• Keratinocyte Growth Factor as a Thymic Stimulant in Stem Cell Transplantation
• The ABCs of BCL2 Expression in Diffuse Large B-Cell Lymphoma
• The Dark Side of Recombinant Factor VIIa
• Angiogenesis and Inflammation Cross Paths at the Blood Vessel Wall
• Targeted Cancer Therapy
• CML Relapse Post-SCT: Is Imatinib a Cure?
• Understanding Acute Promyelocytic Leukemia - What's Special About t(15;17)?
• Prions: Are They Doing Us Any Good?
• Mutated Nucleophosmin (NPM1) Gene in Acute Myeloid Leukemia: A Useful Marker for Monitoring Residual Disease?
• The Tumor Suppressor Menin Plays a Central Role in MLL-Associated Leukemias
• Mobilized Peripheral Blood Stem Cells for Cardiac Regeneration after Acute Myocardial Infarction - Back to the Repair Shop

Keratinocyte Growth Factor as a Thymic Stimulant in Stem Cell Transplantation

Stephen Emerson, MD, PhD

Dr. Emerson indicated no relevant conflicts of interest.

Alpdogan O, Hubbard VM, Smith OM, et al. Keratinocyte growth factor (KGF) is required for postnatal thymic regeneration. Blood 2006;107:2453-60.

Keratinocyte growth factor (KGF) is one of several cytokines that induce epithelial cell growth and differentiation, and it has recently been found to help prevent mucositis following high-dose chemotherapy. In this paper, Alpdogan and colleagues show that KGF also improves T-cell production in mice whose normal thymic function has been impaired by either irradiation or by aging. T-cell production in the thymus declines dramatically after childhood and is also compromised following allogeneic stem cell transplantation (SCT). Older recipients of SCT have the most impaired thymopoiesis, leading directly to both immunodeficiency and worsened graft-versus-host disease in adult recipients of allo-SCT, which together comprise the major barriers to safe allo-SCT. If one could amelerioate or prevent thymic dysfunction following SCT, patients would have far fewer infections, and almost certainly much reduced GVHD as well, resulting in much improved survival.

In this paper, the authors began by studying T-cell production in mice genetically engineered to lack KGF. Under normal, gentle conditions, T cells developed normally in these mice. If, however, the mice were treated with high doses of irradiation or cyclophosphamide, de novo T-cell production through the thymus was impaired in these KGF-/- mice, compared with the recovery of normal mice. This defect in KGF-/- mice was not due to a stem cell defect, but to the non-stem cell microenvironment caused by the KGF deficiency. Finally, the administration of recombinant KGF corrected this defect, thus "closing the loop" to demonstrate that KGF deficiency will lead to non-stem cell dysfunction which prevents normal stem cells from developing into functional T cells. To test whether administering pharmacologic KGF could be applied to the clinic, the authors tested the effects of KGF on normal mice, both at baseline and following XRT, chemotherapy +/- allogeneic transplantation. They found that KGF enhanced T-cell production in normal young and old mice and significantly improved recovery following sublethal XRT or chemotherapy. Most interestingly, KGF increased thymopoiesis in normal middle-aged mice, but the effect was actually not apparent until two to three months following transplantation.

Taken together, these very promising studies point the way to clinical trials of KGF for the prevention of T-cell immunodeficiency, particularly in the setting of stem cell transplantation. Just as G-CSF and Epo have proven to be extremely useful molecular pharmaceuticals for hematopoietic cells over the past two decades, KGF may prove to be the first effective stromal cell stimulant in the years to come. Let the careful trials begin.

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The ABCs of BCL2 Expression in Diffuse Large B-Cell Lymphoma

Michael Williams, MD

Dr. Williams indicated no relevant conflicts of interest.

Iqbal J, Neppalli VT, Wright G, et al. BCL2 expression is a prognostic marker for the activated B-cell-like type of diffuse large B-cell lymphoma. J Clin Oncol 2006;24:961-8.

Diffuse large B-cell lymphoma (DLBCL) is recognized as a heterogeneous disease with distinct subgroups delineated via gene expression profiling. The anti-apoptosis gene BCL2, located at chromosome 18q21, is frequently over-expressed in DLBCL, but there were previously conflicting data regarding its utility as a prognostic marker. Iqbal and colleagues correlated BCL2 protein expression with survival in DLBCL cases previously stratified into the two major subgroups of germinal center B-cell-like (GCB) and activated B-cell-like (ABC). They also investigated whether BCL2 expression occurred via the chromosomal translocation t(14;18)(q32;q21) or via chromosome 18q21 gain or amplification. BCL2 was expressed in half of the GCB cases, usually via the t(14;18), but had no correlation with overall survival. In contrast, BCL2 expression in the ABC cases was demonstrated in 59 percent of cases and was strongly associated with poorer survival (Figure). This correlation held even with use of differing cutoff limits for positivity in BCL2 expression. Unlike GCB, BCL2 expression in the ABC subgroup was associated with 18q21 gain or amplification but in no instance with the t(14;18) (Table).

