• Stem Cell and Gene Therapy to the Rescue of a Sick(le) Mouse! (Cover Story)
• HLA Matching and Unrelated Donor Hematopoietic Cell Transplantation: End of an Old Story?
• From Russia with Blood: An Animal Model of Chuvash Polycythemia
• Beyond Using ZIP Codes for Marketing
• A Better Appreciation of ATM Importance in CLL
• Identity of "Erythroid Regulator" of Iron Absorption Revealed?
• Novel Therapy for Relapsed Myeloma
• The Wasteland of Relapsed Adult ALL
• Caught in the Act: Megakaryocytes Delivering Platelets Into the Circulation

HLA Matching and Unrelated Donor Hematopoietic Cell Transplantation: End of an Old Story?

By Gérard Socié, MD, PhD

Dr. Socié indicated no relevant conflicts of interest.

Lee SJ, et al. High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood. 2007;110:4576-83.

Shaw BE, et al. The importance of HLA-DPB1 in unrelated donor hematopoietic cell transplantation. Blood. 2007;110:4560-6.

In the last issue of Blood in 2007, two articles reported results of transplants from unrelated donors using HLA matching. The journal article by Stephanie Lee and coworkers addresses the relative importance of various HLA loci and the resolution level at which they are matched for unrelated donor transplantation. To address this question, National Marrow Donor Program data from 3,857 U.S. transplantations performed from 1988 to 2003 were analyzed. Patient-donor pairs were fully typed for HLA-A, B, C, DRB1, DQB1, DQA1, DPB1, and DPA1 alleles (see Figure). High-resolution DNA matching for HLA-A, B, C, and DRB1 (8/8 match) was the minimum level of matching associated with the highest survival. A single mismatch detected by low- or high-resolution DNA testing at HLA-A, B, C, or DRB1 (7/8 match) was associated with higher mortality (relative risk 1.25, p<0.0001) and one-year survival of 43 percent compared to 52 percent for 8/8 matched pairs. Single mismatches at HLA-B or HLAC appear better tolerated than mismatches at HLA-A or HLA-DRB1. Mismatching at two or more loci compounded the risk. Mismatching at HLA-DP or DQ loci and donor factors other than HLA type were not associated with survival.

In the second paper, Brownen Shaw, et al. examined the extent to which transplant outcome may be improved with donor matching for HLA-DP. The risks of acute GVHD, relapse, and mortality associated with HLA-DPB1 allele mismatching were determined in 5,929 patients who received a myeloablative HCT from an HLA-A, B, C, DRB1, DQB1 matched or mismatched donor. There was a statistically significantly higher risk of both grade II-IV aGVHD (OR=1.33, p<0.0001) and grade III-IV aGVHD (OR=1.26, p=0.0007) after HCT from an HLA-DPB1 mismatched donor compared to a matched donor. The increased risk of acute GVHD was accompanied by a statistically significant decrease in disease relapse (HR=0.82, p=0.01).

Historically, the selection of potential donors for hematopoietic cell transplantation (HCT) relied on serological tests for donor-recipient identity for classical HLA-A, B, and DR antigens. Later, as molecular methods for typing HLA genes became available, the degree of diversity of this region became apparent. When such typing methods were applied to the analysis of HCT recipients and donors, many serologically identical, unrelated donor recipients were found to harbor mismatches between two unique alleles of the same antigen. Clinical studies have demonstrated that such allelic differences are clinically relevant, increasing the risks of graft failure, acute GVHD, and mortality.

The first study by Lee and coworkers clearly highlights the fact that there is a 10 percent loss of survival with any mismatch (although, of marginal significance for HLA-DQ), but also that classical risk factors including patient age, race, disease stage, and CMV status were as predictive of survival as donor HLA matching. The second study by Shaw and coworkers confirms in a large number of patients that DPB1 functions as a classical transplantation antigen. The increased risk of GVHD associated with HLA-DPB1 mismatching is accompanied by a lower risk of relapse.

