• Computed Tomographic Pulmonary Angiography – Too Good to Be True? (Cover Story)
• Performance-Enhancing Drugs
• Bedside-to-Laboratory Translational Work Identifies Novel Tumor-Specific Antigen
• GVHD Has Proteomics Come of Age?
• Role of miR223 in Myelopoiesis
• Clinical Penetrance of HFE Hereditary Hemochromatosis, Serum Ferritin Levels, and Screening Implications: Can We Iron This Out?
• Combination Proteasome Inhibitor Therapy

Performance-Enhancing Drugs

By Nelson Chao, MD

Dr. Chao indicated no relevant conflicts of interest.

Napolitano LA, Schmidt D, Gotway MB, et al. Growth hormone enhances thymic function in HIV-1-infected adults. J Clin Invest. 2008;118:1085-1098.

Did Mark McGwire use it or not? We all have our opinions, but we do know that his wife used it. The drug is human growth hormone. Human growth hormone (HGH) is known to improve muscle strength and has been claimed as an anti-aging agent because it can increase lean body mass and bone mineral density, although there are limited data to support this claim. However, growth hormone is important in thymopoiesis. Administration of HGH or its proximal mediator, insulin-like growth factor-1 (IGF-1), can reverse thymic involution in aging mice and accelerate immune recovery in immune-compromised animals, including hematopoietic cell transplants (HCTs). HGH is produced by thymocytes, thymic epithelial cells, and mature lymphocytes. Under steady state, HGH and IGF-1 do little, but under periods of stress, they do have a beneficial effect.

There are accumulating data suggesting that following ablative HCT, especially in adults in the setting of T-cell depletion or cord blood transplantation, lack of adequate immune reconstitution is an important source of morbidity and mortality. We know that the thymus is the major site for the generation of new T cells and that functional recovery of the thymus is likely the single most important factor in allowing a patient to recover his or her adaptive immunity. Therefore, methods to improve and accelerate thymic function are an area of great interest and clinical need.

In this article, investigators performed a prospective, randomized study in 22 HIV-1–infected adult patients who were on highly active antiretroviral therapy (HAART). They received either daily subcutaneous injections of HGH for 12 months or were observed and then were crossed over to the other arm. This study confirmed the group’s previous observation that there was an increase in thymic mass and moderate increased numbers of naïve CD4+ T cells. HGH use increased de novo T-cell production as measured by the T-cell receptor excision circles (TRECs) and also increased peripheral T-cell expansion. An increase in thymic mass was also documented by CT scans. It is not clear how long this increase was sustained after the completion of the study.

Can we apply this study to patients receiving an HCT? In many ways, patients receiving an HCT can also have a significant impairment in T-cell numbers and function. CD4+ T cells may take several years to normalize, and peripheral homeostatic proliferation of a limited T-cell repertoire may result in significant skewing of these T cells as demonstrated by TCR spectratyping results. The overall result may be a limited ability of T cells to respond to new challenges. This restricted response can also be seen in aging individuals’ response to viruses, where fewer naive T cells result in a lower frequency of responder cells.

Would HGH or IGF-1 work in HCT? Based on the results reported above, the expectation is that this approach could have a beneficial effect on immune recovery with certain caveats. For example, the effects of prior radiation to the chest or total body irradiation will destroy thymic epithelial cells. Graft-versus-host disease (GVHD) is another complication that has a direct effect on the thymus. In these two circumstances, there may not be sufficient residual thymic tissue for HGH to have an impact in thymopoiesis, although it could still expand the peripheral T cells. There is also the concern for increasing the risk of GVHD and other known side effects of this agent. Understanding how T cells are increased could identify specific patients that could benefit from HGH. Ultimately, we need a clinical trial with HGH transplantation to enhance the immune system’s performance.

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Bedside-to-Laboratory Translational Work Identifies Novel Tumor-Specific Antigen

By John C. Byrd, MD

Dr. Byrd indicated no relevant conflicts of interest.

Fukuda T, Chen L, Endo T, et al. Antisera induced by infusions of autologous Ad-CD154-leukemia B cells identify ROR1 as an oncofetal antigen and receptor for Wnt5a. Proc Natl Acad Sci USA. 2008;105:3047-52.

