Bone Marrow Research

Multiple Myeloma Bone Marrow Research Multiple Myeloma Copyright © 2011 Hindawi Publishing Corporation. All rights reserved.

This is a focus issue published in volume 2011 of “Bone Marrow Research.” All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Editorial Board

Joseph H. Antin, USA Hans Jochem Kolb, Germany PeterJ.Quesenberry,USA Igor W. Blau, Germany Robert B. Levy, USA Shahin Rafii, USA Catherine Bollard, USA Mark R. Litzow, USA Voravit Ratanatharathorn, USA Paolo De Fabritiis, Italy Fausto Loberiza, USA David A. Rizzieri, USA Yigal Dror, Canada David Ma, Australia Patrick J. Stiff,USA Csar O. Freytes, USA Ignazio Majolino, Italy Anna Sureda, Spain A. Ganser, Germany Philip L. McCarthy, USA Guido J. K. Tricot, USA Eva C. Guinan, USA Kenneth R. Meehan, USA Celalettin Ustun, USA Kais Hussein, Germany Paul V. O’Donnell, USA Catherine Verfaillie, Belgium Issa F. Khouri, USA Helen A. Papadaki, Greece Axel R. Zander, Germany Hans Klingemann, USA Finn B. Petersen, USA Guido Kobbe, Germany Luis F. Porrata, USA Contents

Antibody-Based Therapies in Multiple Myeloma, Yu-Tzu Tai and Kenneth C. Anderson Volume 2011, Article ID 924058, 14 pages

A Case of Monoclonal Lymphoplasmacytosis of the Bone Marrow with IgM-Positive Russell Bodies, Hidekazu Kayano, Tsuneyuki Shimada, Naoki Wakimoto, Yuichi Nakamura, Masami Bessho, Hiroshi Yamaguchi, Atsushi Sasaki, and Michio Shimizu Volume 2011, Article ID 814372, 3 pages

Immune Recovery after Cyclophosphamide Treatment in Multiple Myeloma: Implication for Maintenance , Amir Sharabi and Nechama Haran-Ghera Volume 2011, Article ID 269519, 7 pages

Recent Advances in the Pathogenesis and Management of Cast Nephropathy (Myeloma Kidney), Stephanie Stringer, Kolitha Basnayake, Colin Hutchison, and Paul Cockwell Volume 2011, Article ID 493697, 9 pages

Oncolytic Virotherapy for Multiple Myeloma: Past, Present, and Future, Chandini M. Thirukkumaran and Don G. Morris Volume 2011, Article ID 632948, 10 pages

Multiple Myeloma: A Review of Imaging Features and Radiological Techniques,C.F.Healy,J.G.Murray, S. J. Eustace, J. Madewell, P. J. O’Gorman, and P. O’Sullivan Volume 2011, Article ID 583439, 9 pages Hindawi Publishing Corporation Bone Marrow Research Volume 2011, Article ID 924058, 14 pages doi:10.1155/2011/924058

Review Article -Based Therapies in Multiple Myeloma

Yu-Tzu Tai and Kenneth C. Anderson

Department of Medical Oncology, Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA

Correspondence should be addressed to Yu-Tzu Tai, yu-tzu [email protected]

Received 18 October 2010; Accepted 4 January 2011

Academic Editor: Ignazio Majolino

Copyright © 2011 Y.-T. Tai and K. C. Anderson. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The unmet need for improved multiple myeloma (MM) therapy has stimulated clinical development of monoclonal (mAbs) targeting either MM cells or cells of the bone marrow (BM) microenvironment. In contrast to small-molecule inhibitors, therapeutic mAbs present the potential to specifically target tumor cells and directly induce an immune response to lyse tumor cells. Unique immune-effector mechanisms are only triggered by therapeutic mAbs but not by small molecule targeting agents. Although therapeutic murine mAbs or chimeric mAbs can cause immunogenicity, the advancement of genetic recombination for humanizing rodent mAbs has allowed large-scale production and designation of mAbs with better affinities, efficient selection, decreasing immunogenicity, and improved effector functions. These advancements of antibody engineering technologies have largely overcome the critical obstacle of antibody immunogenicity and enabled the development and subsequent Food and Drug Administration (FDA) approval of therapeutic Abs for cancer and other diseases.

1. Introduction -6 (IL-6), vascular endothelial growth factor (VEGF), Receptor Activator of NFκB ligand (RANKL) (also Despite the landmark approval of the anti-CD20 mAb rit- known as osteoprotegerin ligand (OPGL)), and Dickkopf uximab for the treatment of B-cell malignancies, to date, no homolog 1 (DKK1) are among those under clinical evalu- mAb-based therapy has been approved for MM treatment. ation. Specifically, targeting bone-MM cell interactions via ff The development of e ective cytotoxic mAb therapies in MM bone biology modulating factors such as DKK1 and RANKL has been hindered by the lack of uniquely and constitutively is likely to trigger anti-MM effects but also improves bone expressed target molecules on all MM cells. Indeed, studies in disease thereby improving both patient survival as well as early 2000 demonstrated only minimal activity of anti-CD20 patient’s quality of life. and antibodies against plasma cell-specific CD38 In the coming years, the preclinical progress in defining ff antibodies in MM [1–4]. However, numerous e orts to iden- novel MM markers will be continued and subsequently will tify new targets on MM cells including gene expression pro- advance the clinical development of therapeutic mAbs, alone filing and oncogenomic studies are under way. Derived mAbs or in combination with other anti-MM agents, to improve (e.g., against CD40, HM1.24, IGF-1R, CD56, CS1, CD138, patient outcome in MM. CD74, IL-6R, CD38, TRAIL-R1, and the activin receptor type IIA (ActRIIA)) have already demonstrated promising preclinical as well as early clinical activity (Table 1). 2. Mechanisms of Action of Therapeutic Given the importance of the bone marrow (BM) mi- Monoclonal Antibodies croenvironment for MM cell growth, survival, and drug resistance, mAbs have been additionally designed to func- Antibodies of IgG, the most commonly used immunoglob- tionally block both autocrine and paracrine secreted ulin form in cancer therapy, are unique proteins with dual cytokines and growth factors as well as molecules mediating functionality. Therapeutic mAbs use one or more following MM-stromal cell interaction. For example, mAbs targeting mechanisms (Figure 1) to reduce tumor burden in patients. 2 Bone Marrow Research without FGF-2, B1R, or B2R expression in vivo , slows down B38-MM growth rate when both MM ect on myeloma cells and low toxocity in the mice ff in vivo Yang Blood 2007; 110:3028–35. & ClinStrong Cancer apoptotic Res e 2009; 15:951–9. Sekimoto Cancer Res 2007; 67:1184–92. adiabody recombinant 2D7-DB single-chain specifically Fv induces multiple myelomabone cell marrow death environment in the suppports potential use as therapeutic agents Zhou Blood 2008; 111:549–557. humanized 2B6patients MoAb with may systemic target AL-amyloidosis. in It blocksCD32B Fc and engagement may of improve the performancecombined of with other them cancer during Mabs administration when Trudel Blood 2006; 2:4908–4915. The inhibitoryPRO-001, anti-FGFR3 is antibody, cytotoxic to t(4;14) MMthe cells treatment and of deserves FGFR3-expressing further myeloma study for Smallshaw J Immunother 2004; Coleman Jsignificantly Immunother prolongs 2006 the cUV3 survival of SCID/ARH-77 mice Sainz Cancer Immunol Immunother 2006 C11C1tumor mAb growth inhibits its own are implanted together and when mAbintraperitoneally. C11C1 MAb C11C1-treated-MM is showed injected decreased MVD and kininogen binding changes erent stages of preclinical/clinical development. ff preclincial Phasepreclinical Remarks Tassone Blood 2004,104:3688–96 preclincial Lyu et al. Mol Cancer Ther 2007; 6:460–70 preclincial 1 mouse preclincial humanized preclincial Ozaki Blood 1999,93:3922–3930 mouseconverted from mouse IgG2b, single-chain Fv diabody preclincial Type of mAb (conjugate) the maytansinoid immunoconjugate mouse IgG1 mAb B-B4 human IgG1 preclincial Fulciniti Clin Cancer Res 2009,15:7144–52 of an antibody tethered to a toxin Fc-engineered humanized IgG MD Anderson Cancer Center Chugai Pharmaceutical OPi EUSA Pharma; Vaccinex licensed to Glaxo Smith Kline Temple UniversitSchool of Medicine y Chugai Pharmaceutical Co. Ltd. 1: targeted by antibodies in multiple myeloma in di Table 2M mAbs β OP-R003-1, 1339 Elsilimomab, Azintrel anti- humanized HM1.24 Xencor Inc 2- TargetCD138 Brand nameHM1.24 B-B4-DM1 humanized HM1.24 Company/Sponsor IL-6 ImmunoGen HLA-DR 1D09C3kininogen C11C1 HLA class I GPC Biotech, AG 2D7-DB β humanmicroglobulin IgG1CD38 preclincialCD32B MOR202 Carlo-Stella Cancer Res 2007 MGA321(2B6)FGFR3ICAM-1 PRO-001 MorphoSys AG MacroGenicsBLyS cUV3 human IgG1 humanizedTACI IgG1 Prochon BLyS/rGel Biotech Ltd. preclincial human IgG1 preclincial Atacicept (TACI-Ig) Abiogen Tesar et al. J. Clin Oncol ZymoGenetics 2007, Inc. 25(18S): 8106 Targa preclincial Therapeutics fusion protein chimeric IgG1 preclincial preclincial Yaccoby Leukemia 2008 22, 406–413 Bone Marrow Research 3 in vitro S6B45 cell ective at ff in vivo Bertino et al. 2010 AACR abtractdoxorubicin no. to 2596. inhibit M24-DOX the is growth as ofdoxorubicin potent MM by as the cells. free But matriptase target antibody delivery significantlytoxicity of reduced toward the cardiomyocytes that lack matriptase expression NCT00258206 (with cyclophosphamide): NCT00505895. High-dose cyclophosphamide in combination with rituximab inprimary patients refractory, high-risk, with or relapsed myeloma,for also the being treatment studied of peripheral neuropathy inNCT00477815: patients Zevalin with radioimmunotherapy MGUS with high-doseand melphalan stem cell transplant for MM NCT00231166 Dose-finding trial of HCD122relapsed in or MM has patients not that responded is to prior therapy NCT00664898: safety and pharmacology ofcombination SGN-40 with administered in Bortezomib (Velcade, PS-341) inrelapsed patients or with refractory MM. NCT00525447 islenalidomide, the and study dex of in SGN40, MM patients Yoshio-Hoshino Cancer Res 2007; 67:871–5. thecombined NRI with gene adenovirus introduction gene delivery inhibited the Lust 2010 AACR abstract no. 2449. XOMA 052 is highly e NCT00135200: to see whether the treatmentand with possibly Bexxar eliminate will residual myeloma decrease cells resistant to NCT00346255: given as an intravenous infusionconsecutive weekly weeks for every two three weeks toCD56-positive relapsed MM; and NCT00991562: relapsed IMGN901 refractory in combinationlenalidomide with and dexamethasone inhibiting IL-1 induced IL-6 production in myeloma patients growth significantly preclincial II (ongoing) Phase Remarks preclincial preclincial Ryan et al. Mol Cancer Ther 2007; 6:3009–18 preclincial II (ongoing) I (ongoing) 1: Continued. Table immunoconjugate with doxorubicin chimeric with a human IgG1 Fc humanized IgG1 I b (ongoing) Type of mAb (conjugate) humana single-chain fragment format dimerized by fusing to the Fc portion of human preclincialimmunoglobulin G1 Auristatin-BCMA Menoret etmAb al. Blood 2006;132: 1356–62 Human Engineered IgG2 radioactive iodine 131 attaching to anti-CD20; muIgG2a (131) humanized (maytansine DM1 conjugation) Human Genome Sciences Roche Pharmaceuticals UMDNJ—The Cancer Inst. of New Jersey NCI & Memorial Sloan-Kettering Cancer Center NCISeatle Genetics/Genentech Novartis mouse IgG1GlaxoSmithKline I (ongoing) human IgG1 I (ongoing) lexatumumab NRI (engineered ) Zevalin (yttrium Y 90 ibritumomab tiuxetan) HCD122 (Lucatumumab) Bexxar (131-tositumomab) TargetCD70TRAIL- Brand nameR2(DR5) SGN-70IL-6R Company/Sponsor BCMA Seatle Geneticsmatriptase humanized SG1 IgG1 M24-DOX IL-1beta preclincial McEarchern Clin Cancer XOMACD20 Res 052 2008 14, 7763–72 Seatle Genetics CD20 Rituxan XOMA (US) LLC CD40CD40 SGN-40 (Dacetuzumab) CD20 CD56 BB-10901 (IMGN901) ImmunoGen, Inc. 4 Bone Marrow Research ective in the treatment ff ects of giving fludarabine and ff cacy was seen but there is a limitation for the clinical use ffi erent cytoreductive therapies and without further established ectiveness of AMG 162 in reducing urinary N-telopeptide in ff ff of a murine sinceanti-mouse it antibodies frequently (HAMA) induces human NCT00259740: to determine if denosumab is e NCT00428545 (in combination with bortezomib); NCT00410605 (added with and dexamethasone) NCT00401843 (in combination with bortezomib); NCT00911859 (added with Velcade-Melphalan-Prednisone); NCT00402181 (in combination with dexamethason) NCT00574288: to establish safety profile ofmonotherapy HuMax-CD38, in given patients as with MMdi relapsed or refractory to at leasttreatment 2 options NCT00741377: in combination with Zoledronic Acidrelapsed/refractory in myeloma Preliminary e NCT01084252: to determine the maximum tolerated(MTD)/maximum dose administered dose (MAD) advanced cancer subjects with bone metastases;III NCT00330759: Study Phase of Denosumab Compared Withthe Zoledronic Treatment of Acid (Zometa) Bone in Metastases in(Excluding Subjects Breast With and Advanced Prostate Cancer Cancer) or MM of relapsed or plateau-phase MM; NCT00104650:e to determine the NCT00625144: studying the side e busulfan together with followed bytransplant donor and stem to cell see how wellhematological it cancer works or in other treating disease patients with II/III (ongoing) Phase Remarks I (ongoing) NCT00723359 1: Continued. Table murinehumanized II chimerized II humanized II (ongoing) NCT00368121 (in I combination /II with (ongoing) dexamethasone) NCT00742560humanized & IgG1 NCT00726869 (in combination with bortezomib) I (ongoing) Type of mAb (conjugate) humanized II (ongoing) human II (ongoing) NCT00315757 (in combination with bortezomib) chimeric (B-B4-maytansinoid DM4) NCI; Fred Hutchinson Cancer Research Institute Orphan Pharma International and Diaclone SA Roche Pharmaceuticals Human Genome Sciences Imclone; Bristol Meyers-Squibb Facet Biotech; Bristol-Myers Squibb Sanofi-Aventis; ImmunoGen Campath-1H (alemtuzumab) Mapatumumab(TRM-1) Target Brand nameRANKL Denosumab Company/Sponsor VEGFCD52 Amgen Avastin beuacizumab GenentechIL-6 human IgG2 IL-6 humanized CNTO 328IL-6RTRAIL- B-E8 (Elsilimomab) IIR1(DR4) (ongoing) MRA (Tocilizumab) Centocor, IncEGFRCS1 chimerized IgG1 Erbitux(EMMA-1) CD38 I/II (ongoing) elotuzumab/HuLuc63 CD38 HuMax-CD38DKK SAR650984 CD138 Genmab BHQ880 BT062 human IgG1 Novartis I/II (ongoing) Biotest; ImmunoGen human IgG1 I/II (ongoing) Bone Marrow Research 5 and oma in vitro antimyeloma in vivo cacy study ffi erent antibody, thus resulting in a new ff Descamps et al. (B J CancerAntibody 2009; combined with 100:366) Anti-IGF-1R bortezomib Monoclonal for patients with rel/ref MM Lacy et al. (J. Clin Onclotolerated 26:3196) and reported may that constitute a CP-751,871 is novelmyeloma well agent in the treatment of multiple NCT00552396 (ASCO May 30 2009 abstracttolerability 09-AB-3032) study safety for and patients withcharacterization relapsed/refractory of MM. 1-7F9, Preclinical a novel humanthat anti-KIR augments therapeutic NK-mediated antibody killing of tumor cells (Romagne et al. 2009) NCT00421525: in patients with recurrent ormyeloma who refractory have multiple failed at leasttreatments. two Its prior isotope, standard drug, systemic and toxinactivity conjugates in have non-Hodgkin’s high and antitumor multiple myeloma molecule whose properties are unknown. However,seek the a Company partner will for the intellectual property relating to this program Dosing study of anti-GM-2 ganglioside (expressedsurface at of high MM levels cells) on followed by the e in tumor xenograft models. Stein et al. 2007 & 2009 Carlo-Stella et al. 2007 showed thatof IFN-gamma-induced HLA-DR up-regulation results in a potentactivity enhancement of of 1D09C3 the in mice. Initialraised clinical any testing unexpected with or unacceptable 1D09C3 safety hasmaximum concerns not tolerated and dose the has not yetdecided been to reached. not GPC put Biotech further has internaldue resources to into potential developing swapping 1D09C3 of IgG4structure antibody with one the half half of of its a Y-shaped di Phase Remarks I/II (ongoing) , company, and other web sites, but accuracy cannot be guaranteed. 1: Continued. Table http://clinicaltrials.gov/ Type of mAb (conjugate) human IgG1 I/IIa (ongoing) NCT00747123 (in patients with osteolytic lesions with MM) humanized IgG1 or humanized IgG1 doxorubicin conjugate Acceleron Pharma, Inc Immunomedics, Inc. Pfizer human IgG2 I ACE-011 BIW-8962milatuzumab (hLL1, IMMU-110) BioWa, Incorporated humanized I/II (ongoing) 1D09C3CP- GPC Biotech751,871/figitumumab human IgG4 I ort has been made to obtain reliable data from multiple sources including ff Targetthe activin receptor type IIA Brand (ActRIIA) nameIGF-1RGanglioside GM2 AVE1642 Company/Sponsor CD74 (variant MHC II) Alpha-4 Sanofi-Aventis (Tysabri) Biogen Idec humanizedMHC II (HLA-DR) humanized IgG4 I/II (ongoing) I/II (ongoing) NCT00675428: patients with relapsedIGF-1R or refractory multiple myel KIR IPH 2101 Innate Pharma human IgG4 I/IIa (ongoing) Every e 6 Bone Marrow Research