DLBCL is the most common lymphoma in Western countries. While about half of all patients are cured by initial therapy, the remainder either progress after initial response or are primarily refractory. Understandably, there is considerable interest in identifying biomarkers of response or resistance which might improve prognostic and therapeutic stratification for these patients. BCL2 has been extensively evaluated in this regard due to its role in promoting cell survival and chemotherapy resistance, but with inconclusive results. The report by Iqbal et al. clarifies previously conflicting data by associating expression with DLBCL subgroup. Importantly, BCL2 expression in ABC lymphomas is associated with chromosome 18q21 gain or amplification, and perhaps with upregulation of NFkB or other regulatory pathways. It is as yet unclear whether BCL2 expression directly contributes to the observed poorer survival in the ABC group or is instead a marker for other co-amplified genes at 18q21, downstream targets of these molecules, or NFkB itself. This study supports the classification of clinical responses by tumor subgroup in ongoing and future DLBCL clinical trials. Investigations of lymphoma pathogenesis, biomarkers of therapeutic response and resistance, and the identification of potential therapeutic targets now have new leads to pursue.

Figure 1

Table 1

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The Dark Side of Recombinant Factor VIIa

Charles Abrams, MD

Dr. Abrams indicated no relevant conflicts of interest.

O'Connell KA, Wood JJ, Wise RP, et al. Thromboembolic adverse events after use of recombinant human coagulation factor VIIa. JAMA 2006;295:293-8.

In this article, the authors reviewed the serious adverse thromboembolic events attributed to the use of recombinant factor VIIa. From March 25, 1999, through December 31, 2004, a total of 185 thromboembolic events were reported to the FDA via the Adverse Event Reporting System (AERS). In 2004, over 40 thrombotic events were reported in 4520 hospitalized patients treated with recombinant factor VIIa. In an addendum to the article, an additional 61 thrombotic events were reported in the first 10 months of 2005. The vast majority of thromboembolic events were associated with the off-label use of this drug. These thrombotic events occurred when recombinant factor VIIa was given for surgery (either prophylaxis or bleeding), intracranial bleeding, and trauma. Alarmingly, 99 (54.1 percent) of these events were arterial. This includes 39 cerebrovascular accidents, 34 myocardial infarctions, and 26 other arterial thrombotic events involving large- and medium-size blood vessels. Venous clots accounted for another 40.4 percent of the thromboembolic events. The remaining 10 events included occlusion of extracorporal membrane oxygenation lines and dialysis shunts.

Recombinant human factor VIIa (NovoSeven) is FDA approved for controlling bleeding associated with inhibitors in patients with hemophilia A or B. It is also approved for treatment of patients with congenital factor VII deficiency. Through a series of case reports and a limited number of randomized trials, recombinant factor VIIa has been used to treat a wide variety of hemostastic abnormalities. These include coagulopathies associated with liver failure, trauma, hemorrhagic strokes, warfarin overdose, and surgical misadventures. These publications have created an impression that recombinant factor VIIa can function as a universal hemostatic agent, and that it is an appropriate supplement for treating the bleeding patient, regardless of the hemorrhagic etiology. Not only is this financially costly to society, this approach may also be costly to the patient's health.

A randomized placebo-control trial published one year ago in the New England Journal of Medicine¹ tested the hypothesis that recombinant factor VIIa would be beneficial in the acute treatment of hemorrhagic strokes. In this study, it was noted that thromboembolic events were increased in patients randomized to receive recombinant factor VIIa. Most of these thrombotic events were arterial, and, notably, most were observed in patients who received the highest dose of recombinant factor VIIa. Novo Nordisk, the manufacturer of Novo Seven, issued a safety alert describing thromboembolic adverse events in non-hemophilia patients, and the FDA posted a safety alert on December 1, 2005.

Although the inclination is to attempt to use the data presented in O'Connell's article to calculate the incidence of thromboembolic complications resulting from recombinant factor VIIa therapy, it is impossible to use the AERS system for this purpose. The majority of thrombotic events detected under this system were spontaneously reported. As the authors acknowledge, underreporting is undoubtedly an issue when one relies on physician-initiated adverse event reports. Consequently, the true incidence of this dark complication of off-label use of recombinant factor VIIa therapy is unknown.