Numerous studies have looked at the impact of HLA matching and transplant outcomes. However, some older data did not include molecular typing for all HLA alleles, thus bringing into question some of the reported results. The data reviewed here (and reported in a few other articles) have led to the current gold standard of a fully matched "10/10" unrelated donor being one who is allele matched for HLA-A, B, C, DRB1, and DQB1. Few patients have a 12/12 donor (also matched for HLA-DPB1), which means they may have less of a risk of GVHD, but more of a risk of relapse. We already know that even a 10/10 molecularly matched donor/recipient pair have three out of four chances to be haplotype-mismatched¹ and of greater disparities if typed for other non-HLA gene polymorphism or for KIR… Thus, in 2008, it’s probably time to stop using the term matched for an unrelated donor (unless specified by the number of molecularly tested loci). However, compelling evidence coming from large series strongly suggests that transplant from 10/10 molecularly matched donors led to results (survival) similar to those obtained with HLA-identical sibling donors — a finding that, in the end, constitutes the only relevant criterion for physicians.

1. Petersdorf EW, Malkki M, Gooley TA, et al. MHC haplotype matching for unrelated hematopoietic cell transplantation. PLoS Med. 2007;4:e8.

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From Russia with Blood: An Animal Model of Chuvash Polycythemia

By Charles Parker, MD

Dr. Parker indicated no relevant conflicts of interest.

Hickey MM, Lam JC, Bezman NA, et al. von Hippel-Lindau mutation in mice recapitulates Chuvash polycythemia via hypoxia-inducible factor-2alpha signaling and splenic erythropoiesis. J Clin Invest. 2007;117:3879-89.

Chuvashia is in the center of European Russia with the capital, Cheboksary, located on the Volga River, approximately 400 miles east of Moscow. During the 1970s, Russian scientists, principally Dr. Lydia Polyakova, began characterizing patients from Chuvashia with a familial form of erythrocytosis that differed from classical polycythemia vera. During a 10-year period, approximately 100 patients were identified.¹ In 2002, Prchal and colleagues² identified a homozygous cytosine to thymidine transition affecting nucleotide 598 (C598T) of the von Hippel-Lindau (VHL) gene in patients with Chuvash polycythemia. Subsequently, homozygosity of C598T was reported in sporadic and familial congenital polycythemia from a number of diverse ethnic groups, and a cluster of Chuvash polycythemia was identified on the Italian island of Ischia (in the Bay of Naples).³ The haplotype of non-Chuvash patients is the same as that of patients from Chuvashia, supporting the concept of a single founder.

How does VHL C598T cause polycythemia? The VHL protein (pVHL) is the substrate recognition component of an ubiquitin ligase complex that, under conditions of normoxia, targets the hydroxylated form of the α-subunit of hypoxia inducible factors (HIFs) for polyubiquination and proteosomal degradation. But under conditions of hypoxia, HIFs are stable because the α-subunit is ineffectively hydroxylated, preventing recognition by pVHL. C598T is a missense mutation that results in substitution of tryptophan for arginine at amino acid 200 (R200W). This amino acid substitution affects one of the two HIF binding sites of pVHL, ultimately resulting in reduced HIF degradation, thereby mimicking the hypoxic state. Support for this concept is provided by studies showing that, under normoxic conditions, cellular expression of HIF-1α is higher in patients with Chuvash polycythemia than in controls.⁴ HIFs are transcription factors. (HIF-1α and HIF-2α are the primary HIF isoforms.) Among the many important genes that are regulated by HIFs are erythropoietin (EPO), vascular endothelial growth factor (VEGF), plasminogen activator inhibitor-1 (PAI-1), glucose transporter-1 (Glut-1), and transferrin receptor. Patients with Chuvash polycythemia have hemoglobin-adjusted serum erythropoietin concentrations approximately 10-fold greater than controls, thereby providing an explanation for the polycythemia component of the phenotype.¹

To investigate further the biological consequences of the Chuvash mutation, Hickey and colleagues generated mice homozygous for VHL C598T using homologous recombination. Many of the features of the human disease, including polycythemia and abnormally high serum concentrations of erythropoietin and VEGF, were observed in the murine model (see Table). Differences between the human disease and the murine model are also noteworthy (see Table). For example, in the murine model, the difference in serum erythropoietin concentration between the homozygous VHL C598T mouse and wild type was 1.3-fold versus 10-fold greater than controls in patients with Chuvash polycythemia. The spleens of homozygous VHL C598T mice were remarkable for a strikingly greater number of erythroid progenitors and megakaryocytes and for increased in vitro erythroid differentiation. This latter property is also a feature of the erythroid precursors of Chuvash polycythemia. Whether the splenic phenotype observed in the murine model is a feature of the human disease is unknown, although clinical splenomegaly is uncommon in Chuvash polycythemia. While the Chuvash polycythemia has been attributed to aberrant expression of HIF-1α,⁴ HIF-2α principally mediates the process in the murine model. Whether this difference is truly species-specific or due to differences in experimental design is uncertain, although a recent report supports the hypothesis that the erythropoietin gene is an HIF-2α specific target in humans⁵ (see Table). Further studies using the murine model will provide additional insights into the function of pVHL and into the mechanisms that underlie the remarkable Chuvash polycythemia phenotype (see Table).

