• Hemo-Globins Continue to Fascinate and Surprise
• Blood Platelets: Nature's Own Targeted Therapeutic Delivery System
• Progress for Young Patients with Diffuse Large B-Cell Lymphoma
• Are We There Yet? Still Traveling on the Road to Correcting Hematopoietic Cell Deficiencies with Gene Therapy
• Transplantation for Acute Myeloid Leukemia: Making the Best Choice?
• Is "Double Platelet Blockade" Twice as Good?
• Homocysteine, B Vitamins, and Cardiovascular Disease: Lingering Questions and Some Answers

Hemo-Globins Continue to Fascinate and Surprise

Josef Prchal, MD

Dr. Prchal indicated no relevant conflicts of interest.

Roesner A, Hankeln T, Burmester T. Hypoxia induces a complex response of globin expression in zebrafish (Danio rerio). J Exp Biol 2006;209:2129-37.

Fraser J, de Mello LV, Ward D, et al. Hypoxia-inducible myoglobin expression in nonmuscle tissues. Proc Natl Acad Sci USA 2006;103:2977-81.

There have been several important milestones in biology — the first protein mutation to be discovered, the first elucidation of the crystal structure of normal and mutant proteins, the discovery of restriction fragment-linked DNA polymorphisms, the first prenatal diagnosis utilizing DNA sequence changes — stemming from the work of experimental scientists fascinated with learning from studies of human hemoglobins. After the existence of a closely functionally- and structurally-related protein, myoglobin, became apparent, we might have assumed that not much was left to be discovered. However, in 2000 Burmester and colleagues reported in Nature¹ that another globin with remarkable homology to hemoglobin was present in neuronal tissues in mice and man. This ancestrally older gene was thus coined "neuroglobin." Now, Roesner, along with his colleagues at Burmester's laboratory, reports that the family of globins present in vertebrates, flies, and zebrafish has grown and that some are hypoxia-regulated. In zebrafish, there are at least six independent genes of the globin family (see Figure 1) that have evolved from a single ancestor that they shared approximately 700 million years ago. Since fish have developed a remarkable adaptability to different oxygen tensions, the role of these genes to hypoxia adaptation was investigated. In this paper, Roesner et al. report that brain neuroglobin mRNA (but not retinal neuroglobin) was dramatically up-regulated by hypoxia, while myoglobin expression was up-regulated to a lesser extent. In the second paper, Fraser and colleagues analyzed hypoxic regulation in another fish (the carp). They report widespread presence of myoglobins in unorthodox tissues such as kidneys, brain, gills, etc., and their striking regulation by hypoxia.

Since man is not a carp, do these elegant discoveries have any meaning for humans? The functional details, such as oxygen-binding properties, the presence or absence of Bohr effects on these diverse globins, their presence in different tissues and different species², and the subtleties of hypoxia regulation, of these genes await elucidation. Nevertheless, there is growing evidence that the newly discovered globins are also present in mammals and found in unorthodox places such as rectal smooth muscle cells, prostate, lungs, the brain, and endocrine organs³. Their role in oxygen delivery, protection against nitric oxide, oxygen radical toxicity, adaptation to exercise, and hypoxia is also becoming established^2,3. One can also ask if the yet-to-be-explained neurotoxicity and failure to thrive that is characteristic of type 2 congenital methemoglobinemia⁴ may be caused by failure to keep the heme iron in these globins in ferrous state by ubiquitous deficiency of cytochrome b5 reductase. Clearly, we will soon learn more about the importance of these newly discovered globins; hematologists will benefit from Dr. Thorsten Burmester's upcoming lecture, Neuroglobin — Fresh Blood for the Globin Family, at the Red Cell Scientific Committee session at this year's ASH meeting.

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Blood Platelets: Nature's Own Targeted Therapeutic Delivery System

Roy Silverstein, MD

Dr. Silverstein indicated no relevant conflicts of interest.

Lesurtel M, Graf R, Aleil B, et al. Platelet-derived serotonin mediates liver regeneration. Science 2006;312:104-7.