E ector Membrane cells Tumor E ector attack (NK cells/ cell cells complex Tumor cell macrophages/ lysis neutrophiles) CR1 FcγR C1q Antigen (CD16, CD32) Complement C1qR Apoptosis or receptor growth arrest CR3 Tumor via cell 1.Blockade of receptor- ligand interaction 2.Activation of apoptotic signaling pathways 3. Specific targeting and delivering immunotoxins ADCC CDC (a) (b) (c)

Figure 1: Mechanisms of actions associated with therapeutic monoclonal antibodies. (a) Therapeutic antibodies could directly induce apoptosis or growth arrest upon binding to cell surface antigen on tumor cells. Rituximab and Mapatumumab (anti-TRAIL-R1) could induce growth inhibition or apoptosis signaling to directly block tumor cell growth and survival. Such mechanism of action was employed by mAbs conjugated with toxins, that is, maytansinoids (DM1, DM4) for BB-10901 (anti-CD56) and BT062 (anti-CD138), thus directly target and eliminate tumor cells. Most of the approved therapeutic mAbs belong to IgG1 subclass, which has a long half-life and trigger potent immune-effector functions. (b) Following the binding of mAbs to a specific target on a tumor cells, antibody-dependent cellular cytotoxicity (ADCC) is triggered by interactions between the Fc region of an antibody bound to a tumor cell and Fc receptors, particularly FcRI and FcRIII, on immune effector cells such as neutrophils, macrophages, and natural killer cells. MAb-coated tumor cells are phagocytosed by macrophages or undergo cytolysis by NK cells. (c) In the case of complement-dependent cytotoxicity (CDC), recruitment of C1q by IgG bound to the tumor cell surface is an obligatory first step. This triggers a proteolytic cascade that leads to generation of the effector molecule, C3b, and then to formation of a membrane attack complex that kills the target cell by disrupting its cell membrane.

