• AFS Cells – Where Do They Fit On The Continuum? (Cover Story)
• Receptors, Not Clots: Coagulation and Fibrinolytic Enzymes Modulate Stroke Outcome by Targeting Endothelial Cells, Not Cerebral Thrombi
• Allogeneic Stem Cell Transplant Donors: Sibling or Stranger?
• Are TPO-Mimetics Better Than the Real Thing?
• Promise and Obstacles to Gene Expression Profiling in Multicenter Trials
• Prion Transmission by Blood Transfusion: The Report of a Third Infected Recipient, and the Second to Die With Variant Creutzfeldt-Jakob Disease, Intensifies the Focus on Preventative and Therapeutic Strategies
• Does It or Doesn’t It? Can Imatinib Eradicate the Stem Cell Clone?
• A Backhand to Complement

Receptors, Not Clots: Coagulation and Fibrinolytic Enzymes Modulate Stroke Outcome by Targeting Endothelial Cells, Not Cerebral Thrombi

Roy Silverstein, MD

Dr. Silverstein indicated no relevant conflicts of interest.

Cheng T, Petraglia AL, Li Z, et al. Activated protein C inhibits tissue plasminogen activator-induced brain hemorrhage. Nat Med. 2006;12:1278-85.

Using an in vitro brain endothelial cell model of ischemia in which cells were subjected to an environment of oxygen and glucose deprivation, these investigators showed that tissue plasminogen activator (tPA) induced increased expression and activity of MMP9, a matrix metalloproteinase that targets critical components of the blood-brain barrier. They also studied two rodent models of stroke, one induced by transient occlusion of the middle cerebral artery in mice and one by inducing cerebral thromboembolism in rats, and showed that tPA delivered systemically led to increased MMP9 expression and activity in the infarct zone along with significantly increased cerebral hemorrhage. In both the in vitro and in vivo models, MMP9 expression, infarct volume, and cerebral hemorrhage were dramatically and significantly inhibited by giving the animals or cells activated protein C (APC) along with or a few hours after the tPA. With inhibitory antibodies, short hairpin inhibitory microRNAs, and cells from genetically engineered mice they showed that the damaging effects of tPA required expression of the endothelial cell receptor LRP1 and that the protective effects of APC required expression of the endothelial cell receptors EPCR and PAR1.

Very little effective therapy is available to mitigate brain damage and tissue loss once a patient begins to experience symptoms of a stroke. Recombinant human tPA is of benefit, but only if given within a very narrow time window. Furthermore, enthusiasm for its use is tempered by increased risks of cerebral hemorrhage with catastrophic sequelae. These authors have used cell culture and rodent models to define a brain microvascular endothelial signaling pathway activated by tPA that is responsible for some of its untoward effects. They then showed that these effects could be prevented by concomitant delivery of APC. The major “problem” with tPA is apparently not related to clot dissolution or clot instability, but rather to its ability to bind and activate an endothelial cell receptor known as LRP1. This receptor then triggers a cascade of events including activation of the pro-inflammatory transcription factor NFκb, increased expression and activity of matrix metalloproteinase-9 (MMP9), and then MMP9-dependent disruption of the blood-brain barrier with subsequent hemorrhage. APC prevents hemorrhage in this setting by blocking NFκb activation and MMP9 up-regulation. This effect is apparently not due to its ability to slow thrombin generation by proteolytic cleavage of Factor V and VIII, but rather to activation of an alternative signaling pathway mediated by its two endothelial cell surface receptors, PAR1 and EPCR. This is consistent with recent studies from other groups describing the mechanistic basis of sepsis protection by APC and helps define a “new” paradigm by which coagulation and fibrinolytic enzymes mediate systemic and localized effects in the vasculature by acting as paracrine ligands for endothelial receptors, not by “busting” clots or blocking thrombin generation. From these studies emerge several exciting potential avenues for drug development, including simply adding APC (drotrecogin alfa/Xigris®) to tPA infusion during acute stroke. More appealing would be designing drugs that specifically target LRP1 and/or the PAR1/EPCR system without influencing the coagulation and fibrinolytic pathways.

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Allogeneic Stem Cell Transplant Donors: Sibling or Stranger?

Michael Williams, MD

Dr. Williams indicated no relevant conflicts of interest.