The liver is one of the few adult tissues in mammals with the capacity for regeneration. This paper reports a surprising role for platelets in this process. Using a well-established mouse model to study liver regeneration after partial (70 percent) hepatectomy, Lesurtel et al. found that hepatocyte proliferation was dramatically diminished if the mice were rendered thrombocytopenic by either chemotherapy or immune depletion. This effect could be duplicated in mice with normal platelet counts by pharmacologic inhibition of platelet function with clopidogrel, a drug that interferes with platelet secretion by blocking the P2Y12 ADP receptor. Platelet-dependent promotion of hepatocyte regeneration was shown to result from interaction of platelet-derived serotonin (5-hydroxytryptamine) with the hepatocyte serotonin receptors 5-HT2A and 5-HT2B. Expression of these receptors increased three-to four-fold after partial hepatectomy, and their specific pharmacologic blockade produced the same effect as thrombocytopenia or clopidogrel. Furthermore, treating thrombocytopenic mice with a specific 5HT agonist restored hepatocyte proliferation after hepatectomy. The investigators also studied mice rendered null for the tryptophan-hydroxylase-1 gene. These animals lack capacity to generate peripheral 5HT and had a similar decrement in hepatocyte proliferation after resection as did thrombocytopenic mice. This was reversed by injecting 5-hydroxytryptophan to "re-load" platelets with 5HT.

Platelets play key roles in many aspects of vascular function over and above their essential and well-characterized role in primary hemostasis. An abundant literature has linked platelets to atherosclerosis, inflammation, and reperfusion injury, and recent studies have pointed to platelet involvement in angiogenesis. One of the more surprising and interesting characteristics of platelets is the large number of biologically active molecules carried in their granules — the molecules poised to be deposited at sites of vascular injury as part of the platelet release reaction. Among the repertoire of platelet α-granule constituents are peptide growth factors (e.g., PDGF and VEGF), enzymes, enzyme inhibitors, and large multifunctional glycoproteins (e.g., thrombospondin and vWF). In the dense granules are found bioactive nucleotides and amines such as ADP and 5HT. Serotonin was first isolated from blood by Irvine Page (at Cleveland Clinic) in 1948. Shortly thereafter, Marjorie Zucker and others found that nearly all serotonin in blood was associated with platelets. Platelets take up 5HT by an active transporter, and the platelet has been used as a model to study 5HT uptake, contributing greatly to our understanding of neurotransmission and the development of drugs now commonly used to treat depression and other psychiatric disorders. Serotonin functions both as a neurotransmitter and as a hormone, acting through a family of related receptors that are expressed widely but differentially. These regulate vascular tone, cardiac function, gut motility, airway reactivity, and, as shown in this report, hepatocyte proliferation. Since patients receiving liver transplantation often have portal hypertension, splenomegaly, and thrombocytopenia, this work suggests that 5HT might be a useful adjunct to promote successful transplant. These data also provide additional evidence for the concept that platelets act as targeted delivery systems for endogenous regulatory molecules. By adhering to the endothelium of injured organs and tissues and then secreting their granular contents, platelets deposit high concentrations of highly-active molecules in a regulated and localized manner. Unexpected roles for platelets in many other biological systems are likely to be discovered.

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Progress for Young Patients with Diffuse Large B-Cell Lymphoma

Michael Williams, MD

Dr. Williams receives research funding from BiogenIDEC and Genentech.

Pfreundschuh M, Trumper L, Osterborg A, et al. for the MabThera International Trial (MInT) Group. CHOP-like chemotherapy plus rituximab versus CHOP-like chemotherapy alone in young patients with good-prognosis diffuse large-B-cell lymphoma: a randomised controlled trial by the MabThera International Trial (MInT) Group. Lancet Oncol 2006;7:379-91.

Treatment of newly diagnosed DLBCL with the R-CHOP regimen (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) is based upon French (GELA) and U.S. Intergroup (ECOG 4494) trials demonstrating improved survival as compared with CHOP alone in patients > 60 years of age. Although R-CHOP has been generally adopted for treatment of younger patients as well, specific clinical study data to support this approach has been lacking. Pfreundschuh et al. conducted a large multinational randomized prospective trial in which patients age 16-60 with low-risk DLBCL (age-adjusted IPI score of 0-1) received CHOP-like chemotherapy (primarily CHOP or CHOP plus etoposide [CHOEP]), administered q21d with or without concomitant rituximab. Radiation therapy 30-40 Gy was also administered to original bulky sites of disease > 5 cm. As observed in elderly patients, the addition of rituximab was associated with significantly improved event-free and overall survival. Toxicities were similar in both groups. Somewhat surprisingly, prognostic subgroups were identified even within this group selected as low-risk (Table). Those with IPI 0 (i.e., limited stage), non-bulky disease had improved outcomes versus those with bulky disease and/or IPI score of 1 (advanced stage, poor performance status or high LDH).