They can be categorized into direct and indirect actions. whereacascadeofdifferent complement proteins become Three modes of action could be further subcategorized from activated, usually when several IgGs are in close proximity the direct action (Figure 1(a)) of mAb-based cancer therapy, to each other, either with one direct outcome being cell lysis, including blocking the function of target signaling molecules or one indirect outcome being attracting other immune cells or receptors, stimulating apoptosis signaling cascades, and to this location for effector cell function. targeting function to selectively target tumor cells and deliver toxins. The receptor functional blocking can occur by inhibiting ligand binding to inhibit cell cycle progression, 3. Antibodies Targeting Cell Surface Protein DNA repair, or angiogenesis. It could also occur by increasing on MM Cells internalization of receptors or decreasing proteolytic cleavage Several mAbs directed against MM cell surface are being of receptors. In the case of targeting function, mAbs could investigated as potential therapy in MM. Listed below are be conjugated with immunotoxins, that is, antitubulin mAbs against receptor antigens that are currently under agents (DM1/DM4, auristatin), doxorubicin, radioisotopes, clinical development or investigation in MM. or other chemotherapeutic drugs, thus selectively targeting and killing tumor cells. Indirect action of mAb therapy is mediated by the . The elimination of tumor 3.1. Limited Clinical Benefit from Anti-CD20 mAb Rituximab cells using mAbs depends on Ig-mediated mechanisms, in MM. MM is usually not considered as a disease suitable including antibody-dependent cellular cytotoxicity (ADCC) for anti-CD20 therapy due to weak CD20 expression in the and complement-dependent cytotoxicity (CDC), to activate majority of patients. For example, results from a clinical immune effector cells to lyse target tumor cells (Figure 1(b)) phase II trial in relapsed MM showed that Rituximab These two mechanisms are believed to have the greatest treatment yielded significant reductions in circulating B cells impact, although there are conflicting views of which of and serum IgM levels but had no beneficial clinical effect [5]. these two pathways contributes the most to the response. Moreover, rituximab was investigated for maintenance ADCC involves the recognition of the Ab by immune cells therapy in MM following autologous hematopoietic stem that engage the Ab-marked cells and either through their cell transplantation (SCT) [2]. Although the number of MM direct action, or through the recruitment of other cell types, patients was too low to draw definitive conclusions, the use of led to the tagged-cell’s death. CDC (Figure 1(c))isaprocess rituximab in this setting was associated with an unexpectedly Bone Marrow Research 7 high rate of early relapse. The authors therefore hypothesized adenovirus vector encoding NRI was administered to mice a possible role for rituximab in provoking a further decrease intraperitoneally (i.p.) and monitored for the serum NRI in the residual, normal B-cell activity within the context of level and growth reduction property on the xenografted IL-6- the complex network of antitumor immune response. Taken dependent MM cell line S6B45. These findings indicate that together, the resistance of MM cells against rituximab could NRI is a promising agent applicable to the therapeutic gene be due to the level of CD20 expression, dissociated action of delivery approach for IL-6-driven diseases. CDC and ADCC, polymorphism in FGCR3 (CD16) receptor, and an inadequate dose schedule. In contrast, other studies demonstrated that the CD20+ 3.3. Targeting CD40 by SGN-40 or HCD122. Novel mon- phenotype is associated with patients with t(11,14)(q13;q32) oclonal antibodies targeting CD40 activation in MM cells, and with shorter survival [6] and that occasional clinical SGN-40/Dacetuzumab (Seattle Genetics, Genentech) and responses have been achieved in selected patients with HCD122/Lucatumumab (Novartis), have been investigated + CD20 myelomatous plasma cells [7, 8]. [20, 21]. In preclinical studies, SGN-40, a humanized IgG1 Finally, new insights suggest that circulating CD20+ partial agonistic mAb mediates cytotoxicity against CD40- clonotypic B cells act as precursors or “neoplastic stem cells” expressing MM cell lines and patient MM cells via suppres- in MM patients, representing the proliferative compartment sion of IL-6-induced proliferative and antiapoptotic effects, of the disease able to play a role in determining relapse after as well as ADCC [20]. SGN-40 also induced significant effective treatments [9]. Thus, clinical trials using rituximab antitumor activity in xenograft mouse models of human in MM may deserve further investigation. MM and lymphoma [22]. HCD122 (CHIR12.12) (Novartis), anovel,fullyhuman,IgG1 antagonistic mAb specifically blocked CD40L-induced adhesion, cytokine secretion, and 3.2. Monoclonal Antibodies Targeting IL-6R to Overt IL- survival of MM, as well as induced marked ADCC against 6/IL-6R Function. IL-6 is a major growth and survival CD40+ MM cells [21]. In vivo anti-MM activity by HCD122 factor in MM cells whose effects are mainly paracrine [10]. wasdemonstratedinaxenograftmodelof12BMMMplas- Various therapeutic agents which affect IL-6-mediated effects macytoma in mice [23]. Early clinical trials have evaluated have been tested including IL-6-conjugated mAbs directed the pharmacokinetics, safety, and efficacy of dacetuzumab against IL-6R and IL-6 [11]. IL-6R antagonist SANT-7, in monotherapy in patients with relapsed/refractory MM and combination with Dex and all-transretinoic acid (ATRA) other B-cell tumors [24, 25]. Phase I data suggest both or zoledronic acid, strongly inhibited growth and induced agents are well tolerated with no immunogenicity and show apoptosis in MM cells [12–14]. These studies suggest that early evidence of single-agent clinical activity in relapsed and overcoming IL-6-mediated cell resistance by SANT-7 poten- refractory MM and NHL [26, 27]. SGN-40 Phase Ib clinical tiates the effect of glucocorticoides and bisphosphonates trials in combination with lenalidomide and dexametha- on MM cell growth and survival, providing a rationale for sone/or bortezomib are planned based on enhanced anti- therapies using IL-6 antagonists in MM. MM activities when combining SGN-40 with lenalidomide Tocilizumab (MRA, atlizumab, Roche Pharmaceuticals) [28]. is a humanized anti-human IL-6R mAb (rhPM-1, IgG1 class) designed by using genetic engineering technology and the first therapeutic mAb developed in Japan [15]. Tocilizumab 3.4. Targeting CS1 by HuLuc63/Elotuzumab in MM. Using specifically blocks IL-6 actions and ameliorates diseases subtractive hybridization of na¨ıve B-cell cDNA from mem- associated with IL-6 overproduction [16]. For example, ory B/plasma cell cDNA, CS1 (CD2 subset-1, CRACC, besides Castleman’s disease and rheumatoid arthritis (RA), SLAMF7, CD319), a novel member of the signaling lympho- tocilizumab has been shown to be effective in patients cyte activating molecule- (SLAM-) related receptor family, with juvenile idiopathic arthritis and Crohn’s disease [17, was identified to be highly expressed in plasma cells [29, 18]. Tocilizumab treatment is generally well tolerated and 30]. Specifically, CS1 mRNA and protein are expressed in safe. Moreover, blockade of IL-6R may prove effective in CD138-purified primary tumor cells from the majority of limiting MM cell growth. Indeed it is now evaluated in MM patients (>97%), but neither in major body organs open-label Phase I (USA) and II (France) trials to assess nor CD34+ stem cells. To a low extent, its expression was its safety and efficacy as monotherapy in MM patients who also observed in NK cells, a subset of T cells, activated are not candidates for, or who have relapsed after stem cell monocytes and activated dendritic cells. CS1 may contribute transplantation (SCT). to MM pathogenesis by increasing MM cell adhesion, In addition, NRI, another receptor inhibitor of IL-6 clonogenic growth, and tumorigenicity via c-maf-mediated genetically engineered from tocilizumab, is under preclinical interactions with BMSCs [31]. A novel humanized anti- evaluation [19]. NRI consists of VH and VL of tocilizumab in CS1 mAb HuLuc63 (elotuzumab) was selected for clinical a single-chain fragment format dimerized by fusing to the Fc development due to its potent tumor-killing activity in vivo portion of human immunoglobulin G1. The binding activity and in vitro. Specifically, elotuzumab induced significant to IL-6R and the biological activity of the purified NRI ADCC against MM cells even in the presence of BMSCs. were found to be similar to those of parental tocilizumab. Moreover, it triggered autologous ADCC against primary Because NRI is encoded on a single gene, it is easily MM cells resistant to conventional or novel therapies applicable to a gene delivery system using virus vehicles. An including bortezomib and HSP90 inhibitor, and markedly 8 Bone Marrow Research enhanced HuLuc63-induced MM cell lysis when pretreated Max-CD38 inhibited CD38+ tumor cell growth in both with conventional or novel anti-MM drugs [29, 32]. preventive and therapeutic settings. In addition, HuMax- A phase I study of HuLuc63 was well tolerated in MM CD38 inhibits the CD38 ADP-ribosyl cyclase activity in patients [33]. Preliminary PK data reveal that peak serum target cells, which may contribute to the effectiveness of drug levels for the 0.5 mg/kg dosing cohort reached 10 HuMax-CD38 in killing both primary MM and plasma cell mg/mL, which was sufficient to achieve CS1 saturation of leukemia cells. at least 70% on the antigen rich NK cell subset. Drug Similarly, MOR202 (MorphoSysAG), a fully human anti- levels dropped below 1 mg/mL by day 7, however, coinciding CD38 IgG1 mAb produced by a human combinatorial with a decrease in saturation. This indicates that the higher antibody library (HuCAL) platform, also efficiently triggers doses to be used in subsequent cohorts may achieve and ADCC against CD38+ MM cell lines and patient MM cells surpass sustained concentrations in patients above this in vitro as well as in vivo in a xenograft mouse model level. Enrollment is continuing to determine the MTD. [39, 42]. One practical problem in applying anti-CD38 Early results of clinical trials of HuLuc63 in combination therapy is the wide expression on lymphoid, myeloid, and with bortezomib or lenalidomide or dexamethasone were epithelial cells, especially following cell activation. However, reported at the ASH meeting 2009 [34, 35], suggesting that mAbs specifically blocking CD38 might still provide a new elotuzumab may enhance the activity of bortezomib and approach for interfering with deleterious growth circuits, lenalidomide in treating MM with acceptable toxicity. PK therefore increasing the susceptibility of MM and leukemic analysis suggests a serum half-life of 10-11 days at higher cells to conventional chemotherapy. doses (10 and 20 mg/kg). Preliminary analysis of mononu- clear cells of peripheral blood and the BM indicates that 3.7. Targeting HM1.24 on MM Cells. HM1.24 (CD317) was objective responses correlate well with complete saturation originally identified as a cell surface protein differentially of CS1 sites by elotuzumab on BM plasma cells and NK cells. overexpressed on MM cells [43]andlaterwasfoundtobe The combination of elotuzumab with lenalidomide and low- identical to bone stromal cell antigen 2 (BST-2). A role of dose dexamethasone has a manageable adverse event profile HM1.24 in trafficking and signaling between the intracellular and compared to historical data for lenalidomide and high- and cell surface of MM cells was suggested since it is one of dose dexamethasone, the preliminary efficacy data (PR of the important activators of NF-kappaB pathway [44]. The 92%) are very encouraging. humanized anti-HM1.24 mAb (IgG1/kappa, AHM, Chugai Pharmaceutical Co., Ltd.) is able to effectively induce ADCC 3.5. Targeting CD56 with Immunotoxin-Conjugated mAb. against some human myeloma cells in the presence of human HuN901 conjugated with the maytansinoid N2 -deacetyl- PBMCs as effectively as a chimeric anti-HM1.24 mAb [45]. N2 -(3-mercapto-1-oxopropyl)-maytansine (DM1), a potent Single intravenous injection of AHM significantly inhibited antimicrotubular cytotoxic agent may provide targeted deliv- tumor growth in both orthotopic and ectopic human MM ery of the drug to CD56-expressing tumors including MM. xenograft models [46]. Although limited, the only one phase HuN901-DM1 has significant in vitro and in vivo anti-MM I/II clinical study reported that a humanized anti-HM1.24 activity at doses that were well tolerated in a murine model mAb did not cause any serious toxicity when administered to [36]. patients with relapsed or refractory MM [46]. The phase I clinical study of huN901-DM1 (BB-10901) Most recently, we characterized XmAb5592, a novel Fc- in 23 MM patients determined the MTD as 140 mg/m2/week engineered and humanized anti-HM1.24 mAb, and studied dose and demonstrated an overall favorable safety profile mechanisms of its anti-MM activity [47]. XmAb5592, with [37]. Exciting single agent activity was observed in heavily double amino acid substitution in Fc region of the wild- pretreated MM patients, which warrant continued investiga- type IgG1, has approximately 40-fold and 10-fold increases tion of this novel agent in MM patients especially when used in affinity for Fc gamma receptor III (FcRIIIa) and (FcRIIa), in combination with approved anti-MM agents/regimens respectively, expressed on effector cells including NK cells. such as lenalidomide and dexamethasone. It triggers 10–100-fold higher ADCC against these MM cell lines than a native/non-Fc-engineered version (anti-HM1.24 IgG1) of the Ab. XmAb5592 also induced more potent 3.6. Targeting CD38 in Multiple Myeloma. The CD38 anti-MM activity in murine subcutaneous xenograft murine molecule is expressed on cell surfaces in a majority of lym- models using RPMI 8226 cells. These results suggest that phoid tumors, notably MM [38, 39]. However, early studies XmAb5592 is a promising next generation immunothera- using anti-CD38 mAb with or without an immunotoxin peutic for MM. (ricin) have not led to useful clinical applications [4, 40]. Recently, a human anti-CD38 IgG1 HuMax-CD38 (Dara- tumumab) was raised after immunizing transgenic mice 3.8. Targeting TRAIL Death Signaling Pathway. Two human (HuMax-Mouse) possessing human, but not mouse, Ig agonistic mAbs directed against TRAILR1 (HGS-ETR1, genes. Preclinical studies indicated that HuMax-CD38 was TRM-1, Mapatumumab) and TRAILR2 (HGS-ETR2) killed effective in killing primary CD38+CD138+ patient MM cells 68% and 45% of MM cell lines, respectively, [48]. Only 18% and a range of MM/lymphoid cell lines by both ADCC and of MM cell lines are resistant to either antibody. There is no CDC [41]. In SCID mouse animal models, using sensitive correlation between TRAILR expression level and sensitivity bioluminescence imaging, treatment with Hu. to TRAIL-R1 or TRAIL-R2 triggering. Both the extrinsic Bone Marrow Research 9