Chronic lymphocytic leukemia (CLL) is associated with profound suppression of the humoral, innate, and cellular components of the immune system. CLL cells are transformed B lymphocytes but lack classic co-stimulatory molecules typically present on normal B cells. The lack of co-stimulatory molecules on CLL cells, along with the production of soluble cytokines that dampen T-cell function, at least in part explain why autologous or even allogeneic T cells do not promote cytotoxicity toward these "stealth-like" tumor cells. To address this issue, Kipps and colleagues developed a strategy in which an adenovirus encoding the co-stimulatory molecule CD154 is used to infect CLL cells ex vivo. The enhanced CD154 expression on CLL cells produces an activated B-cell phenotype that promotes T-cell activation.¹ This results in T-cell recognition and cytolysis of CLL tumor cells in vivo, even those that did not encounter the virus. After extensive pre-clinical work and regulatory hurdles due to the increased concern over gene therapy trials, this group initiated a phase I study employing this approach that showed promising early and delayed anti-tumor responses.² Additionally, broad evidence of immunologic activation of T cells was noted, together with enhanced sensitivity of CLL cells to activation-induced cell death mediated by p73.^3,4

As a follow-up to this study, Dr. Kipps’ laboratory has now identified that a subset of CLL patients treated with this therapy developed "self" monoclonal antibodies directed at the tumor antigen ROR1A. Following identification of ROR1A as an autologous self antigen, the authors went on to demonstrate that this antigen is not expressed on normal B cells or other tissues in patients with CLL. Additionally, ROR1A appears to signal through WNT5a and NF-κB. Here the story becomes more complex, as CLL cells do not express WNT5a. However, WNT5a is expressed by accessory dendritic cells, which potentially provide stromal support to CLL tumor cells. Evidence for a stromal interaction of ROR1A and WNT5a is provided by demonstration that co-culture of CLL cells with a WNT5a-expressing cell line enhances survival, whereas addition of a ROR1A blocking antibody antagonizes survival. The relevant in vivo accessory cell remains to be identified, but this finding emphasizes the importance of the microenvironment in providing survival signals to leukemia cells. These results also provide a potential mechanism for the death of transformed B cells long after therapy and possibly explains the prolonged disease stabilization experienced by many patients following treatment with CD154 gene therapy.² Furthermore, this work constitutes one of the first demonstrations in CLL of a safe therapy to break immunologic tolerance against a "self tumor antigen."⁵ Without persistent bedside-to-laboratory translational research, this observation would have been lost.

Clinical investigation in the area of gene therapy is quite difficult, and before undertaking such an approach for phase II-III studies, it is clearly important to have a strong indication that this strategy could truly benefit patients long-term. The identification of induction of ROR1A antibodies in CLL patients receiving CD154 gene therapy as described in this paper provides such encouragement and shows that this line of clinical investigation warrants further pursuit.² A clinical trial using the human CD154 gene via a similar adenovirus vector has completed phase I investigation⁵ and will move forward to phase II testing soon. The strategy of enhancing CD154 expression in CLL cells using gene therapy or another method has exciting potential for the treatment of CLL. Furthermore, the use of ROR1A-directed therapeutic antibodies against CLL cells represents an option that should be actively pursued based upon the data presented in this paper and others.⁶ Most importantly, the paper by Kipps and colleagues highlights the great value of rigorous bedside-to-laboratory translational investigations of novel therapies. Such detailed correlative work that allows understanding of the mechanism of action of a new therapeutic agent should be included in virtually all clinical trials of targeted agents, so that expected (and more importantly, unexpected) findings, such as the induction of ROR1A antibodies noted in this report, can be discovered.

1. Kato K, Cantwell MJ, Sharma S, et al. Gene transfer of CD40-ligand induces autologous immune recognition of chronic lymphocytic leukemia B cells. J Clin Invest. 1998;101:1133-41.
   
   
2. Wierda WG, Cantwell MJ, Woods SJ, et al. CD40-ligand (CD154) gene therapy for chronic lymphocytic leukemia. Blood. 2000;96:2917-24.
   
   
3. Chu P, Deforce D, Pedersen IM, et al. Latent sensitivity to Fas-mediated apoptosis after CD40 ligation may explain activity of CD154 gene therapy in chronic lymphocytic leukemia. Proc Natl Acad Sci USA. 2002;99:3854-9.
   
   
4. Dicker F, Kater AP, Prada CE, et al. CD154 induces p73 to overcome the resistance to apoptosis of chronic lymphocytic leukemia cells lacking functional p53. Blood. 2006;108:3450-7.
   
   
5. Wierda WG, Castro J, Aguillon R, et al. A phase I study of immune gene therapy for patients with CLL using a membrane-stable, humanized CD154. Blood (Annual Meeting Abstract). 2007;110:607a.
   