1. Gordeuk VR, Sergueeva AI, Miasnikova GY, et al. Congenital disorder of oxygen sensing: association of the homozygous Chuvash polycythemia VHL mutation with thrombosis and vascular abnormalities but not tumors. Blood. 2004;103:3924-32.
   
   
2. Ang SO, Chen H, Gordeuk VR, et al. Endemic polycythemia in Russia: mutation in the VHL gene. Blood Cells Mol Dis. 2002;28:57-62.
   
   
3. Perrotta S, Nobili B, Ferraro M, et al. Von Hippel-Lindau-dependent polycythemia is endemic on the island of Ischia: identification of a novel cluster. Blood. 2006;107:514-9.
   
   
4. Ang SO, Chen H, Hirota K, et al. Disruption of oxygen homeostasis underlies congenital Chuvash polycythemia. Nat Genet. 2002;32:614-621.
   
   
5. Percy MJ, Furlow PW, Lucas GS, et al. A gain-of-function mutation in the HIF2A gene in familial erythrocytosis. N Engl J Med. 2008;358:162-8.

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Beyond Using ZIP Codes for Marketing

By Nelson Chao, MD

Dr. Chao indicated no relevant conflicts of interest.

Rao K, Darrington DL, Schumacher JJ, et al. Disparity in survival outcome after hematopoietic stem cell transplantation for hematologic malignancies according to area of primary residence. Biol Blood Marrow Transplant. 2007;13:1508-14.

As a community, we have learned much about risk factors for patients undergoing hematopoietic cell transplantation (HCT). Some factors reflect the patient and his or her physique. The most primitive scale has been the Karnofsky or the ECOG performance statuses, both of which are still widely used but are fairly crude measurements. The more recent adoption of comorbidity scales has significantly improved our ability to predict which patients are at very high risk for HCT. Other factors reflect the malignancy, such as the stage of the disease, the remission status, the cytogenetics of the tumor, disease sites, and number of prior therapies, to name a few. These measurements reflect the individual and the biology of the malignancy in that particular person ― more of the nature of the disease. But what about nurture? Specifically, are there factors in the patient’s environment that could make a difference?

Some of the earliest applications of ZIP code data were for use in customer acquisition promotions, direct mail, and telemarketing. If one lived in a high-rent district, it would be a prime area for marketing. In contrast, poor or rural areas were spared. The rationale was obvious in that the high-rent districts had access to disposable income and perhaps better services. This process of targeting specific ZIP codes has continued precisely because it worked well. If this is the case, then the reverse should also be true. Poorer or rural areas could be expected to have less disposable income and poorer services, including access to health care.

A recent paper published in Biology of Blood and Marrow Transplantation explored whether a patient’s area of primary residence is an independent risk factor for overall survival (OS) after HLA-identical sibling or autologous HCT. This was a retrospective cohort study that included patients who had undergone autologous (n=1739) or HLA-identical sibling (n=267) HCT to treat a hematologic malignancy between 1983 and 2004 at the University of Nebraska Medical Center. Primary area of residence, using the patient’s ZIP code, was categorized as either urban or rural (including isolated, small rural, or large rural), according to the Rural Urban Commuting Area Codes (RUCA) classification system. An association between area of primary residence and survival was examined using Cox proportional hazards regression analysis while adjusting for patient-, disease-, and treatment-related variables. Patients from rural areas who received autologous HCT had a higher relative risk (1.18) of death compared to urban patients. Survival rates in patients from rural and urban locations were as follows: one year, 73 percent versus 78 percent (P=.04); five year, 48 percent versus 54 percent (P=.012). There were no differences in the allogeneic population, although the number of patients was more limited.

It is important to recognize that this was a retrospective analysis, and thus there were no controls for important variables such as comorbidities, income, distance traveled, educational status, etc. However, these data raise interesting questions since the disparity in survival was related to treatment toxicity. Many of these toxicities, especially those in the intermediate to long term, are preventable or treatable if detected early. Moreover, some of the toxicities, such as interstitial pneumonitis in autologous HCT, are frequently recognized and treated in transplant centers. However, in a more rural community under the care of a general practitioner, this complication may not be readily recognized and treated. The distance that a patient has to travel may also affect how often or even whether a patient wants to return for a follow-up visit to a transplant center. These data do raise important issues for planning for discharge, and a comprehensive plan for communications and follow-up should be in place for all patients. While health-care inequities are not new, they should not be ignored.