1. Mayer SA, Brun NC, Begtrup K, et al. Recombinant activated factor VII for acute intracerebral hemorrhage. N Engl J Med 2005;352:777-85.

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Angiogenesis and Inflammation Cross Paths at the Blood Vessel Wall

Roy Silverstein, MD

Dr. Silverstein indicated no relevant conflicts of interest.

Fiedler U, Reiss Y, Scharpfenecker M, et al. Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation. Nat Med 2006;12:235-9.

In this paper, Fiedler and colleagues discovered a surprising function for the anti-angiogenic protein angiopoietin-2 (ang2) - modulation of the inflammatory response. Mice rendered genetically null for ang-2 had dramatically diminished ability to recruit neutrophils into their peritoneal cavities after a potent local pro-inflammatory challenge despite normal circulating white blood cell counts. Using the powerful tool of in vivo video microscopy to monitor the vascular component of the inflammatory response, they found that leucocytes in ang-2 null mice exhibited a steady increase in "rolling" down the inner wall of cutaneous blood vessels superfused with the pro-inflammatory cytokine TNF-α, but diminished ability to stick firmly to the endothelium. This indicates that the leukocytes could "sense" the presence of inflammation (i.e., tether and roll down the endothelial lining), but were unable to adhere firmly enough to migrate through the vessel wall and enter the inflamed tissues. What exactly is ang-2 doing in this setting? Previous studies showed that vascular endothelial cells (EC) store ang-2 in granules that are released in response to pro-inflammatory or pro-thrombotic stimuli, including TNF-α. The investigators therefore "knocked down" expression levels of endogenous ang-2 in cultured human EC and showed that adhesion molecule expression in response to low concentrations of TNF-α was abrogated. Furthermore, they showed that exposing exogenous ang-2 by itself to EC did not affect leukocyte adhesion, but significantly enhanced adhesion if the EC were co-treated with small amounts of TNF-α.

Angiogenesis, both embryonic and postnatal, is controlled by a complex signaling network involving factors that regulate growth, migration, and remodeling of nascent blood vessels. An important component of this network is tie-2, a tyrosine kinase receptor on the surface of EC that responds to two related proteins known as angiopoietins. Ang-1 is secreted by peri-vascular smooth muscle cells (pericytes) in a paracrine manner and provides crucial maturation signals to stabilize neo-vessels. Ang-2 is produced by EC in an autocrine manner and was initially thought to serve as an antagonist to ang-1 signaling, leading to capillary disruption. More recently it has been shown that in some vessels, particularly lymphatic, ang-2 has a tie-2-dependent agonist function. Mice with genetic deletion of angiopoietins or tie-2 have severe vascular developmental defects. This paper points to an important role for the angiopoietin system in inflammation as well as angiogenesis. The data suggest that ang-2 "primes" or sensitizes EC to respond to subthreshold concentrations of TNF-α, thereby amplifying the developing inflammatory response. The mouse experiments show that this amplification step is crucial for initiating a sustained inflammatory response. Thus ang-1 and ang-2 seem to function through tie-2 as binary switches to stabilize (ang-1) or destabilize (ang-2) blood vessels and amplify (ang-2) or inhibit (ang-1) inflammation. This cross talk between inflammatory signaling and angiogenic signaling is not altogether surprising given the importance of new vessel formation in certain types of inflammation (e.g., wound granulation tissue). Furthermore, studies of unrelated systems, such as obesity and insulin resistance, revealed that inflammatory signaling pathways are actually critically connected to other seemingly unrelated signaling systems via "cross talk" among receptors and downstream effectors. These studies point to novel pathways other than TNF-α to target for anti-inflammation drug development.

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Targeted Cancer Therapy

Robert Lowsky, MD, FRCPC

Dr. Lowsky indicated no relevant conflicts of interest.

Thorne SH, Negrin RS, Contag CH. Synergistic antitumor effects of immune cell-viral biotherapy. Science 2006;311:1780-4.

Most chemotherapy and radiotherapy regimens exploit a modest window of differential sensitivity between tumor cells and normal host tissue that prohibits dose escalation and limits effectiveness. Targeted biological therapies, therefore, hold tremendous potential for cancer treatment. In this paper, Thorne et al. combined immune effector cell populations that naturally migrate to tumors (cytokine-induced killer, or CIK cells) with an oncolytic virus (vaccinia) to achieve directed delivery to and regression of tumors in mouse models.