(caspase 8, Bid) and the intrinsic (caspase 9) pathways for the treatment of 16 patients with advanced MM. Overall, are activated by anti-TRAIL mAbs. Mapatumumab is well 13 of 16 patients (81.3%) exhibited a response, with a tolerated in a phase I study in patients with advanced solid complete response (CR) seen in 6 patients (37.5%) without malignancies (n = 41) and 12 patients had stable disease for any toxic or allergic reactions. However, the incidence of 1.9 to 29.4 months [49]. These studies encouraged clinical thrombocytopenia and neutropenia increased. Subsequent trials of anti-TRAILR1 mAb in MM. In addition, based on clinical trials of BE-8 concluded that limitations of this enhanced cytotoxicity when combining mapatumumab with regimen are first the amount of BE-8 that can be injected bortezomib in preclinical experiments [50], a randomized due to its short half-life (3-4 days) and second, the continued phaseIIstudywasrecentlystartedcomparingTRM-1plus production of IL-6 in vivo. Most recently, a high-affinity fully bortezomib (Velcade) versus bortezomib alone in patients human version of BE-8, OP-R003-1 (or 1339, Azintrel), was with relapsed or refractory MM. selected through ActivMAb antibody discovery technology. Indeed, it enhanced cytotoxicity induced by dexamethasone, 3.9. Targeting CD74 with Milatuzumab. CD74 is an integral as well as bortezomib, lenalidomide, and perifosine, in a membrane protein that functions as a MHC class II chap- synergistic fashion [57]. Importantly, Azintrel also blocked erone. Milatuzumab is a humanized anti-CD74 mAb con- bone turnover in SCID-hu mouse model of MM, providing structed using the same human backbone as an additional rationale for its use in MM. (anti-CD22), whose safety has been demonstrated in clinical Despite overcoming the safety concerns of human anti- trials of patients with B-cell malignancies and autoimmune mouse antibodies associated with murine anti-IL-6 mAb and disorders [51, 52]. MM cell lines express CD74 (∼60% a long half-life (17.8 days) in circulation, the chimeric mouse of samples) and milatuzumab caused growth inhibition mAb to IL-6 CNTO 328 has been ineffective in producing and induction of apoptosis in CD74-expressing MM cell a meaningful response in MM [58, 59]. Nevertheless, due lines when cross-linked with an anti-human immunoglob- to enhanced anti-MM activities of combined CNTO 328 ulin G secondary antibody [53]. Moreover milatuzumab and bortezomib/or dexamethasone in preclinical models, demonstrated promising therapeutic activity in a CAG-SCID ongoing studies are investigating these regimens for their mouse model of disseminated disease for MM when used clinical value in treating MM [60, 61]. Specifically, results alone or in combination with doxorubicin, dexamethasone, of a small safety analysis (n = 21) done as a run-in to a bortezomib, or lenalidomide [54, 55]. In a phase I trial, larger ongoing Phase II trial showed promising preliminary milatuzumab showed no severe adverse effects in patients efficacy of CNTO 328 in combination with bortezomib in with relapsed/refractory MM, and it stabilized the disease relapsed/refractory MM. CNTO 328 is also being evaluated in some patients for up to 12 weeks [51]. Supporting as part of a combination therapy for initial treatment of MM the data in MM ongoing clinical trials testing different in a Phase II trial which compares the safety and effectiveness treatment schedules of milatuzumab in chronic lymphocytic of CNTO 328 plus Velcade-melphalan-prednisone (VMP) leukemia, non-Hodgkin’s lymphoma, and MM indicate that with VMP alone. milatuzumab shows no severe adverse effects in humans.

4. Antibodies Targeting MM Cells in the Bone 4.2. Targeting MM-Induced Bone Lesion Marrow Microenvironment 4.2.1. Targeting RANK/RANKL/OPG Axis Using Denosumab for MM-Associated Bone Destruction. Receptor activator of MM cells are highly dependent on the BM microen- nuclear factor-kappaB ligand (RANKL) is a cytokine mem- vironment for growth and survival through interactions ber of the tumor necrosis factor family that is the principal particularly with BM stromal cells (BMSCs) and osteoclasts, mediator of osteoclastic bone resorption [62]. Osteoprote- which secrete important MM growth factors and cytokines. gerin (OPG), a natural soluble decoy receptor of RANKL, Importantly, these factors/cytokines are further induced modulates the effectofRANKLandisabletopreventexces- from BMSCs when MM cells adhere to BMSCs [10]. Thus, sive bone resorption in the normal state. RANKL expression mAbs designed to block the binding of MM cell growth and is elevated in patients with MM [63, 64]. Denosumab (AMG survival factors to their cognate receptors have been under 162, Amgen Inc., Thousand Oaks, CA) is an investigational intensive development. fullyhumanmAbwithhighaffinity and specificity for RANKL that mimics the natural bone-protecting actions of 4.1. Blockage of IL-6 Binding to MM Cells. Early work OPG [65]. A phase 1 clinical trial in patients with MM (n = in developing mAb-based for MM has 25) or breast cancer with bone metastases (n = 29) showed been focused on the blockade of IL-6 secretion from BM that following a single s.c. dose of denosumab (0.1, 0.3, 1.0, microenvironment because of its key role in promoting MM or 3.0 mg/kg), levels of urinary and serum N-telopeptide cell growth and survival. Initial studies of mouse mAb to IL- decreased within 1 day, and this decrease lasted through 84 6 (murine BE-4 and BE-8) demonstrated a transient tumor days at the higher denosumab doses [66]. Mean half-lives cytostasis and reduction in toxicities from IL-6 [56]. The of denosumab were 33.3 and 46.3 days for the two highest potential of combination therapy, including BE-8 (250 mg), dosages. Larger trials are ongoing to investigate the effect of Dex (49 mg/day), and high-dose melphalan (220 mg/m2 denosumab for the treatment of cancer-induced bone disease (HDM220)), followed by autologous SCT was demonstrated and other bone loss disorders [67]. 10 Bone Marrow Research

4.2.2. Targeting the Wnt Inhibitor Dickkopf-1 (DKK-1). Moreover, ACE-011 has potential as a novel therapy Dickkopf-1 (DKK1), a soluble inhibitor of wingless (Wnt) for chemotherapy-induced anemia and may be an effective signaling secreted by MM cells contributes to osteolytic alternative to erythropoietin- (EPO-) based treatments. bone disease by inhibiting the differentiation of osteoblasts. The effect of anti-DKK1 mAb on bone metabolism and 4.3. Targeting Angiogenesis by VEGF Inhibitor Bevacizumab tumor growth in a SCID-rab system has been evaluated (Avastin). Vascular endothelial factor (VEGF) is important [68]. The implants of control animals showed signs of for the formation of new blood vessels and plays a key role MM-induced resorption, whereas mice treated with anti- not only in solid tumors but also in hematologic malignan- DKK1 antibodies blunted resorption and improved the bone cies, including MM [74]. Bevacizumab targets and blocks mineral density of the implants. Histologic examination VEGF and VEGF’s binding to its receptor on the vascular revealed that myelomatous bones of anti-DKK1-treated mice endothelium [75]. Anti-VEGF Abs were active alone, and in had increased numbers of osteocalcin-expressing osteoblasts combination with radiation in earlier preclinical studies [75, and reduced number of multinucleated TRAP-expressing 76]. It is currently being studied clinically in many solid and osteoclasts. The bone anabolic effect of anti-DKK1 was blood tumors including primary systemic amyloidosis and associated with reduced MM burden (P<.04). Anti- MM [77, 78]. NCI’s Cancer Therapy Evaluation Program is DKK1 also significantly increased BMD of the implanted sponsoring a phase II study of bevacizumab plus bone and murine femur in nonmyelomatous SCID-rab in MM [78]. mice, suggesting that DKK1 is physiologically an important regulator of bone remodeling in adults. Anti-DKK1 agents including BHQ880 (Novartis) may therefore represent the 4.4. Targeting BAFF/ARPIL Growth and Survival Pathway next generation of therapeutic options for the enhancement by Atacicept (TACI-Ig) or BAFF Inhibitor. Recently, B-cell of bone repair in some malignant and degenerative bone activating factor of the tumor necrosis factor (TNF) family diseases including MM [69, 70]. Although BHQ880 had (BAFF; also known as B lymphocyte stimulator, BLyS) and no direct effect on MM cell growth, BHQ880 increased a proliferation inducing ligand (APRIL), were identified osteoblast differentiation, neutralized the negative effect of as new survival factors for MM [79–81]. In addition to MM cells on osteoblastogenesis, and reduced IL-6 secretion. BMSCs, osteoclasts produce these factors to support MM Furthermore, in a SCID-hu murine model of human MM, cells in the BM microenvironment [81, 82]. Their cognate BHQ880 treatment led to a significant increase in osteoblast receptors are BAFF-R/BR3, transmembrane activator and number, serum human osteocalcin level, and trabecular calcium modulator (TACI), and B-cell maturation antigen bone. Preliminary results from a phase I/II trial in MM where (BCMA) with heterogeneous expression among patient MM BHQ880 was given IV for 28 days was well tolerated when cells. Specifically, RNA expression of BCMA and TACI is given in combination with zoledronic acid. approximately >30-fold and >10-fold higher, respectively, than that of BR3 [81]. BR3 specifically binds BAFF but not APRIL, and has very limited expression in mature B- 4.2.3. Targeting the Activin Receptor Type IIA (ActRIIA). cells plasma cells [83]. In fact, BCMA expression is only ACE-011, a novel bone anabolic agent currently in a Phase acquired in mature B cells and accompanied by loss of BAFF- 2 clinical trial in MM, is a protein therapeutic based on R expression [83], suggesting a key role of BCMA in plasma the activin receptor IIA. In numerous preclinical models cell survival. These studies provide clinical rationale to target of bone loss, ACE-011 has demonstrated beneficial effects BAFF/APRIL survival pathway in MM. on both trabecular and cortical bone [71, 72]. ACE-011 Atacicept (TACI-Ig, ZymoGenetics; Serono) acts as a increased bone mineral density, improved bone architecture, decoy receptor by binding to and neutralizing soluble BAFF increased the mineral apposition and bone formation rates, and APRIL, and preventing these ligands from binding to and improved bone mechanical strength [73]. Results of the their cognate receptors on B-cell tumors, thereby enhancing Phase 1 study in postmenopausal women demonstrated that cytotoxicity. An open-label, dose-escalation Phase I/II study a single dose of ACE-011 caused a rapid, sustained, dose- enrolled 16 patients with refractory or relapsed MM (n = dependent increase in serum levels of bone-specific alkaline 12) or active, progressive Waldenstrom’s Macroglobulinemia phosphatase (BSAP), a marker of bone formation, while (n = 4) [84]. Atacicept was well tolerated and showed clinical a marker of bone resorption, C-terminal type 1 collagen and biological activity consistent with its mechanism of telopeptide (CTX), decreased. In MM an ongoing multi- action. TACI was expressed heterogeneously among patient center Phase 2 trial is conducted in Russian patients which MM cells, which may explain promising results for the are treated with melphalan, prednisone, and thalidomide treatment of TACIhigh MM cells in a trial for atacicept [84, and randomized to receive either monthly doses of ACE- 85]. 011 or placebo for up to three months. Preliminary results In addition, the in vivo antitumor activity of neutralizing show clinical significant increases in biomarkers of bone anti-BAFF mAb in SCID-hu model of human MM provide formation, improvement in skeletal metastases, decreases in the preclinical rationale for its evaluation in the treatment bone pain as well as antitumor activity [54]. In summary, of MM [86]. Moreover, since all MM cell lines and patient these data indicate that ACE-011 is well tolerated and has MM cells express BCMA, BCMA might be a promising significant hematologic activity in MM patients receiving target for monoclonal antibody development against MM. myelosuppressive chemotherapy. Importantly, MM cells in remission postallogeneic transplant Bone Marrow Research 11 due to graft-versus-tumor response have donor derived anti- References BCMA Abs that are tumor lytic in vivo [87]. Thus, BCMA is a target of donor B-cell immunity in patients with myeloma [1] S. P. Treon, Y. Shima, M. L. Grossbard et al., “Treatment of who respond to donor lymphocyte infusion (DLI). Antibody multiple myeloma by antibody mediated immunotherapy and induction of myeloma selective antigens,” Annals of Oncology, responses to cell-surface BCMA may contribute directly to vol. 11, no. 1, pp. S107–S111, 2000. tumor rejection in vivo. Indeed, BCMA antibodies show [2] C. Gemmel, F. W. Cremer, M. 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Case Report A Case of Monoclonal Lymphoplasmacytosis of the Bone Marrow with IgM-Positive Russell Bodies