Yakoub-Agha I, Mesnil F, Kuentz M, et al. French Society of Bone Marrow Transplantation and Cell Therapy. Allogeneic marrow stem-cell transplantation from human leukocyte antigen-identical siblings versus human leukocyte antigen-allelic-matched unrelated donors (10/10) in patients with standard-risk hematologic malignancy: a prospective study from the French Society of Bone Marrow Transplantation and Cell Therapy. J Clin Oncol 2006;24:5695-702.

Allogeneic stem cell transplantation (SCT) provides a curative option for many patients with relapsed hematologic malignancies and for others with high risk of recurrence after initial therapy. Traditionally HLA-matched sibling donors were preferred as the source of stem cells, but the availability of such a donor in only about one-third of patients has led to the use of matched unrelated donors. This French multicenter study assessed the outcomes for patients with standard-risk hematologic malignancies undergoing allogeneic SCT from related versus unrelated donors, utilizing a uniform pre-transplant conditioning regimen of total body irradiation plus high-dose cyclophosphamide and uniform graft-versus-host disease (GVHD) prophylaxis with cyclosporin-A and short-course methotrexate for all patients. The study population included patients with acute leukemia in first or second complete remission, CML, and myelodysplastic syndrome. Bone marrow rather than peripheral blood was used as the source of stem cells for all patients. One hundred and eighty-one patients had an HLA-matched sibling donor, while 55 had an HLA-matched unrelated donor identical at 10/10 allelic loci. Patient characteristics in the two groups were largely comparable, and all but seven patients engrafted. With a median post-transplantation follow-up of 34.6 months, there was no significant difference in time to engraftment, acute GVHD, event-free survival, or overall survival (Figure). A retrospective review of patients in this trial showed increased occurrence of chronic GVHD in unrelated versus sibling transplants. Mortality was increased in CMV seropositive recipients, in those who developed >/= grade II acute GVHD, and in those with stem cell donors > 37 years of age.

This study supports the conclusion that curative outcomes for 10/10 HLA-matched unrelated donors are comparable to matched sibling donors for patients transplanted for standard-risk hematologic malignancies. Additional studies will be needed to determine the impact of alternative conditioning regimens, peripheral blood rather than marrow stem cells, related- versus unrelated-donor nonmyeloablative allogeneic SCT, alternative GVHD-preventive regimens, and the impact of these on and for higher-risk hematologic malignancies.

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Are TPO-Mimetics Better Than the Real Thing?

Charles Abrams, MD

Dr. Abrams is an ad hoc consultant to GlaxoSmithKline.

Bussel JB, Kuter DJ, George JN, et al. AMG 531, a thrombopoiesis-stimulating protein, for chronic ITP. N Engl J Med 2006;355:1672-1681.

This is a phase I/II trial that tests the hypothesis that inducing increased platelet production would be beneficial in ITP. AMG 531 binds and activates the thrombopoietin (TPO) receptor, but bears no structural similarity to TPO. Therefore, it is a TPO-mimetic and, in theory, should increase platelet production in man. In the first phase, 24 patients were exposed to two injections of escalating doses of AMG 531. Doses greater than 3 micrograms/kg induced an increase to some extent in platelet counts in all subjects. All doses up to 10 micrograms/kg were well tolerated. In phase II, 16 long-term ITP patients were enrolled in a double-blind, placebo-controlled trial of AMG 531. Patients were treated with six weekly subcutaneous injections of 1 or 3 micrograms/kg of the study drug. AMG 531 increased the platelet count to greater than 50,000/µl in twelve patients (a 75 percent response rate). Notably, two patients who received 3 micrograms/kg of AMG 531 had platelet counts that increased above 500,000/µl. Interestingly, there appeared to be no relationship between baseline TPO levels and platelet responses. This implies that TPO-mimetics are able to overdrive platelet production even in patients with appropriate (i.e., normal) TPO levels. Reassuringly, the overall toxicity was low.

Although the majority of ITP patients have a compensatory increase in megakaryopoiesis, plasma from some ITP patients can inhibit platelet production ex vivo. These data suggest that, in addition to increased platelet destruction, impaired megakaryopoiesis is another reason for the thrombocytopenia in this disease. The fact that TPO levels in the plasma of ITP patients are not as high as TPO levels in patients with other types of thrombocytopenia suggests that there might be room to overdrive platelet production in ITP patients by administration of exogenous TPO. Several years ago, a small trial demonstrated that platelet counts could indeed be significantly increased in some ITP patients exposed to a truncated form of recombinant thrombopoietin (PEG-MGDF)¹. Unfortunately, PEG-MGDF-induced auto-antibodies against endogenous thrombopoietin, which ultimately necessitated the discontinuation of further product development.