CIK cells are derived from blood or spleen mononuclear cells following a three-week ex vivo expansion with interferon-γ, interleukin-2, and CD3-specific antibody (OKT3). These cells migrate to tumor sites and exhibit non-major histocompatibility complex-restricted killing of tumor targets in vitro and in vivo. Vaccinia virus is a poxvirus that spends its entire lifecycle in the cytoplasm and induces profound cytopathic effects by inhibiting host protein synthesis, resulting in cell death. Modifying the virus by deleting the thymidine kinase gene and the vaccinia growth factor gene enhances tumor selectivity as viral replication becomes limited to dividing cells. The authors demonstrated that CIK cells infected with the modified vaccinia virus retained their ability to kill tumor targets in vitro. The virus remained dormant in these cells for 48-72 hours, roughly the same amount of time CIK cells take to localize to tumor sites. Whole body optical imaging confirmed that infected CIK cells trafficked efficiently to tumor sites in immune-competent and immune-deficient tumor-bearing mice, and delivered a pattern of viral biodistribution limited to the tumor sites with little virus detection in any other organ. The combination of CIK cells and modified vaccinia virus was highly efficacious and significantly extended the median survival in tumor-bearing mice compared with CIK cells or the modified virus alone. In fact, the combination therapy resulted in complete responses even in some cases of mice bearing CIK-resistant tumors.

This study puts forth a new paradigm for the delivery of cancer therapy. Thorne and colleagues used a population of hematopoietic cells, CIK cells, that naturally migrate to tumors to deliver a potent oncolytic virus. The CIK cells transported the virus deep within the tumors to provide a uniform distribution of infection. The viral infection in turn enhanced tumor cell killing by the CIK cells and significantly inhibited tumor growth in mice. Although each component of the therapy had been shown previously to have antitumor activity, their demonstrated synergy now offers the potential of a targeted biological therapy that may in combination prove to be much more effective. Many areas of study remain: Do dying cancer cells release a free virus that can infect other cells? If so, in what tissue? Vaccinia is known to infect a wide range of human tissue (yet not cause human disease), whereas murine tissue is relatively resistant to vaccinia infection. Is the antitumor effect compromised by concurrent or recently administered chemotherapy? The mechanism underlying the enhanced CIK cell antitumor activity following vaccinia infection is unclear; the specific subset within the broad population of CIK cells that transports and delivers the virus to tumor cells remains undefined. Ultimately, hematologists and transplant hematologists need to follow these types of strategies and collaborate with researchers in the field as the era of cellular therapy without hematopoietic cell transplantation for targeted cancer therapy is fast approaching.

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CML Relapse Post-SCT: Is Imatinib a Cure?

Peter Lee, MD

Dr.Lee indicated no relevant conflicts of interest.

Hess G, Bunjes D, Siegert W, et al. Sustained complete molecular remissions after treatment with imatinib-mesylate in patients with failure after allogeneic stem cell transplantation for chronic myelogenous leukemia: results of a prospective phase II open-label multicenter study. J Clin Oncol 2005;23:7583-93.

While imatinib mesylate (IM) has become the frontline treatment for most patients with chronic myelogenous leukemia (CML), it does not represent a cure. Even patients with complete molecular remission (CMR) relapse rapidly upon discontinuation of the drug. Allogeneic stem cell transplantation (SCT) remains the only potentially curative treatment for CML - the putative mechanism is induction of an effective graft-versus-leukemia (GVL) immune response. Still, 5-20 percent of CML patients relapse after SCT. While an effective treatment is donor lymphocyte infusion (DLI), this is associated with substantial risk of graft-versus-host disease (GVHD). Thus, IM represents an attractive alternative treatment for relapsed CML post-SCT. The questions then are: Is IM effective in relapsed CML post-SCT? Is it simply a bridging measure? What should be done next? In a prospective multicenter trial by Hess et al., the investigators followed 44 CML patients who relapsed (18 molecular relapses and 19 cytogenetic relapses) post-SCT and were treated with IM. Subjects were analyzed with quantitative PCR for bcr-abl to monitor molecular responses. Immunosuppression was generally tapered prior to enrollment, and the initial IM dose of 400 mg/day was escalated to 600-800 mg/day for patients who did not achieve at least a major molecular response after three months. Eventually 77 percent of patients achieved CMR (defined as negative nested PCR for bcr-abl in three consecutive samples after an initial positive PCR). As part of this trial, patients could be taken off IM after at least six months if they remained in CMR at three consecutive time points within a period of three months. By these criteria, 10 patients discontinued IM. Intriguingly, four of these 10 patients (40 percent) maintained durable CMR off IM with a median follow-up of 494 days. This is distinctly different from CML patients treated up front with IM (without SCT), who invariably relapse within 90 days.