   
6. Baskar S, Kwong KY, Hofer T, et al. Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia. Clin Cancer Res. 2008;14:396-04.

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GVHD Has Proteomics Come of Age?

By Gérard Socié, MD, PhD

Dr. Socié indicated no relevant conflicts of interest.

Hori T, Naishiro Y, Sohma H, et al. CCL8 is a potential molecular candidate for the diagnosis of graft-versus-host disease. Blood. 2008. [Epub ahead of print]

Although graft-versus-host disease (GVHD) is a life-threatening complication of hematopoietic stem cell transplantation (HSCT), its current diagnosis mainly depends on clinical manifestations and invasive biopsies. Early diagnosis of GVHD, preferably based on unbiased laboratory screening tools, may increase the safety of allogeneic HSCT and thus further broaden its application to even larger patient populations. In the past, many efforts were made to use single-protein biomarkers, which were specific for infection or inflammation after allogeneic HSCT but not specific for acute GVHD. Although some of these reports seem to hold promise, in many cases there was a high probability that a single marker was not specific, thus making differential diagnosis of similar diseases difficult. It is reasonable to believe that the simultaneous monitoring of more than one protein or peptide within a sample holds greater promise for the differential diagnosis of diseases, including GVHD. Recently, the application of proteomic tools allowing screening for differentially expressed or excreted proteins in body fluids is becoming more important.

Using proteomics, a Japanese group from Saporo screened for plasma proteins specific for GVHD in a mouse model. One peak retained a discriminatory value in two diagnostic groups (GVHD and normal controls) with increased expression in the disease, decreased expression during cyclosporine treatment, and was barely detectable in syngeneic transplantation. Purification and mass analysis identified this molecule as CCL8, a member of a large chemokine family. In human samples, the serum concentration of CCL8 correlated closely with GVHD severity. All non-GVHD samples contained less than 48 pg of CCL8 per mL. In sharp contrast, CCL8 was highly up-regulated in GVHD sera. Strikingly, two patients with severe fatal GVHD had extremely high levels of CCL8. Thus, CCL8 seems to be a promising specific serum marker for the early and accurate diagnosis of GVHD.

This study is of major importance for several reasons:

It confirms preliminary results reported by Weissinger and co-workers¹ published in Blood in which authors describe the application of capillary electrophoresis coupled online with mass spectrometry to 13 samples from 10 patients with acute GVHD of grade II or more and 50 control samples from 23 patients without GVHD. About 170 GVHD-specific polypeptides were detected and as a result a tentatively acute GVHD-specific model consisting of 31 polypeptides was chosen, allowing correct classification of 13 of 13 acute GVHD samples and 49 of 50 control samples in a training set. The subsequent blinded evaluation of 599 samples enabled diagnosis of acute GVHD greater than grade II, even prior to clinical diagnosis, with a sensitivity of 83 percent and a specificity of 76 percent.

The study by Hori and colleagues took advantage of a murine model (in which many parameters could be controlled for) to set up the search for specific markers that allows the characterization of proteins following a huge amount of work that would not have been easily feasible from human samples analyzed by Weissinger and colleagues.¹

CCL8 discovery makes the bridge even stronger between chemokines and acute GVHD pathophysiology. Indeed, the migration of cells from vascular to extra-vascular compartments implies a sequential cascade of events, involving interplay between adhesion molecules and chemokines. Acute GVHD requires that effector cells reach their target tissues. Lymphocytes do not enter specific tissues because they "recognize" a given antigen; they enter because they possess the requisite combination of homing receptors and chemokine receptors to engage the endothelium at the target tissue(s). Because GVHD is relatively organ-specific — principally affecting the skin, gut, and liver — our increasing knowledge of the pertinent adhesion molecules and chemokines directing effector-cell trafficking to these sites offers novel therapeutic approaches for prevention or treatment of GVHD.

Finally, the use of proteomics opens the door to exciting developments in the understanding of GVHD, the main one (at least in my opinion) being to use this tool to, finally, try to understand why and how some patients may develop a self-limited disease with an accompanying graft-versus-leukemia effect while others will develop a fatal steroid-resistant disease.

1. Weissinger EM, Schiffer E, Hertenstein B, et al. Proteomic patterns predict acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Blood. 2007;109:5511-9.

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Role of miR223 in Myelopoiesis

By Diane S. Krause, MD, PhD, and Stephanie Halene, MD

Drs. Krause and Halene indicated no relevant conflicts of interest.