Hidekazu Kayano,1 Tsuneyuki Shimada,2 Naoki Wakimoto,2 Yuichi Nakamura,2 Masami Bessho,2 Hiroshi Yamaguchi,3 Atsushi Sasaki,1 and Michio Shimizu3

1 Department of Pathology, Faculty of Medicine, Saitama Medical University, 38 Moro Hongoh, Moroyama, Iruma-gun, Saitama 350-0495, Japan 2 Department of Hematology, Faculty of Medicine, Saitama Medical University, 38 Moro Hongoh, Moroyama, Iruma-gun, Saitama 350-0495, Japan 3 Department of Surgical Pathology, Saitama International Medical Center, 1397-1 Yamane, Hidaka, Saitama 350-1298, Japan

Correspondence should be addressed to Hidekazu Kayano, [email protected]

Received 13 November 2010; Accepted 13 January 2011

Academic Editor: Paolo De Fabritiis

Copyright © 2011 Hidekazu Kayano et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A 71-year-old Japanese male patient infected with HCV was diagnosed with thrombocytopenia. Histological examination of the bone marrow aspirate showed numerous lymphoid aggregates with Russell bodies. Immunohistochemistry and flow cytometric analysis demonstrated clonal expansion of CD5+ CD23+ B cells. Russell bodies were positive for IgM and lambda immunoglobulin light chain. The patient also underwent gastric biopsy, which revealed Helicobacter pylori (HP) infection. Subsequent eradication of the bacteria resulted in improvement of his thrombocytopenia. The clinical course remained uneventful at 15-month follow- up, consistent with monoclonal B-cell lymphocytosis. The observed clonal expansion with plasmacytic differentiation may have occurred under the influence of HCV with HP infection.

1. Introduction chronic lymphocytic leukemia (B-CLL) and a very low chance to progress to overt B-CLL. During histological examination of the bone marrow, lym- Some infective agents, such as hepatitis C virus (HCV) phoid aggregates are occasionally found. Those lymphoid and Helicobacter pylori (HP),arewellknowntodriveB- aggregates may be either neoplastic or reactive, yet the cell proliferation, causing different monoclonal or polyclonal histological distinction can be difficult [1], as it often lymphoplasmacytic diseases, and this is reviewed by Chiba requires ancillary molecular techniques to detect the clonal and Marusawa [4]. In patients infected with HCV, B-cell population of B-cells in the bone marrow. Although malig- aggregates are occasionally observed in the bone marrow nant B-cell populations harbor clonal rearrangements of [1, 5]. Recently, it has been reported that chronic gastritis immunoglobulin (Ig) heavy chain genes, clonality of Ig genes infected with HP may show accumulation of plasma cells is not always sufficient for the diagnosis of malignancy, containing Russell bodies (RBs) in the gastric mucosa because some clinically benign diseases, including mono- known as Russell body gastritis [6]. RBs are spherical clonal gammopathy of undetermined significance (MGUS) inclusion bodies in plasma cells occupying the cytoplasm and [2] and monoclonal B-cell lymphocytosis (MBL) [3], contain compressing the nucleus and are thought to be associated clonal expansion of B-cells. MGUS represents monoclonal with an imbalance between the production and secretion expansion of plasma cells with a very low level of abnormal steps of Ig and deposit within vesicular structures derived serum protein, whereas MBL is characterized by clonal from dilated rough endoplasmic reticulum [7]. Accordingly, expansion of B-cells with phenotypes resembling B-cell RBs may reflect defects in B-cell development. 2 Bone Marrow Research

Figure 1: Low-power view of a clot section of bone marrow aspirate (H&E stain). Several lymphoid aggregates are scattered. Figure 2: High-power view of lymphoid aggregates in the bone marrow (H&E stain). At the periphery, plasma cells containing Russell bodies are observed.

Here, we describe a clonal B-cell population with a CLL- like phenotype aggregated in the bone marrow and show plasmacytic differentiation without clinical progression in a case infected with both HCV and HP.

2. Case Report A 71-year-old Japanese man with chronic hepatitis C infection was referred to our hospital and diagnosed with thrombocytopenia. On admission, physical examination revealed no particular change, such as lymphadenopathy or organomegaly. Laboratory data included white blood cell count, 4, 150 × 106/L; absolute number of lympho- cytes, 1, 170 × 106/L; red blood cell count, 495 × 1010/L; Figure 3: Gastric biopsy showing numerous microorganisms on platelet count, 35, 000 × 106/L; aspartate aminotransferase, the foveolar epithelium (Wright-Giemsa stain). 23 IU/L; alanine aminotransferase, 11 IU/L. Total protein was 7.3 g/dL. Antigen of hepatitis B virus was negative, and HCV antibody was positive. No abnormality of serum or urine ff with numerous Russell bodies at the periphery (Figures 1 protein was noted. The di erentiated nuclear cells from bone and 2) in the background of normocellular marrow with marrow aspirate showed 4% myeloid precursors, 1% myelo- trilineage hematopoiesis and mild erythroid preponderance. cytes, 7% metamyelocytes, 34% neutrophils, 3% eosinophils, Immunohistochemically, the aggregates were composed of 0.3% monocytes, 17% lymphocytes, 0.2% plasma cells, CD20+/BCL2+/lambda Ig light chain+ small B-cells with and 33% erythroblasts. Although clot section from the scattered CD3+T-cells. BCL6-positive large B-cells (centrob- aspirated bone marrow showed numerous lymphoid aggre- lasts) and CD21-positive follicular dendritic cells revealed gates suggesting lymphomatous involvement, the patient germinal centre remnants. At the periphery of the aggregates, was observed at our clinic without therapy for 15 months. almost all of the plasma cells contained Russell bodies Meanwhile, he also underwent gastric biopsy, which revealed positive for IgM (Figure 4) and the lambda Ig light chain, chronic gastritis with HP, but no RB (Figure 3). Subsequent whereas IgD, IgG, IgA, and the kappa light chain were eradication of HP was successful, and his platelet count negative. The plasma cells also expressed CD138 and MUM1 × 6 improved to the level of 130, 000 10 /L. Fifteen months but not CD45, CD20, and CD56. Flow cytometric analysis later, bone marrow was aspirated again, revealing the same with two-color immunofluorescence on CD45-targeted cell findings as in the initial examination. His clinical course population demonstrated monoclonal B-cells with a CLL- has been uneventful 22 months after his administration for like phenotype, namely, CD5 (76%), CD23 (43%), CD20 thrombocytopenia. Bone marrow trephine biopsy was not (71%), CD19 (74%), CD10 (10%), and monotypic restric- performed. tion of the lambda Ig light chain (72%). Conventional cyto- genetic analysis showed no clonal aberration. Genomic DNA 3. Pathological Findings extracted from the paraffin-embedded aspirate was subjected to a seminested polymerase chain reaction (PCR) assay, Clot section from the aspirated bone marrow showed numer- revealing clonality of the Ig heavy chain gene rearrangement ous lymphoid aggregates composed of small lymphocytes (data not shown). Paraffin-embedded bone marrow aspirate Bone Marrow Research 3

and directly modulate cellular function, thus giving cell growth advantages and resistance to apoptosis [4]. In fact, HCV infection is associated with proliferation of B-cells expressing CD5 and/or IgM [8],andlymphoidaggregatesor less frequent plasmacytosis in the bone marrow is found in over 30% of HCV-infected patients [5]. Thus, it is possible that in our case, CD5-positive B-cells had differentiated to IgM plasma cells under the influence of HCV infection. HP infection is also associated with B-cell proliferation such as some gastrointestinal . Recently, RB gastritis, a variant of chronic gastritis with numerous RB, has been reported to be associated with HP infection, and the disappearance of RB upon follow-up biopsy after the eradication of HP has been described, thus supporting the causative role of HP in the development of RB [6]. In the Figure 4: Immunostain for IgM demonstrates intense expression of Russell bodies and some plasma cells. present case, however, the impact of HP infection on the RB formation is yet to be known since the bone marrow aspirate repeated after eradication of HP revealed RB as in the initial examination. was also subjected to fluorescence in situ hybridization (FISH) analysis using two color fusion probes for t(11;14) (Vysis), which revealed no fusion signal of cyclin D1 and the References Ig heavy chain genes. [1] J. Thiele, T. K. Zirbes, H. M. Kvasnicka, and R. Fischer, “Focal lymphoid aggregates (nodules) in bone marrow biop- 4. Discussion sies: differentiation between benign hyperplasia and malignant lymphoma—a practical guideline,” Journal of Clinical Pathol- In the present case, clonal expansion of B-cells with a ogy, vol. 52, no. 4, pp. 294–300, 1999. CLL-like phenotype remained in the bone marrow for [2] R. A. Kyle, J. A. Child, K. Anderson et al., “Criteria for the 15 months without clinical manifestation, consistent with classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma MBL. Of note, each lymphoid aggregate was surrounded Working Group,” British Journal of Haematology, vol. 121, no. by numerous IgM-positive RB, indicating plasmacytic dif- ff 5, pp. 749–757, 2003. ferentiation. Although histological di erential diagnosis of [3]T.D.Shanafelt,P.Ghia,M.C.Lanasa,O.Landgren,andA.C. the present case would include lymphoplasmacytic lym- Rawstron, “Monoclonal B-cell lymphocytosis (MBL): biology, phoma, characterized by admixture of small lymphocytes natural history and clinical management,” Leukemia, vol. 24, and plasma cells with IgM expression, CD5 expression and no. 3, pp. 512–520, 2010. clinical indolence seen in the present case are not typical [4] T. Chiba and H. Marusawa, “A novel mechanism for of lymphoplasmacytic lymphoma. Mantle cell lymphoma, inflammation-associated carcinogenesis; an important role of another CD5-positive B-cell tumor, can be also negated activation-induced cytidine deaminase (AID) in mutation due to the lack of a cytogenetic hallmark or chromosomal induction,” Journal of Molecular Medicine, vol. 87, no. 10, pp. translocation involving the CyclinD1 gene. 1023–1027, 2009. In western countries, more than 4% of the general pop- [5]J.M.Klco,B.Geng,E.M.Bruntetal.,“Bonemarrowbiopsyin patients with hepatitis C virus infection: spectrum of findings ulation over the age of 40 harbor clonal B-cell populations and diagnostic utility,” American Journal of Hematology, vol. 85, with an absolute number of peripheral B-cells of less than no. 2, pp. 106–110, 2010. × 6 5, 000 10 /L, and the majority of such clonal populations [6] S. Paik, S. H. Kim, J. H. Kim, W. I. Yang, and Y. C. Lee, “Russell show a CLL (CD5+/CD23+)-like immunophenotype, yet body gastritis associated with Helicobacter pylori infection: a they will not progress to overt CLL. Such a condition has case report,” Journal of Clinical Pathology, vol. 59, no. 12, pp. been recognized as MBL, for which the diagnostic criterion 1316–1319, 2006. is simply based on the detection of a clonal expansion of [7]R.Ronzoni,T.Anelli,M.Brunati,M.Cortini,C.Fagioli,andR. peripheral B-cells. However, it has been suggested that almost Sitia, “Pathogenesis of ER storage disorders: modulating russell all MBL have some degree of bone marrow involvement, yet body biogenesis by altering proximal and distal quality control,” ffi detailed understanding of the bone marrow in MBL remains Tra c, vol. 11, no. 7, pp. 947–957, 2010. to be clarified [3]. [8]E.D.Charles,R.M.Green,S.Marukianetal.,“Clonal expansion of immunoglobulin M+ CD27+ B cells in HCV- In the pathway of B-cell differentiation, activation- associated mixed cryoglobulinemia,” Blood, vol. 111, no. 3, pp. induced cytidine deaminase (AID) is a physiologic genome 1344–1356, 2008. mutator essential for somatic hypermutation and class switch recombination of Ig genes. AID is induced by nuclear factor kappa B activation via both HCV and HP, causing gene mutations in Ig and non-Ig genes, consistent with the notion that HCV and HP drive B-cell proliferation Hindawi Publishing Corporation Bone Marrow Research Volume 2011, Article ID 269519, 7 pages doi:10.1155/2011/269519