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Understanding Acute Promyelocytic Leukemia - What's Special About t(15;17)?

Nancy Andrews, MD, PhD

Dr. Andrews indicated no relevant conflicts of interest.

Sternsdorf T, Phan VT, Maunakea ML, et al. Forced retinoic acid receptor alpha homodimers prime mice for APL-like leukemia. Cancer Cell 2006;9:81-94.

On the surface, the molecular cause of acute promyelocytic leukemia (APL) appears to be remarkably simple - APL patients carry an aberrant chromosomal translocation that disrupts the chromosome 17 gene encoding a retinoic acid-binding transcription factor, RARα. Almost always, a unique translocation [t(15;17)] leads to the production of a fusion oncoprotein made up of the first part of PML, an unrelated protein, and the last part of RARα (Figure 1A). In the sixteen years since RARα fusion proteins were first described in APL, investigators have tried to answer three questions: Why is RARα always altered? How do the translocations change a normal transcription factor into an oncoprotein? How do pharmaceutical doses of all-trans-retinoic acid (ATRA) overcome the leukemic process? In this report, Sternsdorf and colleagues provide partial answers, following up on many previous studies. They considered what PML has in common with the other proteins that have occasionally been found fused to RARα in APL - not much, other than the ability to self-associate. They asked whether other fusion partners engineered to make RARα multimerize (Figure 1B) might have similar properties. They showed that multimerization makes RARα act as a more potent transcriptional regulator of an expanded repertoire of target genes in vitro. However, when the novel multimerizing fusion proteins were expressed in mice, only a small fraction of the animals developed leukemia after a long latency period, suggesting that at least one other genetic event was necessary. They showed that leukemia could be potentiated in mice that also expressed an activated form of the β_commonsubunit of cytokine receptors, thus providing a second hit. They concluded that multimerization was important, but that the PML-RARα fusion protein must also have new, unknown properties that make it an oncoprotein. It cannot simply be that fusion to RARα prevents PML from doing its normal job, because forced RARα multimerization caused leukemia at the same low frequency in mice lacking PML altogether.

RARα , acting with its normal partner RXR, clearly has a special role as a transcriptional regulator of the myeloid development program. But there can be too much of a good thing. Forcing RARα to self-aggregate seems to make it better at shutting off transcription of its normal target genes and probably other genes as well, thus perturbing normal myeloid differentiation. This happens whether the fusion involves PML or an irrelevant protein that causes RARα to form multimers. It suggests that higher doses of ATRA, a molecule that normally modulates RARα activity, are required to overcome the stronger effects of RARα multimers. But the new protein created by fusion of PML to RARα must have at least one other, as yet unexplained, function that makes cells malignant. There are two important messages from this work. First, a single genetic event, the translocation that fuses PML to RARα, can have complex sequelae - not just multimerization of RARα, but also a new activity that has not yet been elucidated. Second, as the authors point out, it seems likely that drugs could be designed to interfere with RARα multimerization, providing a new option for treating APL.

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Prions: Are They Doing Us Any Good?

Josef Prchal, MD, and Xylina Gregg, MD

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

Zhang CC, Steele AD, Lindquist S, et al. Prion protein is expressed on long-term repopulating hematopoietic stem cells and is important for their self-renewal. PNAS USA 2006;103:2184-9.

Steele AD, Emsley JG, Ozdinler PH, et al. Prion protein (PrPc) positively regulates neural precursor proliferation during developmental and adult mammalian neurogenesis. PNAS USA 2006; [Epub ahead of print].