Johnnidis JB, Harris MH, Wheeler RT, et al. Regulation of progenitor cell proliferation and granulocyte function by microRNA-223. Nature. 2008;451:1125-9.

New avenues of discovery have opened over the last decade with the discovery of microRNAs. MiRNAs are small RNAs of approximately 22 nucleotide length, which are transcribed from genomic DNA like messenger RNAs (mRNAs) but do not encode proteins. Their main function is that of gene regulation by targeting specific sequences in the 3’-untranslated region of mRNAs. It is estimated that the human genome encodes 300 to 500 miRNAs, and that ~30 percent of all genes are regulated by miRNAs. Differential expression of different miRNAs during hematopoiesis was first reported in 2003, and the specific regulatory functions of several miRNAs have since been elucidated.

The expression and processing of miRNAs has been reviewed in detail elsewhere. Initially a long, capped and polyadenylated, primary precursor (pri-miRNA) is transcribed, which is then cleaved into a hairpin-shaped pre-miRNA. The pre-miRNA is further processed into a single-stranded RNA of about 22 basepairs, which associates with the so-called RNA-induced silencing complex (RISC). This way the miRNA guides the RISC to specific mRNAs and thus regulates protein translation by targeting the mRNA for degradation or by translational silencing. The fact that a single miRNA can target multiple genes and a single gene can be targeted by multiple miRNAs allows for complex regulatory networks.

Several miRNAs with distinct roles in cell differentiation during development and in adult tissue maintenance have been discovered. For example, miR181 is expressed predominantly in lymphocytes and its expression promotes B-cell differentiation.¹

In this paper, Johnnidis, et al. focus on miR223, which is expressed at low levels in hematopoietic stem and progenitor cells, and at higher levels in common myeloid progenitors with steadily rising expression with further granulocytic differentiation. In order to investigate the function of miR223, the investigators created mice that lacked expression of miR223 (knockout [KO] mice). These mice showed a surprising finding within the hematopoietic system. Since miR223 expression is upregulated with granulocytic differentiation, it was predicted to promote granulocytopoiesis and hence the mice were expected to lack granulocytes. Instead, these mice actually had higher numbers of granulocytes, which were hyper-responsive causing a hyperinflammatory state in the mice. The neutrophil count was twice that of wildtype (WT) mice, and this increase was found to be due to an increase in the number of granulocyte progenitors and enhanced neutrophil differentiation.

Using bio-informatics, the investigators found more than 100 potential target genes for miR223 but focused on mef2c, a transcription factor known to play a role in myelopoiesis, as it was the only gene with two conserved miR223 complimentary "seed" sites in its 3’UTR (untranslated region). Indeed, when the investigators created mice that lacked both miR223 and mef2c, they found that the mice had normal granulocyte numbers. However, the hyperinflammatory state persisted. Thus, while the increased neutrophil count of the miR223 KO mouse is caused at least in part through loss of downregulation of mef2c by miR223, a distinct mechanism is likely responsible for the hyperinflammatory state.

The investigators have identified a role for miR223 in regulating granulocytopoiesis and granulocyte activation. MiR223 inhibits translation of Mef2c, a transcription factor that promotes myeloid progenitor proliferation and likely other factors, thereby keeping granulopoiesis "in check." It is intriguing that the increasing expression of miR223 with granulocytic differentiation appears to function as a built-in repressor or brake in the system, ultimately to prevent hyperinflammatory states, supporting the importance of the regulatory functions of miRNAs in hematopoiesis.

1. Chen CZ, Li L, Lodish HF, et al. MicroRNAs modulate hematopoietic lineage differentiation. Science. 2004;303:83-6.

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Clinical Penetrance of HFE Hereditary Hemochromatosis, Serum Ferritin Levels, and Screening Implications: Can We Iron This Out?

By Michael Linenberger, MD

Dr. Linenberger indicated no relevant conflicts of interest.

Allen KJ, et al. Iron-overload-related disease in HFE hereditary hemochromatosis. N Engl J Med. 2008;358:221-30.

Waalen J, Felitti VJ, Gelbart T, et al. Screening for hemochromatosis by measuring ferritin levels: a more effective approach. Blood. 2008;111:3373-6.

