of regulatory proteins on the surface of blood cells, leaving them vulnerable to complement attack. This can lead to premature death of the red blood cells, a process called hemolysis, which results in severe anemia and contributes to a high risk of clotting. The availability of the anti-C5 complement inhibitor Eculizumab has dramatically improved the treatment of PNH; however, since a substantial proportion of patients continue to show sign and symptoms of the disease, there is a room for improving the current anti-complement treatment of PNH.

Several novel anti-complement agents are currently in pre-clinical or clinical development; our project aimed to complete the pre-clinical development of the lead analog of a novel class of peptides targeting the key component of the complement cascade C3. In our hands, Cp40/AMY-101 showed an optimal efficacy in vitro, as well as a favorable toxicology profile in animal studies using non-human primates. In comparison to other anti-complement agents in development, Cp40/AMY-101 anticipates a better efficacy for the treatment of PNH because it should prevent the destruction of red blood cells (hemolysis) occurring via different mechanisms (intravascular hemolysis and extravascular hemolysis), possibly resulting in a substantial benefit for PNH patients. With the completion of the pre-clinical work, Cp40/AMY-101 is currently under investigation in human trials, starting with a phase I study in human volunteers. The further clinical translation program of Cp40/AMY-101 in PNH patients is also supported by another AAMDS grant awarded to Prof. Risitano.

Current Position/Title: 
Department of Clinical Medicine and Surgery
Current Institution: 
University of Naples, Italy

Hideki Makishima, MD, PhD

Pubmed Author Name: 
Makishima, H
Lead Photo
Grant Year: 
2013
Original Research Center: 
Cleveland Clinic Foundation
Research Title: 
Clonal Architecture in PNH: Somatic genetic defects facilitating clonal expansion
Summary: 

PNH is a disease in which a mutation in the gene called PIG-A is acquired in the stem cells (mother cells of all blood cells) in the bone marrow of patients.  As a result the blood cells produced by this stem cell are defective.  While previous discover of the PIG-A gene mutation has helped to explain the symptoms in the disease, it remains unclear how PIG-A mutation makes the PNH stem cells outcompete healthy stem cells.  In this project we propose to apply a very efficient sequencing technology to examine all genes in PNH stem cells to see whether additional mutations will explain how PNH develops.  In the initial experiments we have identified such additional mutations.  They may help to devise treatments to eradicate PNH stem cells from the patient’s bone marrow. 

First Year Report: 

Paroxysmal nocturnal hemoglobinuria (PNH) is a blood disease with chronic clinical course. PNH has been considered a genetically simple disease caused by PIG-A gene mutations.  However, according to our next generation search of whole human gene (in comparison between PNH cells and normal blood cells), various kinds of other genetic abnormalities were clearly found in addition to the acquisition of PIG-A mutation. Moreover, we discovered multiple (2 to 3) PIG-A mutations in the different PNH cells from the same PNH patients. Based on such novel results we are first to elucidate the variations of causes and explain the differences in symptoms of PNH for individual patients. For example, newly identified some genetic abnormalities in this project had been already commonly reported also in myelodysplastic syndromes, which is more aggressive blood disease. It makes sense since a part of PNH cases show more aggressive clinical conditions than the other PNH patients. In this first-year progress report, we mention novel findings of new genetic abnormality in addition to PIG-A mutations in PNH. Deeper research for discovered genes in more PNH cases will confirm these conclusions for the feedback to better patients’ care in the clinic. More details of this project were presented in 55th American Society of Hematology Annual Meeting @New Orleans, LA.