DLBCL is the most common non-Hodgkin lymphoma subtype in the western world, and is both clinically and biologically heterogeneous. This important trial confirms and extends earlier studies and establishes R-CHOP as a current standard for younger as well as older patients with DLBCL. There was no clear benefit to those who received R-CHOEP as compared with R-CHOP, suggesting to these authors that rituximab has a "chemo-equalizing" effect or that the more intensive and toxic CHOEP regimen impaired immune effector mechanisms important to rituximab response. The identification of a highly favorable subgroup of very low-risk patients (IPI 0, non-bulky) suggests that they should be treated and studied in future clinical trials separately from DLBCL patients with greater degrees of risk. The addition of rituximab to CHOP has provided an important but incremental advance in DLBCL, and it remains essential to continue to build upon this progress. To this end, current clinical research in previously untreated DLBCL includes dose-modulated chemo-immunotherapy (e.g., dose-adjusted R-EPOCH or dose-dense R-CHOP), R-CHOP followed by radioimmunotherapy consolidation, and risk stratification for early stem cell transplantation based upon clinical IPI score, phenotypic or molecular markers, and PET response after 2-3 cycles of induction chemotherapy.

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Are We There Yet? Still Traveling on the Road to Correcting Hematopoietic Cell Deficiencies with Gene Therapy

Targeting a genetic defect in a defined cellular compartment remains attractive, but recent data that implicate vector insertion with the activation of an oncogene and leukemia have raised some concern about this approach. The true power of this study is in the detailed analysis of the vector insertion sites over time. Detailed and extensive analysis was done that demonstrated that reconstitution was due to clones that had vector insertion into three discrete genes which encode zinc-finger transcription factors. All of these genes have been associated with translocations in patients with leukemia and raise a concern about the long-term fate of the hematopoietic precursor cells in these patients. At the time of the study, it is evident that the expression of the vector is restricted to the myeloid compartment. It is clear that the insertion into these sites has conferred a selection advantage on these clones with other insertion sites disappearing with time. The major question that is raised from these findings is whether the expression and control on expansion will remain a feature of these cells with time. Expressing NADPH oxidase, even though not to the level of wild type and not in all cells, benefits patients with this disease. It may well be that the vector choice has made the difference in the insertion site. The gradual restriction to such a discrete set of clonal insertion sites that are genes associated with leukemia, and the growth of the select group of clones raises concern that a small population of cells with a capacity for self renewal may remain in the pack to repopulate with an immature population. The results so far support that insertion into this site still leads to a restricted differentiated population of cells that reconstitute a critical protein function. The data presented continue to add to the questions of targeted gene therapy, but the type of detailed analysis in this work may also be used to direct vector choices and the cell population in which the gene is introduced.

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Transplantation for Acute Myeloid Leukemia: Making the Best Choice?

The interesting point of this study is that it presents data of a large series of cases collected from various institutions with a long follow-up. There are obvious methodological limitations inherent to the retrospective design of the study. First, as the study is not based on a prospective comparison between autologous and alternative donor transplantations, it is quite likely that patients with different risk profiles were selected for each of the treatments. Indeed, the authors show that patients with unfavorable cytogenetics were more likely to be offered URD transplantation. In addition, URD recipients were more likely to have a reduced performance score and have leukemia with unfavorable cytogenetics, and they had more difficulty (i.e., required more time) attaining CR1. These adverse features of the URD patient group may have negatively influenced the URD transplant results. Furthermore, in this study important cytogenetic information was available only in a minority of cases. It would have been of clinical interest to know about outcome in the distinct subsets of patients with high-risk AML and intermediate prognostic risk. The numbers did not allow for such an analysis. Finally, in recent years molecular HLA matching has improved considerably. The degree of HLA match between unrelated donor-recipient pairs was insufficiently defined in this study. Nowadays, high-resolution HLA typing allowing for better donor selection appears to favorably affect the outcome of URD transplants.

What can we conclude? Both autologous and URD transplantation represent realistic and clinically meaningful therapeutic options. As a next step, additional studies involving (cyto)genetically characterized patient cohorts and using modern donor selection methods are warranted in order to more fully define the comparative values of autologous and URD transplantation modalities in patients with AML.