Several investigators proposed the hypothesis that molecules that bear no structural resemblance to TPO, but still bind and activate the TPO receptor (so called TPO mimetics), might be useful for the treatment of ITP without the risk of inducing anti-TPO antibodies. Since that time, a few groups have been able to identify peptides that bind the TPO receptor with high affinity. AMG 531 is the most developed pharmaceutical in the TPO mimetic category. It is composed of several copies of a TPO receptor-binding peptide spliced into a recombinant antibody. This peptide mimetic competes with thrombopoietin for binding to the TPO receptor and activates the receptor in an identical fashion to endogenous thrombopoietin. As shown in this paper, AMG 531 produces a dose-dependent increase in platelet counts in ITP patients.

A similar approach has been used to identify other small molecules that bear little structural similarity to TPO, yet are still capable of binding and activating the TPO receptor. Screening small-molecule libraries for compounds that have TPO-like activity identified these so-called TPO nonpeptide mimetics. The most developmentally advanced of this category is Eltrombopag. Like subcutaneously administered AMG 531, oral Eltrombopag also produces a dose-dependent increase in the platelet counts of both healthy volunteers and patients with ITP. Although less far along in clinical development, orally administered AKR-501 also functions by a similar mechanism. A final class of drugs that stimulate the TPO receptor are agonist antibodies, although these as well are not far along in clinical development.

Although the data on TPO mimetics are currently tantalizing, the long-term effects of these compounds remain to be established. Of concern is the development of reticulin bone marrow fibrosis in one patient who took AMG 531 for an extended period of time. Thus far, this specific toxicity has been reversible after discontinuation of the medication. Also notable is that TPO can increase the sensitivity of platelets to standard platelet agonists in vitro. Therefore, whether these agents will lead to thrombotic complications over time remains to be seen. But, for now, the news is good. Trials of TPO-mimetics appear to be heralding a new era of non-immunosuppresive therapy for ITP.

1. Nomura S, Dan K, Hotta T, et al. Effects of pegylated recombinant human megakaryocyte growth and development factor in patients with idiopathic thrombocytopenic purpura. Blood 2002;100:728-30.

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Promise and Obstacles to Gene Expression Profiling in Multicenter Trials

Kenneth Anderson, MD

Dr. Anderson indicated no relevant conflicts of interest.

Mulligan G, Mitsiades C, Bryant B, et al. Gene expression profiling and correlation with outcome in clinical trials of the proteasome inhibitor Bortezomib. Blood 2007[Epub ahead of print].

Gene expression profiling offers the promise to identify novel therapeutic targets, form the basis for new prognostic classification systems, assess mechanisms of action versus resistance to therapies, and ultimately identify those patients most likely to respond to a given therapy. In order to achieve these goals, correlative genomic studies need to be performed on samples obtained from uniformly treated patients with rigorous clinical annotation. Often this will require large multicenter randomized trials, which add complexity and obstacles ranging from adequate sample acquisition to appropriate bioinformatic analysis and interpretation for meaningful clinical application.

In this paper, Mulligan et al. correlated the baseline transcriptional profiles with clinical response to the novel proteasome inhibitor Bortezomib in two large phase II clinical trials of Bortezomib treatment for patients with relapsed and refractory disease, as well as a large multicenter phase III clinical trial comparing Bortezomib with Dexamethasone treatment for patients with relapsed myeloma. In this study, a pre-treatment gene expression profile associated with response to Bortezomib was identified. Most importantly, however, there are several lessons from this study regarding the challenges that implementation of similar research studies may face in the present and future.

In these multicenter trials, uniform protocols were applied to obtain informed consent to acquire bone marrow samples for genomics analysis, separate myeloma cells from bone marrow samples, and prepare tumor cell RNA. However, isolated RNA samples were deemed of inadequate quantity or quality for analysis in nearly half of the patients enrolled in the trials. This is a major potential confounder of the correlation of transcriptional profiles with clinical outcome, highlighting the need for methodologies which assure adequate sample collection in the majority of patients enrolled to allow for meaningful analysis.