In this paper, Zhang et al. studied the role of the much maligned and feared prions in hematopoiesis. Prions are expressed in many tissues, including hematopoietic cells and neurons. When the authors examined mouse bone marrow, they found that many marrow cells expressed prion protein (PrP) and that the erythroid progenitors were particularly likely to express PrP (more than 80 percent of cells bearing the erythroid surface antigens were PrP+). They then focused on the possible function of PrP in hematopoietic stem cells, which are particularly prevalent in the side population (identified by exclusion of Hoechst Dye 33342¹). About half of the hematopoietic stem cells also expressed PrP. The authors then concentrated on the ability of PrP+ versus PrP null cells (using PrP knockout mice) to generate hematopoietic progenitors in clonogenic assays using BFUE, CFU-G, and CFU-GEMM. They found that the relative proportion of the progenitors and the appearance of the colonies derived from both PrP+ and PrP null stem cells were normal, suggesting no difference in the number and proliferating capacity of PrP+ versus PrP stem cells. Zheng and colleagues then investigated whether PrP has an effect on self-renewal of pluripotent stem cells. For this they used a well-described competitive repopulation transplantation model. In the first four months after transplantation, they found that PrP null versus PrP+ donor cells exhibited equal engraftment and reconstitution of peripheral blood. After the first four months, the PrP+ hematopoietic stem cells clearly out-competed the PrP null cells - a finding even more striking in secondary and tertiary transplantation. Further, the long-term repopulating stem cells were only in PrP+ side population. The conclusion that PrP is augmenting the yet-to-be fully elucidated self-renewal properties of hematopoietic stem cells was further confirmed by the superiority of PrP+ hematopoietic stem cells after the stress of 5-FU therapy in reconstituting the marrow of transplanted recipients, as well as by the rescue of PrP null cells after retroviral transfection-induced ectopic PrP expression. In a paper published two weeks later, Steele and colleagues also examined the function of the PrP in regulation of neural proliferation and differentiation. In this paper, the authors showed that, in neuronal tissue, PrP plays an essential role in neurogenesis and neuron progenitor differentiation.

What are the implications of these findings? First, that PrP is anchored on the surface of the cells and likely has a receptor or co-receptor function which affects hematopoietic stem cell activity. It likely interacts with a yet-to-be-determined ligand. The elucidation of the function and identity of the postulated ligand would greatly further our understanding of hematopoietic stem cells and may be important in the future for in vitro and in vivo stem cell manipulation. Furthermore, PrP is a GPI-linked protein² and has been shown to be defective in paroxysmal nocturnal hemoglobinuria³. Thus, the PrP defect may be the long-sought-after link for understanding the frequent bone marrow failure and aplasia seen in patients with somatic PIG-A mutations, resulting in failure of GPI-linked protein expression, i.e., the pathognomonic lesion of paroxysmal nocturnal hemoglobinuria. Additionally, since copper is required for expression of PrP, it may also explain the pancytopenia described in patients with copper deficiency⁴.

1. Goodell MA, Brose K, Paradis G, et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183: 1797-1806.

2. Stahl N, Borchelt DR, Hsiao K, et al. Scrapie prion protein contains a phosphatidylinositol- glycolipid. Cell 1987; 51: 229-240.

3. Durig J, Giese A, Schmucker U, et al. Decreased prion protein expression in human periph- eral blood leucocytes from patients with paroxysmal nocturnal haemoglobinuria. Br J Haematol 2001;112:658-62.

4. Gregg XT, Reddy V, Prchal JT. Copper deficiency masquerading as myelodysplastic syndrome. Blood 2002; 100: 1493-1495.

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Mutated Nucleophosmin (NPM1) Gene in Acute Myeloid Leukemia: A Useful Marker for Monitoring Residual Disease?

Bob Löwenberg, MD, PhD

Dr. Löwenberg indicated no relevant conflicts of interest.

Chou WC, Tang JL, Lin LI, et al. Nucleophosmin mutations in de novo acute myeloid leukemia: the age-dependent incidences and the stability during disease evolution. Cancer Res 2006;66:3310-6.

Gorello P, Cazzaniga G, Alberti F, et al. Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations. Leukemia 2006; [Epub ahead of print].

Acute myeloid leukemia (AML) is a highly heterogeneous disease both from a clinical and genetic point of view. Mutations in the nucleophosmin gene (NPM1) in AML were discovered in 2005. These mutations are seen in as many as one-third of cases, representing the most prevalent gene mutations in AML today. NPM1 mutations are reported to occur in 45 percent of cases of AML with normal karyotypes. NPM1 mutations are highly prevalent (60 percent) in association with the second-most-frequent category of FLT3 mutations in AML, the mutations of the so-called fms-like receptor tyrosine. FLT3 mutations express negative prognostic value (relapse, survival), but, in contrast, NPM1 mutations express favorable prognostic impact. NPM1 is a phosphoprotein that shuttles between nucleus and cytoplasm with physiologic functions that are thought to be multifold. Recent studies in NPM1 genetic mouse models show that its loss of function leads to loss of tumor suppression because NPM1 is involved in the p53 and p19^ARF pathway and maintenance of genomic integrity. The mutations affect exon 12 and usually lead to frame shifts that dislocate NPM from the nucleus, which determines the abnormal cytoplasmic retention of mutated NPM1.