Review Article Immune Recovery after Cyclophosphamide Treatment in Multiple Myeloma: Implication for Maintenance Immunotherapy

Amir Sharabi1, 2 and Nechama Haran-Ghera1

1 Department of Immunology, the Weizmann Institute of Science, Rehovot 76100, Israel 2 Department of Internal Medicine B, the Tel Aviv Sourasky Medical Center, Tel Aviv 64239, Israel

Correspondence should be addressed to Amir Sharabi, [email protected]

Received 13 December 2010; Accepted 7 February 2011

Academic Editor: David A. Rizzieri

Copyright © 2011 A. Sharabi and N. Haran-Ghera. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Multiple myeloma (MM) is a progressive B-lineage neoplasia characterized by clonal proliferation of malignant plasma cells. Increased numbers of regulatory T cells (Tregs) were determined in mouse models and in patients with MM, which correlated with disease burden. Thus, it became rational to target Tregs for treating MM. The effects of common chemotherapeutic drugs on Tregs are reviewed with a focus on cyclophosphamide (CYC). Studies indicated that selective depletion of Tregs may be accomplished following the administration of a low-dose CYC. We report that continuous nonfrequent administrations of CYC at low doses block the renewal of Tregs in MM-affected mice and enable the restoration of an efficient immune response against the tumor cells, thereby leading to prolonged survival and prevention of disease recurrence. Hence, distinctive time-schedule injections of low-dose CYC are beneficial for breaking immune tolerance against MM tumor cells.

1. Introduction mass, and draining lymph nodes from patients of a wide spectrum of cancers. A strong correlation exists between Treg Multiple myeloma (MM) is a progressive B lineage neoplasia levels and the progression of cancer. The increased number characterized by clonal proliferation of malignant plasma of Tregs was reported to reflect poor prognosis [2] and is cells that localize in the bone marrow (BM) replacing associated with suppression of proliferation, down- the normal BM population. A reduced level of polyclonal regulation of proinflammatory cytokines, and involvement immunoglobulins is a consistent feature of active MM in tumor tolerance to self antigens. Thus, new anticancer reflecting the suppression of CD19+ lymphocytes that corre- strategies involving interference in Treg biology by means of late inversely with the disease stage. The relationship between functional disruption or numerical depletion are of critical myeloma plasma cells and the BM microenvironment is importance. Treg depletion can lead to decreased tumor critical for the maintenance of the disease. Tumor cells cell growth both directly by enabling anti cancer cytotoxic and stromal cells interact via adhesion molecules and effects or indirectly by inducing cellular immune responses cytokine networks to simultaneously promote progression of against cancerous cells. General strategies to reduce Treg the disease leading to bone destruction, vertebral collapse, functions include depletion of Tregs by chemotherapeutic hypercalcemia, renal failure, hypogammaglobulinemia, and drugs, blockade of Treg function by target known receptors, peripheral neuropathy. The disease is associated with both blockade of Treg trafficking, and combing depletion of Tregs cellular and humoral immune deficiencies [1]. Recent studies with tumor vaccination. have revealed that CD4+CD25highFoxp3+ regulatory T cells Recently, we showed a correlation between increased (Tregs), which are physiologically engaged in the mainte- ratios of functional Tregs and disease progression in a unique nance of immunological self-tolerance, play critical roles for mouse model of MM that mimics the human disease [3, 4]. the control of antitumor immune responses. Increased num- Low-dose cyclophosphamide (CYC) that selectively depletes bers of Tregs were documented in peripheral blood, tumor Tregs reduced MM progression. Treatment of tumor-bearing 2 Bone Marrow Research mice with repeated administrations of low-dose CYC at specific such as CD4 and CD8 cells, B cells, NK cells, natural longer time intervals (coinciding with the blocked renewal killer T cells (NKT), and DCs [19–21]. Both types of T cells, of Tregs) resulted in reduced tumor load and prevention or CD4 and CD8 cells, are considered key components against delay of disease recurrence. The break of immune tolerance tumors [22, 23]. Further, NK cells destroy tumor cells that against MM tumor cells by prolonged maintenance of have reduced the expression of MHC class I but still express transient Treg depletion will be reviewed. ligands for activating receptors of NK cells [24]. These cells are prone to inhibition by Tregs, which might lead to the continuation of tumor development and proliferation. 2. Immune Abnormalities in MM Patients 3.1. Tregs and Malignancy. Tregs, naturally occurring and The number and function of T cells subsets are aberrant in inducible, play a key role in the regulation of antitumor patients with MM [5, 6]. The CD4 : CD8 ratio is inverted immune response in patients with cancer. It was shown and the helper T-cell type 1 to type 2 (Th1 : Th2) ratio that within the tumor microenvironment the tumor itself among CD4 cells is abnormal [7]. In addition, the levels induces na¨ıve CD4 cells to convert into antigen-specific Tregs of expression of CD28 costimulatory molecules required for [15]. Many of the immune cells that reside in the tumor T cell activation are downregulated in T cells derived from milieu are affected by Tregs, and thus Tregs help tumors to MM patients [8]. The elevated levels of transforming growth escape detection and elimination by the immune system. It factor (TGF)-β, in addition to the impaired accessory signals was shown that depletion of Tregs in tumor-bearing mice from Th cells, contribute to the presence of dysfunctional resulted in the enhancement of antitumor immunity and B cells [9]. Defective natural killer cells (NK) have also the reduction of tumor growth [25–29]. In patients with been noted in MM patients [10]. Circulating dendritic cells ovarian cancer, the infiltration of CD3+ effector T cells (DCs) from MM patients were shown to be dysfunctional, was associated with a better prognosis [30], whereas the failing to upregulate costimulatory molecules required for accumulation of Tregs was suggestive of poor prognosis [2]. DCs activation, which led to reduced phagocytic activity These observations were also confirmed in non-small-cell and antigen presentation [11]. It was reported that the lung cancer [31]. The accumulated data strongly suggested impaired activity of DCs may be linked to the upregulation a new therapeutic strategy in the field of cancer aimed at of Tregs [12]. Interestingly, in vivo expansion of Tregs eliminating Tregs or disrupting their activity. could be induced in the presence of MM-specific antigens in association with reduced immune effector functions. 3.2. Treg and MM. There is now sufficient evidence that Immunologically active compounds that promote tumor-cell CD4+CD25highFoxp3+ Tregs actively impede the antitumor survival are produced by myeloma cells. The latter include immune response in cancer patients. In vivo peripheral TGF-β, IL-10, IL-6, vascular endothelial angiogenic growth expansion of natural na¨ıve CD4+CD25highFoxp3+ Tregs were factor (VEGF), Fas ligand, Mucin 1, cyclooxygenase (Cox)- observed in large cohorts of 195 MM patients [32, 33]. 2, and related prostanoids and metalloproteins [13]. The Increased numbers of naturally occurring Tregs correlated increased numbers of Treg and the impairment and mod- with disease burden (coinciding with paraprotein levels) and ulation of immune functions in myeloma patients suggest increased morbidity. The frequency of double negative T cells that breaking tolerance by chemoimmunotherapy involving − − (CD3+,CD4 , and CD8 αβTCR+)wasreducedinpatients transient Treg depletion and recruitment of compatible with MM. CD4+CD25high T cells from MM patients were immune-derived cells could perhaps reduce tumor load and shown to coexpress markers associated with Treg phenotype delay or prevent tumor development. such as CTLA-4, GITR, and CD69. High serum levels of IL-10 and TGF-β were also observed. In both humans and 3. CD4+CD25highFoxp3+ Tregs animal models of MM, Tregs have been described as anergic cells, fully functional in early and late stage MM, exerting Subpopulations of T cells with suppressive capacities against strong suppression after T-cell receptor stimulation [34, 35]. antitumor activity of the immune system were first described Contradictory findings concerning Treg levels and activity in in the early 1980s [14]. In the last decade, these subsets MM patients were published by Prabhala et al. [36], namely, of cells were found to be naturally occurring or adoptively reduced CD4+Foxp3+ T cells as well as Treg dysfunction, induced. They constitute 5–10% of na¨ıve CD4+ T cells in the indicating that Tregs were unable to suppress anti-CD3+ periphery and are anergic cells with suppressive capabilities. mediated T-cell proliferation. Whether these tested cells were These cells are induced early during tumor development and coexpressing CD25high was not determined in this study. were shown to contribute to tumor tolerance [15]. These Notably, it seems that differences in research strategies may cells were characterized as CD4 cells that express CD25 and account for the contradictory data in the area of MM the transcription factor Foxp3 (forkhead/winged helix tran- malignancy. scription factor). The latter is essential for the suppressive The response of Tregs to tumors is illustrated in mice that phenotype of the cells [16, 17]. Also, Tregs constitutively lack Tregs and effectively reject tumors [26]. The involvement express CTLA-4, and CTLA-4 deficiency in Tregs results of Tregs in MM progression was also studied in a unique in enhanced antitumor immunity [18]. The mechanisms mouse model of MM (5T2MM) that developed sponta- underlying the suppressive effects of Tregs include inhibiting neously in BM of a very old mouse of the C57BL/KalwRij the activity of a variety of immune cells that are tumor strain [37, 38]. The tumor designated 5T2MM can be Bone Marrow Research 3

State of disease Low-dose CYC

CD4 Window tregs opportunity

NKT cells

CD4+CD25+Tregs Injection NKT cells CD4+CD25+Tregs NKT cells Mq Activation of DCs 1 45 90 135 day DCs Tcells Tcells CYC CYC CYC CYC Bcells Bcells Plasma cells Plasma cells