Secondly, this study analyzed genomics data from several clinical trials, including two phase II trials in which more heavily pretreated relapsed refractory patients were enrolled, and a phase III trial targeting patients earlier in the disease course with relapsed myeloma. Therefore, patients were not uniform in disease status, further confounding clinical interpretation and application.

Finally, despite the rapid translation of Bortezomib from the bench to the bedside, more than four years elapsed between collection of initial samples in the phase II trials and the analyses of updated survival data from the phase III trial. During this interval, changes in methodologies for collection and processing of samples (e.g., hybridization using different batches of arrays for analysis) may add further complexity to meaningful data interpretation.

Mulligan and colleagues have successfully identified a gene signature of response to the novel proteasome inhibitor Bortezomib to be validated in future studies. Importantly, they have identified several practical obstacles to be overcome if the promise of correlative genomic studies is to translate to improved clinical practice.

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Prion Transmission by Blood Transfusion: The Report of a Third Infected Recipient, and the Second to Die With Variant Creutzfeldt-Jakob Disease, Intensifies the Focus on Preventative and Therapeutic Strategies

Michael Linenberger, MD

Dr. Linenberger indicated no relevant conflicts of interest.

Wroe SJ, Pal S, Siddique D, et al. Clinical presentation and pre-mortem diagnosis of variant Creutzfeldt-Jakob disease associated with blood transfusion: a case report. Lancet 2006;368:2061-2067.

The first studies linking a variant form of Creutzfeldt-Jakob disease (vCJD) to transmission of bovine spongiform encephalopathy (BSE) were published a decade ago. At that time, measures were taken to prevent exposure to contaminated cattle-derived food products, and programs were implemented to both minimize the theoretical risk of prion transmission by transfusion and to monitor recipients of blood products from donors who were subsequently diagnosed with vCJD. This paper describes a third case of autopsy-confirmed prion infection, and the second case of clinical vCJD, identified within a cohort of 66 at-risk transfusion recipients followed by the U.K. Transfusion Medicine Epidemiological Review (TMER) study. The patient was a 31-year-old man with ulcerative colitis who developed neurological symptoms six years after receiving non-leukodepleted red cells from an individual who was diagnosed with vCJD at 20 months after donation. The patient suffered progressive cognitive impairment, leg dysesthesias, ataxia, and dysarthria. An MRI scan eventually revealed changes in the posteromedial thalamus typical of the pulvinar sign associated with vCJD. The patient died in hospice after a 32-month course, having failed a therapeutic trial of quinacrine. Pre-mortem sequencing of PRNP, the gene encoding the prion precursor protein (PrP^c), ruled out mutations associated with inherited prion disease. He was homozygous for methionine (M/M) at codon 129, a polymorphism found in all vCJD cases and implicated in disease predisposition and pathogenesis. Post-mortem analyses confirmed the presence of misfolded prion protein (PrP^Sc) in tissue homogenates of tonsil and brain. Abnormal PrP was identified in immunohistochemical sections of the tonsil and within areas of gliosis and plaques in brain cortical sections.

This case, along with the post-mortem detection of infection in two other recipients from the TMER cohort (one of whom died with neurodegenerative symptoms consistent with vCJD), strongly reinforces the concern that pathogenic prion proteins may be transmitted through blood transfusions. The donors in these cases developed their clinical symptoms of vCJD at 18, 20, and 40 months following donation. The two recipients with vCJD were homozygous for M/M at codon 129 (the asymptomatic, infected recipient was heterozygous for methionine and valine), and they developed symptoms at six and 6.5 years after transfusion. Notably, of the 32 individuals in the original TMER cohort who survived beyond five years after transfusion, two developed clinical vCJD. Thus, pre-clinical infection appears to be sufficient for blood transmission; the “incubation period” for transfusion-associated prion disease is likely shorter than that for food-borne vCJD (which is estimated to be decades); infectivity rates may be high, at least under certain donor and/or recipient conditions; and recipient genotype at codon 129 may be an important disease-modulating factor. Given the lack of a blood assay for infection, uncertainty about prion tropism and host susceptibility, and incomplete knowledge of disease pathogenesis, a number of preventative measures have been in effect, and additional interventions are in various stages of development. These include (see Figure): (A) elimination of potentially contaminated cattle feed; (B) removal of older and diseased animals from the food chain; (C) generation of cloned cattle that lack the normal PrP^c protein¹, which is required to propagate misfolded PrP^Sc prions; (D) treatment of infected individuals with agents that reduce Pr^Pc production, such as lentiviral vector-mediated transfer of small interfering RNA^s2; (E) screening and deferral policies to prevent horizontal transmission from potentially infected blood donors; and (F) removal of infectious prions from blood-derived products by leukodepletion, filtration, resin adsorption³, and/or biophysical purification. Although prion diseases are a growing concern worldwide, the urgency for progress is greatest for the U.K. and France, which account for 85 percent and 10 percent, respectively, of all the vCJD cases reported to date. The blood donor populations in those countries are estimated to include the highest numbers of individuals with asymptomatic, undetected prion infections.