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A Better Appreciation of ATM Importance in CLL

By John C. Byrd, MD

Dr. Byrd indicated no relevant conflicts of interest.

Austen B, Skowronska A, Baker C, et al. Mutation status of the residual ATM allele is an important determinant of the cellular response to chemotherapy and survival in patients with chronic lymphocytic leukemia containing an 11q deletion. J Clin Oncol. 2007;25:5448-57.

Chronic lymphocytic leukemia (CLL) represents one of the most common types of leukemia in adults and has a widely variable natural history in terms of time to development of symptoms and overall survival. Several well-designed studies using classic karyotype analysis and then interphase cytogenetics demonstrated that patients with either of two genetic abnormalities, del(11q22.3) or del(17p13.1), have a reduced time to developing symptoms, abbreviated remission following therapy, and ultimately shorter survival. Because del(11q22.3) is more frequently observed, much attention has been focused on determining which gene(s) are lost at the 11q22.3 locus that lead to poor prognosis. While several coding and non-coding genes are found in this minimally deleted region of del(11q22.3), the ataxia telangiectasia mutated (ATM) gene has generated the most interest due to its known importance in double-stranded DNA repair as well as p53 activation with subsequent cell cycle arrest and/or apoptosis. ATM is a very large gene (more than 150 kb and 62 coding exons) that makes detailed study of single nucleotide polymorphisms (SNPs) and mutations challenging. Furthermore, loss of gene function in the absence of a dominant mutation generally requires loss of the alternative allele through mutation or epigenetic silencing; thus, cases with genetic defects may or may not have complete loss of protein function. Despite this, several groups have examined CLL cells for evidence of ATM mutations and found these to be present in up to 30 percent of patients. Until recently, we have been left with the question of whether ATM mutations and del(11q22.3) represent an actual mechanism of pathogenesis and progression in CLL, or are instead only surrogate markers of genomic instability. In a recent issue of the Journal of Clinical Oncology, the authors advance our understanding of the importance of del(11q22.3) in CLL by taking a unique, comprehensive approach to address the importance of loss of one versus two alleles of ATM. Their data demonstrate that ATM mutations are quite common (36 percent) in patients bearing the del(11q22.3) abnormality, and that the loss of one allele by deletion and the other by functional mutation results in complete loss of ATM function. Most importantly, they demonstrate that this loss of function occurs with a detrimental phenotype, with these patients having a significantly reduced survival. As would be expected with silencing of a tumor suppressor gene important in the pathogenesis of a disease, allele-specific deletions occurred at the greatest percentage when mutations were present in the alternative allele, and with disease progression in a subset of patients, mutations and increased percentage deletions developed concurrently. Unlike many other adverse prognostic factors [i.e., del(17p13.1), ZAP-70 over-expression, unmutated IgVH gene status], ATM mutations appear to be independent in this analysis. This paper, therefore, provides both an important biomarker for predicting CLL outcome and firm evidence that ATM is an important gene in the pathogenesis of CLL.

Beyond describing "just another biomarker," the authors of this paper are to be commended for performing detailed functional assessment of ATM in which they show that CLL patient cells bearing both ATM mutations and deletions respond to DNA-damaging agents differently at a protein level with respect to functional pathways. For many years, it has been known that irradiation and select DNA-damaging agents activate the ATM pathway and that cells deficient in this pathway are more sensitive to both apoptosis and also eventual transformation presumably through acquisition of additional genomic mutations. The finding of true loss of ATM function in a subset of CLL with an associated phenotype provides a justification to generate novel therapies for this patient population. CLL cells with dysfunctional ATM should be unique from all other cells in the body, potentially making it possible to develop agents that target only these cells. Additionally, this work raises other questions that now should be aggressively pursued with respect to ATM investigation. For example, are there alternative pathways such as epigenetic silencing by promoter methylation or over-expression of miRNAs that silence translation of this important protein? Might there be specific SNPs in ATM that produce variable transcription, translation, or splicing and ultimately lead to protein dysfunction? As we move forward with investigative studies in CLL, Dr. Stankovic’s extensive work clearly supports detailed study of ATM as well as therapies that can target cells with ATM defects.