Since the discovery of the homozygous C282Y mutation of the HFE gene as the major cause of hereditary iron overload in Caucasians of Northern European descent, numerous studies have attempted to define the risk of iron-overload-related disease and a practical rationale for screening. The interpretation of cross-sectional population and family-based studies has been controversial,^1,2 related to concerns about ascertainment bias, observer bias, insufficient control groups, and variable definitions of disease penetrance. Longitudinal cohort studies have involved relatively few HFE homozygotes and mostly women, thus raising issues about gender bias and other confounding variables. The U.S. Preventive Services Task Force recommendation against genetic screening cited the need for more information on the clinical penetrance of HFE-associated disease, factors affecting phenotypic expression, and outcomes from prospective follow-up and therapeutic intervention trials.³

Allen, et al. analyzed 12-year biochemical and clinical data on 1,438 adults of Northern European ancestry (99 percent 40 to 69 years old) participating in a longitudinal health study. The 203 C282Y homozygotes and matched controls with other HFE genotypes or no mutations underwent baseline and follow-up interviews, blinded physical exams, and laboratory studies. Liver biopsies were performed on 16 of 33 men and one of seven women HFE homozygotes who had at least one serum ferritin >1,000 µg/L. Among those with documented iron overload, clinical disease occurred in 21 of 74 men (28.4 percent) and one of 84 women (1.2 percent); including hepatocellular carcinoma (n=2), liver fibrosis or cirrhosis (n=12), elevated transaminases (n=6), abnormal metacarpophalangeal joints (n=5), or other hemochromatosis-related symptoms (n=11). Serum ferritin ≥1,000 µg/L was significantly associated with fatigue, liver disease, use of arthritis medication at baseline, and elevated transaminases, but not associated with diabetes or joint arthropathy. One non-C282Y homozygote had clinical disease. Waalen, et al. observed a serum ferritin level >1,000 µg/L in 59 of 29,699 white adults (0.2 percent) in the Scripps-Kaiser hemochromatosis study, including 20 C282Y homozygotes and four with other HFE genotypes, only one of whom had liver cirrhosis. (The number of biopsies was not reported.) Hyperferritinemia in 30 of the 35 without HFE mutations (86 percent) was attributable to excess alcohol, cancer, liver disorders, or hemolytic anemia. The researchers concluded that a single serum ferritin determination could serve as a cost-effective screen for the most threatening HFE mutation-associated complication (liver cirrhosis). Ferritin was also valuable in identifying non-HFE disorders, similar to observations among multi-ethnic populations.⁴

The report by Allen, et al. supports prior observations that iron-overload-related disease, when broadly defined, affects roughly a quarter of male HFE C282Y homozygotes.1 Both reports confirm data from others that liver cirrhosis develops at a serum ferritin >1,000 µg/L. Importantly, however, pre-cirrhotic fibrosis is found in 28 percent of asymptomatic C282Y homozygotes with a ferritin of 500 to 1,000 µg/L (mostly middle-aged males) and phlebotomy reverses this process.⁵ Thus, using a ferritin of >1,000 µg/L for screening, rather than the conventional thresholds of >200 µg/L for premenopausal women and >300 µg/L for other adults, may fail to identify some high-risk homozygotes before irreversible liver injury develops. Despite new understanding, a risk-adapted screening and management approach to HFE hemochromatosis still awaits answers to the following questions:

• How do genetic, lifestyle, and other co-factors affect progression or "nonexpression" of iron overload and liver disease?

• Are other tissues (joint, endocrine, cardiac) at risk? If so, by what mechanisms?

• What clinical and/or biochemical parameters (e.g., ferritin level) should be used to initiate therapeutic phlebotomy?

1. Ajioka RS and Kushner JP. Clinical consequences of iron overload in hemochromatosis homozygotes. Blood. 2003;101:3351-3.
   
   
2. Beutler E. Rebuttal to Ajioka and Kushner. Blood. 2003;101:3354-7.
   
   
3. U.S. Preventive Services Task Force. Screening for hemochromatosis: recommendation statement. Ann Intern Med. 2006;145:204-8.
   
   
4. Adams PC, Reboussin DM, Barton JC, et al. Hemochromatosis and iron-overload screening in a racially diverse population. N Engl J Med. 2005;352:1769-78.
   
   
5. Powell LW, Dixon JL, Ramm GA et al. Screening for hemochromatosis in asymptomatic subjects with or without a family history. Arch Intern Med. 2006;166:294-301.

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Combination Proteasome Inhibitor Therapy

By Kenneth Anderson, MD

Dr. Anderson indicated no relevant conflicts of interest.

Chauhan D, Singh A, Brahmandam M, et al. Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma. Blood. 2008;111:1654-64.