As The Hematologist was going to press, a fourth case of transfusion-associated prion infection, and a third case of probable vCJD, was reported among a surviving member of the TMER recipient cohort (see http://www.hpa.org.uk/infections/topics_az/cjd/vCJDBloodDonors.htm#nfn).

1. Richt JA, Kasinathan P, Hamir AN, et al. Production of cattle lacking prion protein. Nat Biothechnol 2007;25:132-8.
2. Pfeifer A, Eigenbrod S, Al-Khadra S, et al. Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice. J Clin Invest. 2006;116:3204-10.
3. Gregori L, Gurgel PV, Lathrop JT, et al. Reduction in infectivity of endogenous transmissible spongiform encephalopathies present in blood by adsorption to selective affinity resins. Lancet 2006;368:2226-30.

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Does It or Doesn’t It? Can Imatinib Eradicate the Stem Cell Clone?

Lilli Petruzzelli, MD, PhD

Dr. Petruzzelli indicated no relevant conflicts of interest.

Roeder I, Horn M, Glauche I, et al. Dynamic modeling of imatinib-treated chronic myeloid leukemia: functional insights and clinical implications. Nat Med 2006;12:1181-4.

A central question that arose from the successful use of the BCR-ABL targeted agent imatinib in CML is whether the leukemic stem cell can be eradicated. Rapid relapse that is observed upon cessation of the drug suggested that the leukemic stem cell was at most only mildly affected by drug therapy. In this paper, the investigators present a model based upon the effects of imatinib on BCR-ABL transcripts in patients treated with the drug. This model hypothesizes that imatinib has an effect on the proliferating BCR-ABL positive cells by inducing degradation and then has a declining percentage of transcript positive cells by reducing stem cell numbers. The model predicts that many of the stem cells are in a dormant state, possibly because of their interaction with the microenvironment, and are then not susceptible to the drug. Their prediction is that relapse is associated with rapid release and growth of the leukemic stem cell from this population. Their model allows for a prediction that moving cells into a more frequent cycling state may make them more responsive to imatinib and that by reducing the stem cell pool, the rapid relapse that is observed on removing the drug may be abrogated. Resistance forms part of their model as well — it fits with introduction of resistant clones early as well as late emergence of imatinib-resistant clones. The investigators propose that the leukemic stem cell can eventually be eradicated by continuous long-term treatment with imatinib unless mutations that result in drug resistance occur. The investigators propose that stem cells may be eradicated by moving stem cells into a proliferative compartment. Resistance remains the main foe, and it may appear early so that some stem cells cannot be eradicated.

Directing future targets in a disease such as CML remains important because of continual need for the drug and relapse in its absence. The model generated from the two patient populations examined in this manuscript present a contrast to the hypothesis on the behavior of the BCR-ABL positive CML in the manuscript by Michor et. al¹. Both manuscripts put forth models for behavior of hematopoietic stem cells based upon in vivo data and observations that focus on the biphasic decline in BCR transcripts on the initiation of imatinib therapy. Where the two manuscripts differ is on the question of whether the hematopoietic stem cell can be a target for imatinib. In the manuscript by Michor, the rapid relapse rate is attributed to the stem cell not being sensitive to chemotherapy. In the model presented in this paper, the investigators propose that it is not chemotherapy insensitivity but rather location of the stem cell in an environment or stage that is quiescent so that it is only slowly responsive to therapy. This distinction is important because moving toward therapy that targets the stem cell is different in these two models. The model here would favor inducing proliferation or movement from one niche to another to more rapidly affect the stem cell, whereas the manuscript by Michor and colleagues suggests that a different approach may be required to target the stem cell. The proof may be in the introduction of an additional therapy, and it remains to be seen whether this should be early, or later after initiation of imatinib.