Figure 1: Cellular responses to low-dose CYC in mice with MM. maintained only by transfer of BM cells from sick mice to drug were the inhibition of Tregs expansion and sustaining young syngeneic mice. The 5T2MM mouse model resembles the potency of cytotoxic CD8 T cells [42]. Patients with the human disease in its main localization to the BM and in CLL treated with fludarabine responded with a preferential the development of hind limb paralysis (due to spinal cord depletion of Tregs mainly as a result of apoptosis induction compression) that occurs as an early manifestation of the [43]. Lenalidomide is a derivative of thalidomide that was disease. Further, as the disease progresses, signs of paraplegia approved by the FDA for treating myelodysplastic syndrome and bone lesions take place. Increased accumulation of Tregs and MM. In vitro assays showed that lenalidomide affected is observed in peripheral lymphoid organs, including lymph Tregs by reducing their expansion and function [44]. CYC nodes, BM, and peripheral blood during disease progression is an alkylating agent that is commonly used in various [3, 4]. The suppressive functions of Tregs were retained, antitumor protocols. In addition to the general cytotoxic indicating that the cells were functional in their capacity to effects of CYC, it was reported that doses may influence regulate immune activity. A correlation between increased selectivity of this drug against Tregs. As a result, the clinical Treg levels and disease morbidity was observed. Adoptive relevance of CYC in oncologic therapy has become stronger. transfer of Tregs separated from thymuses of sick 5T2MM Its use in this regard is discussed below. mice into 5T2MM-bearing mice serving as Treg recipients, increased severity of MM and enhanced tumor progression. Severe bone destruction, fractures of vertebra, and massive 5. CYC in Cancer Therapy tumor cell infiltration into skeletal muscles were observed. The transfer of Treg-free thymocytes did not mediate the Therapeutic approaches for breaking tolerance towards tumor-enhancing effects [3]. These results clearly indicated tumor cells have been studied extensively. The depletion of that Tregs can specifically support in vivo tumor progression. Tregs is the most investigated strategy and CYC was found to have specific effects on T cells with tumor inhibiting proper- ties [45]. In the liver, CYC is converted to the active metabolic 4. Common Chemotherapeutic Drugs aldophosphamide and phosphamide mustards which bind to Affecting Treg Depletion DNA, thereby inhibiting DNA replication and causing cell death. CYC has a broad antitumor spectrum, a low niche A number of chemotherapeutic agents that affect Tregs are of inducing chemoresistance, and a limited hematopoietic used for preventing tumor protection by the immune system. toxicity [46]. High doses of CYC have direct tumoricidal Gemcitabine is a nucleoside analogue that inhibits DNA effects, cause by lymphoablation, and synthesis. In patients with colon cancer that were treated with is used also in the clinic with the primary aim of damping gemcitabine, the depletion of Tregs occurred concomitantly ongoing immune responses in patients with autoimmune with an increase in cytotoxic CD8 T cells [39]. However, diseases. Administration of low-dose CYC (in the range when this drug was administered in pancreatic cancer, it did of 20–150 mg/kg body weight) can paradoxically augment not deplete Tregs although it led to activation of na¨ıve CD4 immune responses (demonstrated in many experimental T cells [40]. Mitoxantrone is an anthracenedione that causes animal models) leading to immunostimulation. Low-dose DNA strand breakup and unraveling. Its administration to CYC acts on cycling Tregs and inhibits their suppressor patients with breast cancer resulted in reduced numbers of function by enhancing apoptosis and decreasing homeostatic Tregs, but this effect was not correlative with the tumor proliferation. The net outcome of CYC immunomodulation response [41]. Fludarabine is a drug used for treating depends on the balance between immune suppressive Treg chronic lymphocytic leukemia (CLL). In vitro effects of this response and non-Treg effector responses. Expression of 4 Bone Marrow Research

GITR and Foxp3 which are involved in the suppressive frequency of Tregs or to modulate their function; in fact, activity of Tregs is downregulated by CYC administration the best reduction of Tregs in this trial was observed in [47]. Studies have shown the presence of a large number of patients who were treated with the lowest dose of three Foxp3+ Tregs in a variety of tumors and the enhancement of tested, for instance, 250 mg/m2. In the metronomic regimen natural as well as vaccine-induced antitumor T cell responses studies [55], it also was stressed that only the low daily by systematically or locally removing Foxp3+ Tregs [48]. A oral dose of 100 mg (in contrast to the nonactive 200 mg single low-dose CYC causing Treg depletion was shown to dose) selectively depleted Tregs cells. Another clinical trial be curative by potentiating tumor-specific immunotherapy that followed after the frequency and function of Tregs against established tumors [49]. High doses of CYC were less in patients with advanced hepatocellular carcinoma who effective in rejecting tumor growth [50]. Treatment of neu-N were treated with CYC at doses 150, 250, or 350 mg/m2 mice with vaccine and low-dose CYC chemotherapy rejected on day 1 and day 29 found that only the lower doses of tumor challenge due to the recruitment of latent pools of CYC (150 and 250 mg/m2) could impair Treg number and CD8+ cells to the antitumor immune response [51]. Mice function and unmask α-fetoprotein-specific T cell responses. bearing poorly immunogenic B16 melanoma did not evoke In fact, a better and more prolonged suppression of Treg immunity; however, the depletion of CD4+CD25+Tregs function was observed in patients treated with 250 mg/m2. resulted in CD8+ T-cell mediated rejection of secondary In contrast, the reduction of Tregs in response to the high challenged B16 tumors [52]. CYC was shown to induce IFN- CYC dose (350 mg/m2) was not significant [58]. There is α production, thereby augmenting lymphocyte proliferation limited information about the kinetics and function of Tregs including CD44high memory T-cell phenotype that might following CYC induced transient Treg depletion (including account for the increased antibody responses and the recovery to pretreatment levels) in patients with cancer. The persistence of memory cells [53]. Low-dose CYC, in addition duration of the blocked renewal of Tregs following CYC to its detrimental effects on Tregs, was shown to augment treatment might be important when considering repeated the antitumor immunogenicity of DC vaccine tested in B16 administrations of low-dose CYC at longer intervals using melanoma or C26 colon cancer [54]. the window opportunity [4, 59]. Increased Tregs in the peripheral blood and tumor microenvironment were observed in many human malig- 5.1. Effects of a Single Low- or High-Dose CYC on 5T2MM nancies. However, the current clinical application of the latter Progression. High doses of CYC are effective in tumor in cancer chemoimmunotherapy is limited. Oral adminis- therapy because of their direct cytotoxic effect and inhibitory tration of metronomic low-dose CYC regimen (100 mg at activity against cycling cells, thus killing tumor cells but regular intervals) in advanced cancer patients induced a along with the depletion of immune cells. In contrast, low profound and selective reduction of Tregs and restoration doses of the drug have been shown to selectively reduce of T-cell proliferation and innate killing activity by NK the number and function of Tregs and induce antitumor- effector functions. An increased dose of CYC (200 mg mediated effects. Within 24 hours after CYC administration taken daily) failed to deplete Tregs or to restore immune to 5T2MM-bearing mice at the clinical phase of the disease functions [55]. These studies validated CYC ability to induce (60–70 days after tumor cell challenge), both low- and transient Treg depletion in humans. The combination of high-dose CYC (100 and 200 mg/kg body weight, resp.) chemotherapy and immunotherapy was tested in advanced resulted in normalization of serum paraprotein level. In the pancreatic cancer patients. The vaccination was induced BM cavity, plasma tumor cells were replaced with normal using allogeneic GM-CSF secreting cell lines alone or in cell populations, in association with prolonged survival. sequence with CYC (250 mg/m2) before vaccination. Further, However, a higher MM incidence (80–85%) was observed patients with metastatic pancreatic cancer treated with CYC in those treated with the high-dose in comparison with the followed by GM-CSF had CD8+ T cell responses to HLA class low-dose CYC (40–50%) [4]. Further, mice treated not with I-restricted mesothelium epitope, and the patients’ survival low-dose but with high-dose CYC still had residual 5T2MM was prolonged [56]. cells, because adoptive transfer of BM cells from grossly A phase I clinical trial in patients with metastatic carci- appearing mice 170 days after initiation of treatment into noma and a high Treg level in their peripheral blood tested young syngeneic recipients resulted in the development of the effect of a single intravenous infusion of CYC in three dif- disease in the BM of the latter 3-4 months afterwards [59]. ferent doses (e.g. 250, 500, or 700 mg/m2) when administered Inhibitory effects of CYC on Tregs have been shown concomitantly with a nonspecific immunotherapy using in previous studies [46, 47, 51]. Mechanistic pathways intratumoral Bacilli Calmette-Guerin (BCG). It was found to explain the susceptibility of Tregs to CYC includes in this trial that CYC at the doses tested did not modulate downregulated expression of the survival molecule Bcl-xL significantly Treg numbers and function, and the authors and of costimulatory CTLA-4 in CD4+CD25high cells, and concluded that CYC may not represent an optimal therapy a significant decreased production of IL-2 by CD4 effector for eliminating Tregs [57]. Nevertheless, it has been reported cells [4]. The inhibitory effects of low-dose CYC on Tregs are (in many animal models) that only CYC at low doses (20– essential for accomplishing antimyeloma activities, because 150 mg/kg body weight) could enhance immunostimulation coadministration of Tregs 24 hours following the injection due to the selective killing of Tregs [4, 46, 47, 51]. Hence, of low-dose CYC (when the cytotoxic effects of the drug were it should not come as a surprise that high doses of 500 substantially diminished [46]), to 5T2MM-bearing mice, and 700 mg/m2 in the reported trial failed to reduce the abrogated the beneficial effects of CYC on MM [4]. Selective Bone Marrow Research 5 depletion of Tregs by low-dose CYC resulted in upregulated of MM depends on dosing and time schedule of drug numbers of IFN-γ producing NKT cells that are capable administration. The use of high-dose of CYC is less effective of controlling tumor growth in vivo [60]andareaffected in preventing the disease, since the cytotoxic effect then is less reciprocally by Tregs [61, 62]. The antitumor properties of selective, reducing both tumor cells as well as immune cells NKT cells are linked to their ability to produce large amounts with potential antitumor properties. In contrast, treatment of IFN-γ upon activation of NKT cells [63–65]. Further, the with low-dose CYC is associated with selective Treg deple- expression of MHC class II and CD86 stimulatory molecules tion, which leads to the restoration of peripheral effector T in DCs was up-regulated, which enhanced their function as cell proliferation and immune function and to a reduction in antigen presenting cells [4, 66–68]. Once the concept of low- MM incidence. Repeated administrations of low-dose CYC dose CYC was proven feasible for potentially enabling an at longer intervals (coinciding with the period of blocked effective immune response against MM cells, it was essential renewal of Tregs) enable on the one hand the elimination of to find the most effective protocol of treatment by means of tumor cells and on the other hand the breakdown of immune satisfactory and durable antimyeloma effects. tolerance as a result of recovery in effector T cells, NKT cells, and mature DCs that would react against the residual tumor cells (Figure 1). 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Review Article Recent Advances in the Pathogenesis and Management of CastNephropathy(MyelomaKidney)

Stephanie Stringer,1, 2, 3 Kolitha Basnayake,1, 2, 3 Colin Hutchison,1, 2, 3 and Paul Cockwell1, 2

1 Renal Institute of Birmingham, University Hospital Birmingham, Birmingham B15 2TH, UK 2 Department of Nephrology, University Hospital Birmingham, Birmingham B15 2TH, UK 3 School of Immunity and Infection, University of Birmingham, Birmingham, B15 2TT, UK

Correspondence should be addressed to Stephanie Stringer, [email protected]

Received 18 November 2010; Accepted 2 March 2011

Academic Editor: Guido Kobbe

Copyright © 2011 Stephanie Stringer et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Multiple myeloma is an incurable plasma cell malignancy that is often accompanied by renal failure; there are a number of potential causes of this, of which cast nephropathy is the most important. Renal failure is highly significant in myeloma, as patient survival can be stratified by the severity of the renal impairment. Consequently, there is an ongoing focus on the pathological basis of cast nephropathy and the optimal treatment regimens in this setting, including effective chemotherapy regimens to reduce light chain production and emerging extracorporeal techniques to remove circulating light chains. This paper bridges recent advances in the pathogenesis and management of cast nephropathy in multiple myeloma.