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Is "Double Platelet Blockade" Twice as Good?

In this paper, Bhatt et al. describe the findings of the Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance (CHARISMA) trial. This study was designed to test the value of adding clopidogrel to aspirin in patients who have multiple risk factors for atherothrombotic events. Previous trials have demonstrated some value of dual platelet inhibition therapy for patients with unstable angina, myocardial infarctions, and for patients undergoing angioplasty.

The CHARISMA trial enrolled 15,603 patients with either documented cardiovascular disease or multiple atherothrombotic risk factors. Patients were randomized to receive low-dose aspirin along with either clopidogrel or placebo, and monitored for an average of 28 months. Although there was a trend toward benefit, the combined incidence of myocardial infarctions, strokes, and cardiovascular deaths was not statistically significant between patients treated with combined modality anti-platelet therapy and those treated with aspirin alone. Similarly, there was a trend toward a higher incidence of severe bleeding in the double treatment group, but again, this was not a statistically significant difference.

Like ticlopidine, clopidogrel is a member of a class of compounds called thienopyridines. Metabolites of these drugs directly inhibit P2Y12, one of two types of ADP receptors found on platelets. The CAPRIE study demonstrated that clopidogrel was at least as good as aspirin in preventing cerebrovascular and cardiovascular events in 19,185 patients with known atherosclerosis¹. Since aspirin and clopidogrel poison platelets by different mechanisms (Figure 1), it is suggested that these agents might have additive effects. In theory, this could be beneficial in the treatment of diseases associated with platelet activation such as ischemic heart disease, peripheral vascular disease, and ischemic strokes. This theory was tested in the CURE trial that analyzed the outcome of 12,562 patients with the acute coronary syndrome². In this study, the addition of clopidogrel to aspirin decreased the combined incidence of cardiovascular deaths, myocardial infarctions, and strokes from 11.4 percent to 9.3 percent. The benefit of double therapy was partially offset by an increase in severe bleeding from 2.7 percent to 3.7 percent.

The findings of the CURE trial spawned a new series of trials investigating whether double platelet blockade was better than single anti-platelet therapy in a variety of patients at risk for arterial thrombi. Additional studies of patients who had myocardial infarctions or angioplasty demonstrated and appeared to confirm a benefit to double platelet blockade that outweighed the associated bleeding risk. However, in the MATCH study, the bleeding complications of patients who received dual anti-platelet therapy for the prevention of strokes offset any benefit³.

The trial published by Bhatt and colleagues was designed to determine whether long-term treatment with both clopidogrel and aspirin was better than aspirin alone in a broad population at risk for cardiovascular events. As found in the other trials, any benefit for double anti-platelet therapy was at best modest. In total, there were 94 less ischemic events in patients treated with both clopidogrel and aspirin, but at the expense of 93 more moderate or severe bleeding events. Except in special circumstances such as angioplasty, it appears that the added benefit of double anti-platelet therapy for most patients is small, and, at times, dangerous.

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Homocysteine, B Vitamins, and Cardiovascular Disease: Lingering Questions and Some Answers

The results of these two studies, together with primary observations of the Vitamin Intervention for Stroke Prevention (VISP) trial¹, indicate that down-modulation of homocysteine with B-vitamin supplementation does not protect against primary or recurrent arterial vascular complications. By comparison, other recent data suggest that certain vascular complications² or patient subgroups³ may derive benefit from B vitamins. The trend toward worse outcomes in the NORVIT study, along with observations of increased risk of in-stent restenosis among vitamin-supplemented patients⁴, have spawned hypotheses that supraphysiologic vitamin levels "shunt" homocysteine toward metabolic pathways that adversely affect gene expression (via hypermethylation of DNA), protein synthesis, cell proliferation, and/or methylation of atherogenic molecules. It is not yet known whether these observations or mechanisms might be relevant to homocysteine modulation with vitamin supplementation for the prevention of venous thromboembolic disease. The current widespread use of dietary folate fortification and the relatively modest associations between homocysteine, B vitamins, and vascular disease limit the ability of these studies to assess a possible benefit or adverse effect of vitamin supplementation. A meta-analysis of pooled data from the many current clinical trials may offer important insights for clinical practice and public health policy. Future prospective studies will need well-defined stratification parameters, greater subject recruitment, and extended duration of treatment in order to achieve adequate statistical power for subgroup analyses.

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