1. Michor F, Hughes TP, Iwasa Y, et al. Dynamics of chronic myeloid leukaemia. Nature 2005;435:1267-70.

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A Backhand to Complement

Charles Parker, MD, and Josef Prchal, MD

Drs. Parker and Prchal indicated no relevant conflicts of interest.

Hillmen P, Young NS, Schubert J, et al. The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria. N Engl J Med 2006;355:1233-43.

The alternative pathway of complement (APC) is in a state of continuous, low-grade activation, and host erythrocytes are normally protected against complement-mediated injury by two glycosyl phosphatidylinositol (GPI)-anchored membrane proteins (Figure). Decay accelerating factor (DAF, CD55) inhibits the formation and stability of the APC C3 and C5 amplification convertases and membrane inhibitor of reactive lysis (MIRL, CD59) block the assembly of the cytolytic membrane attack complex (MAC) (Figure). Coombs-negative, complement-mediated intravascular hemolysis, the clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH), is a consequence of deficiency of CD55 and CD59. A logical strategy for controlling the hemolysis of PNH is to compensate for deficiency of CD55 and CD59 by blocking the APC pharmacologically. In this paper by Hillmen and colleagues, this approach was employed in a phase III, randomized, placebo-controlled trial in which a humanized monoclonal antibody (eculizumab) that binds the fifth component of complement (C5) was used to inhibit formation of the MAC (Figure). Treatment with eculizumab (43 patients vs. 44 in the control group) resulted in a dramatic, sustained decrease in the concentration of plasma LDH (a sensitive surrogate marker for intravascular hemolysis). A marked reduction in red cell transfusion requirement was also observed in the eculizumab-treated group. Patients in the treatment group, however, remained anemic, and an earlier phase II study showed essentially no change in reticulocyte count in patients treated with eculizumab¹. The persistent anemia may be the result of extravascular hemolysis of C3-coated erythrocytes, as the anti-C5 antibody eclulizumab does not inhibit formation of the amplification C3 convertase (Figure). Quality-of-life measurements suggested that inhibition of complement-mediated intravascular hemolysis ameliorates the fatigue associated with PNH, but the effects of eculizumab therapy on such PNH-associated symptoms as dysphagia/odynophagia and abdominal pain² were not specifically addressed in the study. Neither was the study designed to determine the effects of eculizumab on thromboembolic complications of PNH; however, only one thrombotic event (affecting a patient in the placebo group) was reported. Four serious adverse events (none infectious and none considered treatment-related) were reported among patients in the eculizumab group.

Although not yet FDA-approved, eculizumab appears to be an effective, safe treatment for controlling the complement-mediated intravascular hemolysis of PNH. Patients most likely to benefit are those with classical PNH². These patients have large PNH clones, and their symptoms are primarily a consequence of uncontrolled hemolysis. As with infusional therapy treatments developed for other orphan diseases, the cost will certainly be high, and treatment must continue indefinitely as eculizumab has no apparent effect on the underlying PIGA-mutant clonal hematopoiesis or on the marrow failure component of the disease. Whether eculizumab therapy will have an impact on thrombosis, the major cause of mortality in PNH, is uncertain. Treatment with eculizumab allows us a glimpse of PNH without its trademark intravascular hemolysis. What we see so far is a relatively benign clonal myeloid disorder³, distinct from myeloproliferative and myelodysplastic processes. Continued investigation is needed to understand the basis of the clonal selection and clonal expansion of PNH so that strategies can be developed to treat the disease, not just the symptoms.

1. Hillmen P, Hall C, Marsh JC, et al. Effect of eculizumab on hemolysis and transfusion requirements in patients with paroxysmal nocturnal hemoglobinuria. N Engl J Med 2004;350:552-559.
2. Parker C, Omine M, Richards S, et al. Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood 2005;106:3699-3709.
3. Inoue N, Izui-Sarumaru T, Murakami Y, et al. Molecular basis of clonal expansion of hematopoiesis in two patients with paroxysmal nocturnal hemoglobinuria (PNH). Blood 2006;108:4232-4236.

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