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Identity of "Erythroid Regulator" of Iron Absorption Revealed?

By Sabina I. Swierczek, PhD, and Josef T. Prchal, MD

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

Tanno T, Bhanu NV, Oneal PA, et al. High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nat Med. 2007;13:1096-101.

Our understanding of the regulation of iron homeostasis has been greatly facilitated by the discovery of the iron-regulatory protein hepcidin. This small peptide regulates cellular iron export by binding and triggering internalization and degradation of the iron export protein ferroportin. This alters the availability of iron by shifting the serum iron away from erythropoiesis to macrophages and by inhibiting iron absorption by the duodenum.¹ As such, an understanding of the regulation of hepcidin has been central to the pathophysiology of anemia of chronic disease (inflammation induces hepcidin expression by the IL-6 pathway) and various types of hemochromatosis (wherein hepcidin is low).² However, while understanding hepcidin regulation largely clarified its interaction with iron control mechanisms that were coined by a seminal paper of Clem Finch as a "stores regulator," "hypoxia regulator," and "inflammatory regulator," it failed, however, to explain the increased iron absorption seen in thalassemias and sideroblastic anemias. This mysterious crosstalk between hyperactive inefficient erythropoiesis and anemia was termed the "erythroid regulator."³ Interestingly, in some anemic states with hyperactive erythropoiesis, such as in sickle cell disease, hepcidin is not decreased, and clinically significant iron overload is uncommon, while in thalassemia major and intermedia hepcidin is markedly decreased with a tendency to iron overload that is unrelated to red cell transfusions.⁴

The study from Jeff Miller’s laboratory is an important step in solving the identity of the erythroid regulator. In their paper, Tanno and colleagues propose that iron overload in ß-thalassemia patients may result from inhibition of hepcidin by high levels of growth differentiations of factor 15 (GDF15), a member of the transforming growth factor-ß (TGFB) superfamily. They followed up on a well-established fact that in patients with ß-thalassemia, hepcidin expression is decreased, speculating that the thalassemic erythroid precursors produce a hepcidin repressor during their hyperactive and largely apoptotic maturation. Based on this hypothesis, the authors examined transcriptional profiles of TGFB members in primary erythroblasts and detected increased expression and secretion of GDF15 during erythroblast maturation in normal controls. The investigators then hypothesized that elevated numbers of apoptotic erythroblasts in patients with thalassemia might produce increased levels of GDF15. Indeed, they report that in some ß-thalassemia patients, serum levels of GDF15 are dramatically increased and that the serum concentration of GDF15 correlated with elevated erythroblast numbers and iron overload in ß-thalassemia. To examine the regulation of hepcidin expression by GDF15, the authors measured hepcidin expression in primary hepatocytes (cells producing hepcidin) that were exposed to the sera with normal and elevated levels of GDF15, and found that hepcidin production was suppressed by high levels of GDF15. Very high levels of GDF15, however, could not completely suppress hepcidin expression. They also noted that depletion of GDF15 significantly increased hepcidin expression in ß-thalassemia serum. These observations suggest that erythroblast-produced GDF15 is involved in the suppression of hepcidin production and thus leads to augmented iron absorption in ß-thalassemia. However, these studies also suggest that GDF15 is not the sole regulator of hepcidin expression by hyperactive erythropoiesis.

Other disorders involved with ineffective erythropoiesis or unexplained iron-loading pathology are being tested for GDF15 overexpression. Tanno and colleagues have also evaluated a small number of MDS patients and have shown a much lower level of GDF15 compared with levels seen in ß-thalassemia. These data, and data that conflicted with the central role of GDF15, presented by Photis Beris at this year’s ASH annual meeting based on studies of other anemic disorders associated with iron overload and ineffective erythropoiesis, suggest that the identity of the erythroid regulator of hepcidin expression is still not fully resolved. However, in our judgment, this paper is an important step toward the eventual elucidation of the still mysterious erythroid regulator of iron absorption.

1. Papanikolaou G, Tzilianos M, Christakis JI, et al. Hepcidin in iron overload disorders. Blood. 2005;105:4103-5.
   
   
2. Hentze MW, Muckenthaler MU, Andrews NC. Balancing acts: molecular control of mammalian iron metabolism. Cell. 2004;117:285-97.
   