Current Position/Title: 
Project Staff, Department of Translational Hematology and Oncology Research
Current Institution: 
Cleveland Clinic Taussig Cancer Institute

Jeffrey Pu, MD, PhD

Pubmed Author Name: 
Pu, J
Lead Photo
Grant Year: 
2012
Original Research Center: 
Pennsylvania State University School of Medicine
Research Title: 
Relevance of PIG-A mutations in acquired bone marrow failure diseases
Summary: 

Acquired bone marrow failure diseases include myelodysplastic syndrome (MDS), acquired aplastic anemia (AA), and paroxysmal nocturnal hemoglobinuria (PNH). PNH is a clonal disorder originating from a multipotent hematopoietic stem cell (HSC) acquiring a PIG-A gene mutation. PIG-A mutations lead to the absence of glycosylphosphatidylinositol-anchor proteins (GPI-AP), which contributes to many manifestations of PNH. About 25% of MDS patients and 60% of AA patients also harbor small populations of PNH-like cells (0.01-10%). It was observed that: 1) 10-20% of AA patients harboring PNH-like cells eventually transform into PNH, but MDS patients seldom evolve to PNH; 2) AA patients harboring PNH-like cells may have a better response to immunosuppressive therapy; 3) PIG-A mutation significantly increased in some human cell lines with genomic instability. However, the clinical significances of these PNH-like cells in MDS patients and AA patients are still unclear. Our preliminary data from a pilot study showed that PIG-A mutations in AA arise from multipotent HSC; PIG-A mutations in MDS are initiated at progenitor level and are transient. The PIG-A mutation frequency in MDS patients harboring PNH-like cells were 10 to 100 times higher than healthy controls. Those MDS patients harboring PNH-like cells rapidly transformed into acute myelogenous leukemia. This AA&MDS Research Grant will allow us to test our hypotheses that 1) PIG-A mutations in MDS arise from progenitors and PIG-A mutations in AA arise from multipotent HSC, this may explain why AA often evolves into PNH, but MDS seldom transform into PNH; 2) PIG-A mutant cells in MDS is a marker of genomic instability and may predict a risk of leukemic transformation. This AA&MDS Research Grant will provide us much needed fund to further explore the clinical significances of those small populations of PNH-like cells in acquired bone marrow failure diseases.

First Year Report: 

Bone marrow is a sponge-like tissue inside the bones. Most blood stem cells sit in bone marrow and are capable to differentiate into red blood cells, white blood cells, and platelets. Bone marrow failure occurs when stem cells are suppressed or damaged. Diseases such as myelodysplastic syndrome (MDS), acquired aplastic anemia, and paroxysmal nocturnal hemoglobinuria (PNH) are acquired bone marrow failure diseases. PNH arises from a stem cell that acquires PIG-A gene mutation. PIG-A mutation further interferes with biosynthesis of an important cell surface molecule called glycosylphosphatidylinositol (GPI) and make cells vulnerable to complement attack. Acquired aplastic anemia is usually caused by an immune attack on bone marrow stem cells, which makes bone marrow unable to produce enough red blood cells, white blood cells, and platelets. Immunosuppressive therapy is highly effective in treating patients with aplastic anemia. MDS is also a disease in which the damaged bone marrow stem cells fail to generate enough healthy blood cells. Because of their common clinical presentation, it is often hard to distinguish these disorders. For example, many patients with MDS and aplastic anemia also contain small populations of PNH-like cells in their blood. Moreover, 0.002% white blood cells in healthy individual are PNH-like cells; however, these PNH-like cells develop from more mature cells called progenitors, which are not able to reproduce themselves and therefore do not cause disease.

It has been found that some aplastic anemia can transform to PNH with time, and both PNH and aplastic anemia can progress to MDS, but MDS almost never leads to aplastic anemia or PNH. However, the clinical significances of these small PNH-like cells in MDS and aplastic anemia are still unclear. The goal of this project is to understand the relevance of PIG-A mutation in MDS patients and aplastic anemia patients, and to explore unique clinical implications of these small PNH-like cell populations in managing patients and in predicting disease progression/transformation. Our preliminary data suggest that the PIG-A mutations that occur in aplastic anemia are similar to those occurring in PNH in that they arise from stem cells. In contrast, the PIG-A mutations that occur in MDS arising from progenitors, similar to those found in healthy persons. This likely explains why MDS patients seldom, if ever, evolve into PNH. It suggests that finding a small PNH-like cell population in MDS patients is not a reliable surrogate for needing immunosuppressive therapy. In addition, the novel assays we have developed to detect PIG-A mutations may help distinguish MDS from aplastic anemia. More importantly, our preliminary data reveal that harboring small population of PNH-like cells in MDS may be a marker of genomic instability and may predict leukemic transformation. We are working hard to recruit more eligible patients to verify those preliminary results. We are expecting to translate those research findings into a clinical trial in the near future.