The proteasome inhibitor bortezomib, which primarily targets the chymotryptic-like (CT-L) proteolytic activity of the proteasome, is an effective therapy for patients with relapsed refractory multiple myeloma (MM) and is superior to high-dose dexamethasone therapy for relapsed MM.^1,2 Excitingly, when combined with dexamethasone it has increased frequency and extent of response both before and after high-dose melphalan and autologous stem cell transplantation.³ In older non-transplant patients, initial therapy with bortezomib combined with melphalan and prednisone achieved significant increases in overall and extent of response, associated with prolonged progression-free and overall survival.⁴ More recently, several next-generation proteasome inhibitors have shown promise at overcoming bortezomib resistance in preclinical models and are under clinical evaluation. Carfilzomib more potently inhibits the CT-L proteolytic activity⁵ and is under evaluation in two phase II clinical trials in MM, having shown early signs of responses in phase I studies.^6,7 NPI-0052, a second-generation proteasome inhibitor targeting CT-L, tryptic-like (T-L), and caspase-like (C-L) proteolytic activities,⁸ is also in phase I clinical trial in MM. Finally, CEP-18770, which is also entering clinical trials, is an oral inhibitor of CT-L proteolytic activity.⁹ At present, the qualitative or quantitative extent of proteasome inhibition associated with clinical efficacy in MM remains to be defined.

Conventional therapies for cancer have been combined to both increase tumor-cell cytotoxicity and decrease attendant toxicity, frequently allowing for use of lower doses of therapy. Chauhan and colleagues provide preclinical evidence suggesting that similar principles may also apply with novel targeted therapies. In particular, combining bortezomib with NPI-0052 induced synergistic activity against MM cell lines in the bone marrow milieu in vitro, as well as in vivo in a human plasmacytoma xenograft model. The biologic sequelae triggered by the combination included activation of caspase-8, 9, 3, and PARP; induction of endoplasmic reticulum stress and JNK; suppression of CT-L, C-L, and T-L proteolytic activities; and blockade of NF-κB signaling.

Immunostaining showed growth inhibition, apoptosis, and decrease of human MM cells, as well as decreased associated angiogenesis, in treated mice. Most importantly, these effects were observed when combining these inhibitors, which are inactive when they are used alone, even at low doses. With these low doses, combination therapy was very well tolerated. These studies suggest, as with conventional combination chemotherapy, that combining proteasome inhibitors may enhance efficacy, delay or overcome drug resistance, lessen attendant side-effect profile, and ultimately improve patient outcome in MM.

1. Richardson PG, Barlogie B, Berenson J, et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med. 2003;348:2609-17.
   
   
2. Richardson PG, Sonneveld P, Schuster MW, et al. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med. 2005;352:2487-98.
   
   
3. Harousseau JL, Mathiot C, Attal M, et al. VELCADE/Dexamethasone (Vel/D) versus VAD as induction treatment prior to autologous stem cell transplantion (ASCT) in newly diagnosed multiple myeloma (MM): updated results of the IFM 2005/01 trial. Blood (Annual Meeting Abstract). 2007;110:138a.
   
   
4. San Miguel JF, Schlag R, Khuageva N, et al. MMY-3002: A phase 3 study comparing Bortezomib-Melphalan-Prednisone (VMP) with Melphalan-Prednisone (MP) in newly diagnosed multiple myeloma. Blood (Annual Meeting Abstract). 2007;110:121.
   
   
5. Kuhn DJ, Chen Q, Voorhees PM, et al. Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma. Blood. 2007;110:3281-90.
   
   
6. Orlowski RZ, Stewart K, Vallone M, et al. Safety and antitumor efficacy of the proteasome inhibitor carfilzomib (PR-171) dosed for five consecutive days in hematologic malignancies: phase 1 results. Blood (Annual Meeting Abstract). 2007;110:127a.
   
   
7. Alsina M, Trudel S, Vallone M, et al. Phase 1 single agent antitumor activity of twice weekly consecutive day dosing of the proteasome inhibitor carfilzomib (PR-171) in hematologic malignancies. Blood (Annual Meeting Abstract). 2007;110:128a.
   
   
8. Chauhan D, Catley L, Li G, et al. A novel orally active proteasome inhibitor induces apoptosis in multiple myeloma cells with mechanisms distinct from Bortezomib. Cancer Cell. 2005;8:407-19.
   
   
9. Piva R, Ruggeri B, Williams M, et al. CEP-18770: A novel, orally active proteasome inhibitor with a tumor-selective pharmacologic profile competitive with bortezomib. Blood. 2008;111:2765-2775.

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