   
3. Finch C. Regulators of iron balance in humans. Blood. 1994;84:1697-702.
   
   
4. Nemeth E, Ganz T. Hepcidin and iron-loading anemias. Haematologica. 2006;91:727-32.

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Novel Therapy for Relapsed Myeloma

By Kenneth Anderson, MD

Dr. Anderson indicated no relevant conflicts of interest.

Dimopoulos M, et al. Lenalidomide plus dexamethasone for relapsed or refractory multiple myeloma. N Engl J Med. 2007; 357: 2123-32.

Weber DM, et al. Lenalidomide plus dexamethasone for relapsed multiple myeloma in North America. N Engl J Med. 2007; 357: 2133-42.

The immunomodulatory drug lenalidomide is more potent than thalidomide in preclinical studies, targeting multiple myeloma (MM) cells in the bone marrow (BM) and overcoming cell-adhesion-mediated drug resistance to conventional therapy by directly inducing apoptosis, even of MM cells resistant to conventional and novel therapies; downregulating adhesion molecules and binding of MM cells to the BM milieu; inhibiting secretion of cytokines mediating MM cell growth, survival, and drug resistance; inhibiting angiogenesis as well as upregulating natural killer and cytolytic T-cell responses against autologous tumor cells.¹ Phase I trials rapidly established the maximal tolerated dose of 25 mg for 21 of 28-day cycles, the low incidence of neuropathy, somnolence, and constipation attendant to thalidomide use, and, even more remarkably, evidence of anti-MM activity in the majority of patients.² Phase II trials then established single daily dosing and the addition of dexamethasone to optimize tolerability and anti-tumor activity, respectively,³ setting the stage for these two randomized phase III trials comparing lenalidomide plus high-dose dexamethasone versus high-dose dexamethasone plus placebo in 353 and 351 patients with relapsed or refractory MM.

Remarkably, response frequency and extent, as well as time to progression and overall survival, were statistically significantly improved in the lenalidomide-plus-dexamethasone-treated cohorts, with nearly identical data in both studies strengthening the validity of the data. Specifically, in the North American study, rates of partial response or better, complete response, time to progression, and overall survival were statistically significantly improved (p<0.001) in patients treated with lenalidomide plus dexamethasone at 61 percent, 14.1 percent, 11.1 months, and 29.6 months, respectively, versus 19.9 percent, 0.6 percent, 4.7 months, and 20.2 months, respectively, in the dexamethasone-treated cohort. In the other study, rates of partial response or better, complete response, and time to progression were statistically significantly improved (p<0.001) in patients treated with lenalidomide plus dexamethasone at 60.2 percent, 15.9 percent, and 11.3 months, respectively, versus 19.9 percent, 0.6 percent, and 4.7 months, respectively, in the dexamethasone-treated cohort. Overall survival was also significantly improved, with a hazard ratio for death of patients treated with lenalidomide of 0.66 (p=0.03). Side effect profiles in both studies were also remarkably similar, with grade 3 or 4 thrombocytopenia and thromboembolism more common in the lenalidomide groups than in the placebo groups.

These studies represent a major advance, further validating the paradigm of targeting the MM cell in its BM microenvironment to overcome drug resistance, and have established a new treatment option for these patients. They are a remarkable example of rapid, collaborative bench-to-bedside research. Responses occurred in patients who had received prior thalidomide and high-dose therapy and stem cell transplant. Most importantly, both the FDA and EMEA have approved lenalidomide plus dexamethasone for treatment of patients who have had one prior therapy for their MM. Remaining issues now to be defined include the utility of high versus low dexamethasone plus lenalidomide, given the survival advantage of the latter when this combination is used as initial therapy for newly diagnosed MM patients, as well as optimal anticoagulation prophylaxis. Going forward, lenalidomide is already showing great promise when used in combination with conventional agents to treat newly diagnosed patients, including its combination with dexamethasone for transplant candidates and melphalan and prednisone for non-transplant patients. In addition, it is being combined with novel agents predicated upon preclinical rationale, with bortezomib to induce dual apoptotic signaling or with monoclonal antibody therapy to enhance antibody-dependent cellular cytotoxicity. Finally, given that it is an oral and well-tolerated agent, multiple studies are now evaluating its utility as a maintenance therapy.

1. Hideshima T, Mitsiades C, Tonon G, et al. Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nat Rev Cancer. 2007;7:585-98.
   
   
2. Richardson PG, Schlossman RL, Weller E, et al. Immunomodulatory drug CC-5013 overcomes drug resistance and is well tolerated in patients with relapsed multiple myeloma. Blood. 2002;100:3063-7.
   