The fact that NPM1 mutations are so frequently seen in AML has raised the question of the potential usefulness of NPM1 mutations as a marker for disease activity. Before the publication of these papers, a few recent studies had already shown stable mutations in direct comparisons of AML specimens at diagnosis and relapse, but those studies had not provided serial follow-up measurements.

Two recent papers by Chou et al. and Gorello et al. offer longitudinal monitoring with NPM1 quantitative polymerase chain reactions (PCR). The results of both studies suggest that NPM1 may furnish a convenient and stable marker for quantitative disease measurements during follow-up. In the first, Chou and colleagues found that none of 28 patients without NPM1 mutations acquired new mutations during a median follow-up interval of 16 months, while among 13 patients with AML and mutant NPM1, the mutations disappeared in all cases during complete remission. In all five NPM1-positive patients who relapsed, the same NPM1 mutations reappeared. Thus, NPM1 disappears during complete remission and comes back as the same abnormality at the time of relapse. The authors also found that in one patient with AML and NPM1 mutation, the mutation was no longer detected at second relapse. In the second paper, Goello et al. found that of 10 patients attaining complete remission, real-time quantitative PCR of NPM1 cDNA revealed a sharp decline of NPM1 mutated copies. Furthermore, in four of these, a subsequent increase in NPM1 copies closely correlated with relapse. The conclusions from the two studies are still preliminary, as the numbers of patients with adequate longitudinal follow up are small. The monitoring of minimal residual leukemia throughout treatment during morphological complete remission might provide an important strategy in clinical practice for decisions regarding treatment discontinuation and a timely treatment switch. Clearly, these early observations in two small series of patients need confirmation and extension.

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The Tumor Suppressor Menin Plays a Central Role in MLL-Associated Leukemias

Lilli Petruzzelli, MD, PhD

Dr. Petruzzelli indicated no relevant conflicts of interest.

Yokoyama A, Somervaille TC, Smith KS, et al. The menin tumor suppressor protein is an essential oncogenic cofactor for MLL-associated leukemogenesis. Cell 2005;123:207-18.

The mixed-lineage leukemia (MLL) protein is a histone methyl transferase that covalently modifies chromatin. In leukemia it is found in an array of chromosomal translocations, and its overexpression leads to enhanced Hox gene expression. This, in part, may contribute to the development of the leukemic phenotype and disruption of normal hematopoiesis. Interestingly, MLL, when it is not fused to another gene, acts by forming a complex with a set of proteins that are similar to a family of proteins that interact with histone methyl transferases in yeast; this complex is associated with a specific methylation event that results in the maintenance of gene expression. The MLL-fusion proteins that are seen in leukemia are no longer able to associate with this complex of proteins, but still result in the activation of gene transcription. In this paper, the investigators probe the molecular basis by which fusion proteins of MLL modulate gene expression and result in leukemic transformation.

Menin is known to bind to MLL, and this event is unique when compared to the highly-conserved binding partners of MLL that are shared with yeast. Menin is the protein product of Men-1, the tumor suppressor gene that is lost in tumors of heritable and sporadic endocrine disorders. Previous work demonstrated that menin still associates with the MLL-fusion protein even though other members of the complex do not. The experiments presented in this manuscript demonstrate that MLL and menin associate on Hox gene promoters. The proteins interact through specific motifs in the MLL portion of the fusion protein, and when specific regions are mutated, menin-MLL association is lost and cellular transformation is disrupted. The need for continued expression of menin to initiate and maintain transformation was specific for MLL - other fusion proteins that induce transformation did not have this requirement. Disruption of the interaction of an oncogenic fusion protein (MLL) with a tumor suppressor (menin) results in differentiation, and disrupts proliferation of leukemic blasts. Lastly, if menin expression is disrupted, Hox gene expression is diminished and cells undergo differentiation.

This work ties together several critical observations that characterize the mechanism for the formation and maintenance of a subset of leukemias. Although MLL is known to be a methyl transferase that activates Hox genes through the interaction of a series of highly conserved proteins, the activating fusion proteins that are seen in leukemia no longer act through this large protein complex but, rather, are able to set things in motion through the interaction with a single protein, menin. This event is so potentially interesting because it not only sheds mechanistic light on the regulation of cellular growth by a fusion protein, but also may lead to unique therapeutic options in at least this subset of leukemias.