It was a very exciting year working on this project. We are looking forward moving into the second year of this study.

Current Position/Title: 
Assistant Professor
Current Institution: 
The Pennsylvania State University School of Medicine

Jeffrey J. Pu, MD, Ph.D. is an assistant professor of medicine at the Penn State Hershey Cancer Institute and Penn State College of Medicine. Prior to this position, Dr. Pu was a senior clinical/research fellow of Hematology at the Sidney Kimmel Comprehensive Cancer Center of Johns Hopkins University. He also did an NIH-supported experimental hematology fellowship and a clinical transfusion medicine/blood banking fellowship at the Lindsley F. Kimball Research Institute of New York Blood Center after finishing an internal medicine residency at Mount Sinai School of Medicine.

David Araten, MD

Pubmed Author Name: 
Araten, D
Grant Year: 
2012
Original Research Center: 
New York University
Research Title: 
Secondary mutations and thrombosis in PNH
Summary: 

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a disorder in which  bone marrow failure is an important feature. The bone marrow failure in PNH is similar or identical to that which occurs in aplastic anemia. However, PNH has a few other features, some of which are well understood and some of which are not . One of the features that is not well understood in PNH includes the  expansion of an abnormal stem cell clone in the bone marrow (called clonal expansion) which can by itself provide a very large percentage of the patient's blood cells. This clone has as its feature the presence of a mutation in a gene called PIG-A. Different patients have different types of mutations in this gene, but it is always in this gene.  It has long been suspected that there may be other genes with mutations, but with the exception of two patients reported years ago, no other mutations have been found in patients with PNH. The other feature that is not well understood is a tendency of the blood to form clots in patients with PNH. We have recently found 4 patients with PNH who have a mutation in genes other than PIG-A--  the JAK2 and the TET2 genes. Three out of these 4 patients have had blood clots, and all of the them have a relatively high number of platelets (the type of cell that starts the clotting process) as well as large percentage of cells that come from the abnormal clone. We are now planning, with the generous support of the Aplastic Anemia and MDS International Foundation, to test a larger number of patients for mutations in these two genes to see how often mutations in TET2 and JAK2 contribute to clonal expanion. We are also going to test the idea that clotting in PNH patients is related to the total number of platelets that come from this abnormal clone as well as the idea that mutations in genes such as JAK2 and TET2 may be driving up the number of abnormal platelets and therefore contributing to the risk of clotting.

First Year Report: 

Paroxysmal Nocturnal Hemoglobinuria can be thought of as a genetic disease because it is associated with mutations (a permanent change in the DNA sequence of a gene). However, the mutations in PNH are not inherited and are not passed down to the next generation. Rather, they are acquired, later in life, and they occur only in cells of the bone marrow, rather than in every cell of the body (as in inherited diseases). It is widely believed that aplastic anemia is the driving force that promotes the expansion of PNH cells in patients with this disorder. While it is understood that mutations in the PIG-A gene are the hallmark of PNH, mutations in other genes may be driving the expansion of PNH cells in some patients. We previously found 3 patients who had a mutation in a gene called JAK2. This prompted us to look at a larger set of patients (19 patients) and we did not find any additional patients with a JAK2 mutation. However, we have found one patient who has a mutation in a gene called TET2. Recently, we have found another unusual patient who has a JAK2 mutation and a mutation called a “BCR-ABL fusion” in addition to PNH. The mutations involving JAK2, TET2 and the BCR-ABL genes are involved in a set of conditions called myeloproliferative diseases (MPD). MPD is different from PNH (though there are some similarities), but MPD really seems to be just the opposite of aplastic anemia, in that patients with MPD typically have high blood counts rather than low blood counts. Our findings raise the possibility that in a small subset of patients with PNH, there are features of MPD rather than aplastic anemia and that this is driving the expansion of PNH cells in this subset of patients.