   
3. Richardson PG, Blood E, Mitsiades CS, et al. A randomized phase 2 study of lenalidomide therapy for patients with relapsed or relapsed and refractory multiple myeloma. Blood. 2006;108:3458-64.

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The Wasteland of Relapsed Adult ALL

By Jerald P. Radich, MD

Dr. Radich indicated no relevant conflicts of interest.

Fielding AK, Richards SM, Chopra R, et al. Outcome of 609 adults after relapse of acute lymphoblastic leukemia (ALL); an MRC UKALL12/ECOG 2993 study. Blood. 2007;109:944-50.

Goldstone AH, Richards SM, Lazarus HM, et al. In adults with standard-risk acute lymphoblastic leukemia (ALL) the greatest benefit is achieved from a matched sibling allogeneic transplant in first complete remission (CR) and an autologous transplant is less effective than conventional consolidation/maintenance chemotherapy in ALL patients: final results of the international ALL trial (MRC UKALL XII/ ECOG E2993). Blood. 2007. [Epub ahead of print]

The advance of therapy for pediatric ALL is a success story that oncology can revel in; it also places the bar very high for the rest of the oncology landscape. Whereas pediatric ALL therapy can cure upward of 80 percent of patients, success in adult ALL is stuck at a rate of ~40 percent. Indeed, some pediatric trials are using risk stratification to give less therapy to some cases. This is a mind-boggling luxury for those physicians who work in the adult arena.

Two recent papers have direct relevance to how we should be treating adult ALL. The first, by Fielding, et al., examines the fate of adult ALL cases that relapse after therapy. The paper is a clear, illuminating, and utterly depressing read. The overall five-year survival for cases who relapsed was only 7 percent. This bleak result was not influenced by the type of therapy the patients initially received (i.e., chemotherapy or transplantation). For those who received chemotherapy following transplant, only 4 percent survived. Patients who were "salvaged" with allogeneic transplant fared slightly better, with a survival of 23 percent. The paper’s overwhelming conclusion is that there is one good chance to cure patients, and that more toxicity up front might be a reasonable tradeoff for a decreased relapse rate.

The recent results of the MRC/ECOG trial of front-line therapy for ALL suggest that allogeneic transplantation might offer the best chance for cure. In this trial, patients who had a donor were eligible for transplant in first CR; those without a donor were potentially randomized to autologous or chemotherapy. Of the 1,929 patients entering the trial, 1,031 were HLA typed, and 443 patients had an HLA-matched related donor, while 588 did not. Analysis was performed based on the donor status. The study suggests that, for Ph-chromosome-negative patients, more intense therapy is associated with greater success, with overall survival in allogeneic >autologous> chemotherapy (~53 percent vs. 45 percent vs. 37 percent, respectively). The advantage of allogeneic transplantation did not appear to be as strong in high-risk ALL, due to an unusual amount of non-relapse mortality among the transplant recipients. There are, of course, the usual caveats attached to such complex studies: worries about dropout rate, differences in the percentages of patients acceptable to randomization, analysis by "intent" rather than actual treatment, etc.

Overall survival in relapsed adult ALL patients, divided by salvage therapy. Figure from Fielding AK, Richards SM, Chopra R, et al. Outcome of 609 adults after relapse of acute lymphoblastic leukemia (ALL); an MRC UKALL12/ECOG 2993 study. Blood. 2007;109:944-50.

Overall survival of Ph-negative adult ALL cases divided by the presence or absence of a donor. Figure from Goldstone AH, Richards SM, Lazarus HM, et al. In adults with standard-risk acute lymphoblastic leukemia (ALL) the greatest benefit is achieved from a matched sibling allogeneic transplant in first complete remission (CR) and an autologous transplant is less effective than conventional consolidation/maintenance chemotherapy in ALL patients: final results of the international ALL trial (MRC UKALL XII/ECOG E2993). Blood. 2008;111:1827-33.

A reasonable interpretation of the data would suggest:

1. Adult ALL patients should be offered a clinical trial whenever possible. The prevailing therapy is not good enough to be a "standard" therapy.
   
   
2. Allogeneic transplantation should be considered in patients who are at good risk. Given that, at many centers, unrelated transplant results are similar to related, both avenues of donors should be considered.
   
   
3. Physicians should employ the most rigorous methods of minimal residual disease detection, either flow cytometric or molecular (i.e., immunoglobulin heavy-chain VDJ rearrangements). Physicians should demand that their centers adopt technology that can detect at least one leukemic cell in a background of >1,000 normal cells, since this level of detection has been shown to reliably predict relapse.
   