Although one has to be concerned that targeting a tumor suppressor gene known to be associated with an array of endocrine tumors may lead to trouble, the investigators were able to finely map the interaction domains and found that at least two sites on MLL were involved in menin binding. Since there are multiple sites that can potentially be targeted, the potential therapeutic options in this interaction remain broad. Furthermore, high-affinity binding through a specific site on MLL was required for cellular transformation. A reduction in affinity of the MLL-menin interaction may be sufficient to abrogate the transformed phenotype. Agents that aim to diminish affinity of these proteins for each other may be sufficient to result in loss of maintenance of the leukemia cell phenotype that appears to be dependent on the continued presence of the MLL fusion protein-menin interaction, but may not affect other key regulatory components of the cellular homolog. With this insightful manuscript, the investigators advance understanding of the connection between a fusion protein product and its effect on cellular gene expression and growth, and also expand the horizon for potential drug targets.

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Mobilized Peripheral Blood Stem Cells for Cardiac Regeneration after Acute Myocardial Infarction - Back to the Repair Shop

Michael Linenberger, MD

Dr. Linenberger indicated no relevant conflicts of interest.

Zohlnhöfer D, Ott I, Mehilli J, et al. Stem cell mobilization by granulocyte colony-stimulating factor in patients with acute myocardial infarction: a randomized controlled trial. JAMA 2006;295:1003-10.

A great deal of excitement was generated five years ago when murine studies demonstrated that purified hematopoietic stem cells or cytokine-mobilized peripheral blood stem cells (PBSC) could engraft into ischemic myocardial tissue, regenerate functional myocardium, and thereby limit injury after acute myocardial infarction (MI). Phase I and I/II human studies quickly followed, with post-MI patients or patients with chronic ischemic heart disease receiving autologous progenitor cells isolated by various means and administered by a variety of different approaches (Table). Early trials with marrow-derived cells delivered by intracoronary infusion reported significant improvements in global left ventricular function and in regional perfusion and contractile function downstream of the infused vessel, suggesting a direct local effect of the cells and/or their trophic factors. The potential benefits of G-CSF administration after acute MI have been studied in two contexts: (1) as a means to collect high numbers of PBSC (by apheresis) for subsequent intracoronary infusion, and (2) to continuously perfuse the ischemic myocardium with a high level of PBSC to facilitate homing and engraftment of the cell types necessary for myocardial repair. In vitro studies also suggest that G-CSF can directly enhance cardiomyocyte survival, induce proliferation, and stimulate the release of proangiogenic mediators. Three phase I/II studies demonstrated the feasibility and relative safety of G-CSF when given for four to 10 days after acute MI, and each reported significant improvements in left ventricular function at three to six months after G-CSF. However, those trials were limited by lack of appropriate randomization, placebo controls, double-blind design, and/or adequate patient numbers.

In this paper, Zohlnhöfer et al. used a randomized double-blind, placebo-controlled design to assess the safety and benefit of G-CSF after an ST-segment elevation acute MI following successful reperfusion by percutaneous coronary stent placement. Fifty-six patients received G-CSF at 10 μg/kg/day for five days, starting five days after the MI, and 58 received placebo. G-CSF successfully mobilized progenitors, with a mean peak peripheral blood CD34+ cell count of 72/μL on day 5, and no major adverse events occurred. Blood CD34+ cell counts in the placebo group were also increased above baseline and ranged from 5 - 9/μL. Both groups showed a similar decrease in left ventricular infarct size at four and six months post-MI and a similar modest increase in left ventricular ejection fraction, with no significant difference between placebo and G-CSF. The rates of angiographic restenosis were also the same in both groups, suggesting that G-CSF was not prothrombotic in this setting.

This study illustrates the importance of a carefully designed, randomized clinical trial to address the benefit of a therapeutic intervention. Of note, a recently completed randomized double-blind, placebo-controlled trial from Denmark also found no benefit of G-CSF when given for six days after acute MI. Together, these observations suggest that the previous encouraging data reflected inadequate study design or critical differences in technical or biological variables. A number of fundamental issues need to be addressed including the optimal number, phenotype, and composition of the stem cell inoculum; the ideal timing of cell delivery; the most effective and safe route of administration; and the most relevant and specific end points. An additional consideration is that local paracrine factors, such as vascular endothelial growth factor-2, might significantly enhance the regenerative effect of CD34+ cells in ischemic myocardium. Thus, one might envision combination therapies using hematopoietic stem cells with systemic agents and/or specialized intracoronary stents that elute specific factors. Since cardiovascular disease is still the leading cause of death in the U.S. and Europe, and one million U.S. adults suffer an MI annually, continued aggressive pursuit of cell therapy approaches for acute and chronic ischemic heart disease appears justified. Despite the disappointing results of this trial, the collective evidence to date sustains the hope that the promise of regenerative medicine will first be realized for cardiovascular disease.

Figure 1

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