One feature that PNH does have in common with MPD is that there is a tendency to form blood clots inappropriately in both disorders. The patient that had the TET2 mutation as well as 2 of the 4 patients with the JAK2 mutation had serious clotting problems, and they tended to have much higher platelet counts than typically seen in patients with PNH. This led us to speculate that the concentration of platelets in the circulation that have the PNH abnormality may be the most important factor in determining who develops a blood clot. This number can be estimated from the platelet count and the percentage of PNH white cells. In the past there have been tests to identify platelets with the PNH abnormality directly by flow cytometry, but there have been some technical difficulties with this. We have recently had some progress in developing this assay using more recently developed reagents. We think that the technique we have developed will better allow us to address this interesting question as to why some patients with PNH develop blood clots and others do not.

Final Report: 

PNH is a benign disease that is closely related to aplastic anemia but only rarely progresses to other more serious bone marrow diseases. The other diseases that PNH will very rarely turn into include myelodysplasia, leukemia, and myeloproliferative diseases. We wanted to determine whether two genes that are very commonly mutated in these more serious diseases—TET2 and JAK2—are commonly mutated in PNH. We looked only at patients who had large PNH clones. Although we had previously found the JAK2 mutation in rare patients with PNH, in a larger set of patients we did not find any with this mutation. We did find one patient with a mutation in the TET2 gene, out of 19 patients with large PNH clones. This one patient was interesting in that almost all of the cells had the PNH abnormality but the TET2 mutation was present in a much smaller percentage of her white cells and was detectable in a fraction of red blood cells that had retained their nucleus. By this analysis, we could determine that the TET2 mutation was not necessary for the establishment of the PNH clone. Rather, we believe that bone marrow failure, such as in aplastic anemia, selects for PNH clones without second mutations being critical for the development of the disease . These findings suggests that these mutations that commonly drive leukemia and other more serious blood diseases are only rarely important in driving the expansion of PNH cells in patients with large PNH cell populations.

We are also interested in developing a test to determine the percentage of platelets in patients with PNH that have the same abnormality as the red cells and granulocytes. This is important because we do not understand why some patients with PNH develop blood clots and we suspect that it may be related to the number of circulating PNH platelets. We have found that a combination of the FLAER reagent and anti-CD59 as well as a special technique for separating the platelets from blood samples may allow a routine measurement of PNH platelets, something which previously has been technically difficult. We have been able to overcome previous difficulties by treating the platelets with aspirin, and by using a gel that separates them from the clotting proteins, and both of these steps helps prevents the platelets from sticking together. We have analyzed patients with various size PNH clones, including those with aplastic anemia and PNH as well as normal samples, and we have found that the percentage of PNH platelets often goes together with the percentage of PNH red cells and granulocytes, but not always. We compared patients who had clots with those who did not have clots. In a group of 16 patients who had had clots the percentage of PNH  red cells, white cells,  and platelets, on average was and 23% ,82%, and 65%. In a group of 32 patients who never had a clot, the percent PNH cells, on average 24% for the red cells, 86% for the white cells, and 76% for the platelets. We do not think that any differences between these two groups was significant.  We identified  two patients with almost no detectable PNH red cells and over 90% PNH white cells, an unusual situation that does not come up often. For these, the proportion of PNH platelets was over 90%, suggesting that they would be at risk of blood clots even though they did not have much break down of red cells. Both were had been on preventive medicines and neither had thrombosis. It is predicted that this technique may be useful for determining thrombosis risk in patients such as this. Studies are ongoing to determine the relationship between the results obtained with this technique and the risk of thrombosis and other features of patients with PNH and bone marrow failure.