   
4. More money needs to be spent studying the biology of ALL. Except for Ph+ ALL, the "targeted" therapeutic revolution has passed over ALL. Much discovery is needed in adult ALL if we wish to ever approach the success of our pediatric colleagues in this disease.

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Caught in the Act: Megakaryocytes Delivering Platelets Into the Circulation

By Robert Flaumenhaft, MD, PhD

Dr. Flaumenhaft indicated no relevant conflicts of interest.

Junt T, Schulze H, Chen Z, et al. Dynamic visualization of thrombopoiesis within bone marrow. Science. 2007;317:1767-70.

Just over a 100 years ago, James Homer Wright, observing that platelets shared tinctorial properties with giant bone marrow cells (now termed megakaryocytes), postulated that platelets were fragments of these large cells. Two models have since evolved to explain how platelets are derived from megakaryocytes. The flow model proposes that megakaryocytes elaborate long pseudopodia that bend, branch, and bulge. The bulges that develop along the length and ends of these pseudopodia form proplatelets, which mature into platelets. The platelet territory model proposes that megakaryocytes fragment along internal demarcation membranes to form platelets. These models were based largely on either analysis of high-resolution snapshots of thrombopoiesis or by observations of cultured megakaryocytes. But how does thrombopoiesis actually occur in the bone marrow?

In a recent article in Science, Junt and colleagues address this question by viewing thrombopoiesis directly in living mice. The cranial marrow cavity was visualized in transgenic mice expressing a targeted enhanced yellow fluorescent protein chimera fused to CD41, an integrin that is expressed exclusively in megakaryocytes and platelets. The investigators found that most megakaryocytes were perivascular and sessile. Treatment with thrombopoietin, however, increased the number of mature megakaryocytes demonstrating irregular shapes and exhibiting fragmented protrusions. Imaging also showed cellular processes extending into microvessels and releasing heterogeneous fragments resembling immature proplatelets into the bone marrow microvasculature. In addition, megakaryocytes extended plump perivascular pseudopodia into the microvessels. Shedding was accomplished primarily by large megakaryocytes with branched, irregular shapes. Quantitation of the frequency of shedding events indicated that this mechanism of platelet production accounted for the majority of circulating platelets.

Junt, et al. observed how mature megakaryocytes in mouse bone marrow routinely extend protrusions (proplatelets) into blood vessels. Sheer stress from blood flow fragments these protrusions, generating platelets. Legend and figure from Geddis AE, Kaushansky K. Immunology: The root of platelet production. Science. 2007;317:1689-91. Illustration: Preston Huey. Reprinted with permission from AAAS.

The observation that megakaryocytes extended into bone marrow microvessels to release immature proplatelets predicts that immature platelet forms will be found in the circulation. Indeed, evaluation of peripheral blood demonstrated multiple beaded proplatelets and barbell-shaped platelets. Previous studies have found that proplatelet counts are increased in prepulmonary arterial vessels while platelet counts are increased in post-pulmonary vessels. These observations indicate that platelet maturation continues following release of platelet fragments into the circulation.

In addition to demonstrating that megakaryocytes form proplatelets from extended pseudopodia in vivo, these studies suggest a role for shear force in thrombopoiesis. The observation that fragmentation of megakaryocytes occurred within blood vessels implicates shear force as a contributing factor. Furthermore, the investigators showed that megakaryocytes cultured with agitation demonstrated increased proplatelet formation compared with those cultured under static conditions.

Visualization of megakaryocytes within bone marrow of living mice demonstrates that pseudopodia extend into the bone marrow vasculature and release fragments that subsequently mature into circulating platelets. These observations validate earlier studies that showed pseudopodia and proplatelet formation in cultured megakaryocytes. This work also implicates shear force as a contributing factor in megakaryocyte fragmentation and proplatelet maturation. The ability to visualize thrombopoiesis in vivo will provide for a more comprehensive analysis of thrombocytopenic states than has previous been possible. The effects of inflammatory conditions, infections, and congenital disorders on megakaryocyte maturation, proplatelet development, and shedding can now be evaluated directly and in live mammals. The in vivo model may also be useful in the characterization of thrombopoiesis-stimulating agents. The ability to visualize the events leading to platelet production in vivo is a landmark achievement and will, in a very real sense, change the way we look at thrombopoiesis.

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