Current Position/Title: 
Assistant Professor
Current Institution: 
New York University School of Medicine

Dr. David Araten is an assistant professor in the Division of Hematology at the NYU School of Medicine in New York City. Prior to holding this position, he worked as an assistant professor and instructor in hematology at Memorial Sloan-Kettering Cancer Center, also in New York City. His residency was in internal medicine at Columbia-Presbyterian Medical Center and received his medical degree from Harvard Medical School in Boston. Dr. Araten’s research has focused largely on PNH and he has published in a wide variety of peer reviewed journals on this topic.

Keith McCrae, MD

Pubmed Author Name: 
McCrae, KR
Grant Year: 
2011
Original Research Center: 
Cleveland Clinic Lerner Research Institute
Research Title: 
Circulating microparticles in PNH
Summary: 

Dr. McCrae's study explores why patients with PNH are at increased risk for the development of thrombosis, or blood clots, that may affect arteries or veins and cause events such as pulmonary emboli or stroke.

First Year Report: 

One mechanism that may contribute to the development of thrombosis in PNH is the formation of microparticles. Microparticles are small cell-derived membrane fragments that are constitutively released from cells, but released in increased amounts as a consequence of cellular activation or damage. Several studies have reported that elevated levels of microparticles circulate in patients with PNH. Unlike previous studies, we are examining the levels of circulating microparticles in freshly-obtained blood samples (within two hours of sample collection) from patients with PNH. This is important as we have found that freeze/thawing of microparticles can significantly and unpredictably alter microparticle levels compared to fresh samples. To date, we have examined the microparticle levels in eleven PNH patients and 29 normal controls. We have analyzed the levels of circulating endothelial cell, platelet, monocyte, and red blood cell-derived microparticles and also examined whether these microparticles express tissue factor.

Our preliminary studies suggest that red blood cell-derived microparticle and tissue factor levels may be higher in PNH patients compared to normal controls. The endothelial cell, platelet, and monocyte-derived microparticle levels do not appear significantly different from control levels. This is an unexpected finding, but must be considered in light of the fact that all but one of our PNH patients have been treated with eculizumab (Soliris®), which would be expected to prevent complement-mediated cellular damage and minimize microparticle release. It is indeed interesting that in many several PNH patients, microparticle numbers appear lower than the levels detected in normal controls. We hypothesize that this is due to the fact that the alternative pathway (AP) of complement activation is constantly activated even in normal individuals, and inhibition of this pathway by eculizumab actually reduces AP activation to a level below that in control patients. Further analysis and collection of samples, as originally proposed, is needed to refine these conclusions.

We are also currently examining whether circulating microparticles in PNH patients express tissue factor. Tissue factor plays a critical role in the initiation of coagulation and thrombosis. During the past year, we have developed methodology for measuring tissue factor on microparticles. Preliminary data suggests that tissue factor is indeed expressed in the circulating microparticles. We predict that patients with the highest microparticle tissue factor expression will be at greatest risk for the development of thrombosis. It is possible that measurements of this nature may be of use in dictating dosing intervals of eculizumab. Again, further analysis is warranted before conclusions can be drawn.

Current Position/Title: 
Staff
Current Institution: 
Department of Cell Biology. Cleveland Clinic Lerner Research Institute

Originally from Maine, Dr. McCrae earned his undergraduate degree from Dartmouth College and his MD degree from Duke University. After his residency in Internal Medicine at Duke, Dr. McCrae completed his fellowship in Hematology/Oncology at the University of Pennsylvania. During this time, he did a postdoctoral research fellowship that initiated his interest in studying the biology behind antiphospholipid syndrome (APS), a clinical disorder characterized by blood clotting in both arteries and veins and recurrent fetal loss.

Share with addtoany.com.