1. Introduction 2. Multiple Myeloma and the Kidneys The kidneys are a common target for injury in a large Multiple myeloma (MM) is a haematological malignancy number of acute and chronic diseases that initially develop characterised by clonal proliferation of plasma cells and at nonrenal sites. Where renal injury develops as a secondary associated with end-organ damage. There is production of consequence of nonrenal disease, then (i) the factors that a monoclonal immunoglobulin (Ig) in over 80% of those cause this injury are often complex; (ii) the involvement of affected; each intact monoclonal Ig will include either a the kidneys and the development of subsequent acute kidney kappa light chain (LC) isotype or a lambda LC isotype; in injury (AKI) or chronic kidney disease (CKD) has a delete- the large majority of cases of intact monoclonal Ig in MM rious effect on subsequent clinical outcomes. The effective there is excess production of the relevant monoclonal LC, management of kidney disease is an important factor in and in around 20% of cases there is LC monoclonality with improving the long-term outcomes of the affected individ- no detectable intact immunoglobulin [1]. This detectable Ig ual. Multiple myeloma (MM) is commonly complicated by LC is often referred to as free light chain (FLC). kidney injury with a major impact on long-term outcomes; The incidence of new-onset (first diagnosis) MM in the therefore understanding the nature of the kidney damage United Kingdom is around 6.5 per 100,000 in Caucasians, and the treatment options that are available are crucial for 10-9-18.2/100,000 in Blacks, and 3.6–6.4/100,000 in Asians improving outcomes for people with MM and kidney injury. [2]. At presentation at least 50% of patients will have AKI In this paper we will overview the current status of basic and of a variable degree, and around 10% have severe AKI from clinical science in this area and show how increasing knowl- which they will die within weeks unless they are treated with edge in both these areas is leading to improvements in clinical renal replacement therapy by dialysis [3]. The prognosis for outcomes. patients with MM and AKI is worse than that for those with 2 Bone Marrow Research

glomerulus because of their size (22.5 kD for Kappa (κ)and 45 kD for Lambda (λ)) and cationic net charge; the glomeru- lar sieving coefficient for free Kappa LC has been calculated at 0.09 [9]. As there are detectable levels of polyclonal FLC in the serum of normal individuals (κ at 3.3 to 19.4 mg/L; λ at 5.7 to 26.3 mg/L), it can be extrapolated that in health between 100–600 mg/24 hr of FLC are presented at the renal tubule; as there are minimal levels of FLC present in the urine this indicates a high capacity for reabsorption of FLC by the tubules [10]. This process of FLC reabsorption takes place at PTEC through receptor-mediated interactions, and free LCs in tubular ultrafiltrate are taken up by the tandem receptors cubilin/megalin, with subsequent endocytosis via the endosomal/lysosomal pathway; some FLC clones bind preferentially to megalin and some to cubilin [11–13]. After endocytosis the FLCs undergo vesicular trafficking, which is Figure 1: Renal biopsy showing cast nephropathy: distal tubular dependent upon acidification of the vesicle. However not all casts and interstitial inflammation and fibrosis. FLC digestion is confined to lysosome, and there may be some degradation of FLC bound to the PTEC membrane [12]. There is no evidence to date that endocytosis of poly- normal renal function [4]. This directly relates to the severity clonal FLC activates PTEC; however there is substantial of AKI at time of presentation, such that those who require data that show a profound proinflammatory and cytotoxic and receive dialysis treatment have a median survival of less potential of monoclonal FLC. There is a differential capacity than 12 months [5]. of any given clone of FLC to activate PTEC to produce Thereareanumberoffactorsthatcancontributeto proinflammatory cytokines through activation of NFκB; this the development of AKI in MM; these include dehydration, may contribute to the inflammatory cell infiltration and hypercalcaemia, and the ingestion of nephrotoxic drugs [6]. accelerated fibrosis that is seen in cast nephropathy [14]. It is important to note that cast nephropathy may not The production of these cytokines also involves mitogen- be the sole pathology in patients with MM and AKI; for activated protein kinases (MAPKs) ERK 1/2, JNK 1/2, and example, there may be concomitant amyloid or another p38 [11]. renal pathology unrelated to MM. All these factors have the A central component of the pathogenicity of monoclonal potential to trigger the development of cast nephropathy, FLC towards PTEC is mediated through the formation of whichisthecommonestcauseofsevereAKI.SevereAKIin hydrogen peroxide (H O ,); in vitro this is generated after MM is a major medical emergency, as it is associated with a 2 2 FLC endocytosis and indicates a high level of oxidative high risk of early death. Historically, less that 20% of people stress [15]. It has subsequently been established that H O with MM require dialysis at or shortly after presentation 2 2 production by monoclonal FLC mediates the oxidation and recover independent kidney function [5]. As a consequence, activation of c-Src (a redox sensitive, nonreceptor tyrosine there is a major focus on understanding the biological basis kinase), an obligate step in this setting for the production for the development of AKI in MM. In addition, the efficacy of MCP-1/CCL2 [16]. These and other studies show that of novel for promoting better outcomes is monoclonal FLCs have a greater inflammatory effect on under evaluation. Finally, with the introduction of protein- PTECs than other freely filtered proteins [14, 17]. permeable dialysers into clinical practice, there is continuing The dynamics of serum FLC (sFLC) levels and renal interest in the clinical utility of extracorporeal removal of clearance is important for the interrelationship between light chains to improve renal recovery. FLC processing by PTEC and the development of cast nephropathy. As MM evolves, the amount of clonal FLC that 3. The Pathogenic Basis of Cast Nephropathy in is present in glomerular ultrafiltrate progressively increases, Multiple Myeloma ultimately to levels that overwhelm the reabsorptive capacity of PTEC. Therefore increasing amounts of FLC are present Cast nephropathy is characterised by the presence of frac- in filtrate in the loop of Henle and the distal tubule. There tured, waxy casts formed of FLC and uromodulin (Figure 1) is then a differential capacity of any given clone of FLC to [7, 8]. These casts precipitate out in the distal tubules aggregate with uromodulin (Tam-Horsfall protein) to form resulting in tubular obstruction; there is also associated casts [18]. One of the key determinants of cast formation is tubulointerstitial inflammation, which may in part be related the isoelectric point (pI) of the protein. to a direct proinflammatory effect of FLC on proximal When the ambient pH of tubular filtrate is close to the tubular epithelial cells (PTEC) [7, 8]. FLC pI, the protein carries no net charge and will tend To fully understand the pathogenesis of cast formation to precipitate out of solution; proteins with a pI of 5.6– it is necessary to understand the renal handling of FLC 7.3 tend to precipitate in the ascending limb of the loop (Figure 2). Serum FLCs are relatively freely filtered at the of Henle or early distal tubule while those with a pI < 5.1 Bone Marrow Research 3

Excess LCs

Freely filtered at glomerulus

Light chains pass to proximal tubule

Light chains pass Scavenged by to distal tubule proximal tubular cubilin/megalin

LCs precipitate out in acidic tubular Endocytosis fluid

LCs combine with Vesicular uromodulin tra cking

Cast formation H2O2 production

Tubular Inflammation via obstruction chemokine and cytokine production

Accelerated fibrosis

Figure 2: Renal handling of LCs.

precipitate more distally (the pI of FLC has been shown to antibody against this peptide sequence was utilised there was range from 3.5 to 9.9) [18, 19]. This reflects the progressive a reduction in FLC binding to uromodulin, and there is acidification of tubular fluid as it flows through the distal great interest therefore in the potential for small molecule nephron; this consequently facilitates the precipitation of blockers to reduce cast formation [23, 24]. Uromodulin proteins with progressively lower pIs. This may have direct may also have an immunomodulatory role via the induction clinical relevance in the management of the acid-base of inflammatory cytokines such as TNF-α and IL-6 from status of the individual patient which is supported by monocyte/macrophages and neutrophils [25, 26]. disease model studies, where the alkalinisation of the urine In conclusion, current concepts around the pathogenesis ameliorated the effects of FLC on inulin clearance in rats of AKI in MM can be summarised as follows: (i) cast [20]. nephropathy may be precipitated by dehydration, hyper- After precipitation of FLC, cast formation occurs when calaemia, or nephrotoxic drugs; (ii) the precipitating factors uromodulin traps the FLC and a gel-like cast is formed. for cast nephropathy may themselves cause AKI in the Uromodulin is secreted at the thick ascending limb of the absence of cast formation; (iii) the direct pathogenicity of loop of Henle; it is the main urinary protein in healthy monoclonal FLC on PTEC may also precipitate AKI; this individuals and has the ability to self-aggregate into a gel may occur in combination with cast nephropathy or (less like substance and bind with many low molecular weight commonly) through causing acute tubular necrosis (ATN) proteins [21, 22]. Uromodulin has been shown to have a in the absence of cast formation. Renal impairment is more specific binding site for FLC; this has been shown in animal likely to be reversible with supportive measures alone when models to be a peptide sequence [23]. When a monoclonal it is not related to cast formation [3]. 4 Bone Marrow Research

Our understanding of the biology of clonal FLC in achieving a serum creatinine of <130 μmol/l [38]. All patients producing AKI in MM indicates that aggressive early received Thalidomide at the same dose, in combination with reduction of FLC levels may be an important factor in a range of other agents. They reported a 67% renal recovery promoting the recovery of kidney function. This concept is rate, although neither of the dialysis patients recovered now being supported by clinical studies that clearly show a independent renal function [38]. More recently Thalidomide relationship between an early fall in sFLC levels and renal use has been reported in larger numbers of patients who have recovery [27, 28]. It is now clear that novel chemotherapeutic been dialysis dependent at presentation; in a study of high cut agents that are having a substantial impact on improving off haemodialysis 14/19 patients received Thalidomide and overall outcomes for patients with MM may be particularly recovered renal function [27]. important in the setting of MM and AKI, in part through early disease response. 4.2. Lenalidomide. Lenalidomide is a derivative of Thalido- mide but has a different side effect profile, it was introduced 4. Novel Agents and Outcomes of Patients with in 2004, and there has been subsequent interest in its Renal Impairment role in the treatment of MM. There have been several large studies of Lenalidomide in relapsed or refractory MM There are two crucial considerations in the assessment that show excellent clinical outcomes with response rates of the utility of novel chemotherapeutic agents in MM (defined variously as complete, near complete, partial, or and AKI: firstly, the efficacy and safety of these agents in very good partial responses) in the region of 50–60% [47– this setting and, secondly, the potential for these agents 50]. However there has been little work examining the to reverse renal impairment. Whilst it is self-evident that role of Lenalidomide in patients with renal impairment, as an agent that rapidly reduces tumour burden should also traditionally trials excluded patients with renal impairment result in an improvement in renal outcome, little work (to varying degrees), largely because early pharmacokinetic has been done to specifically address this question. This studies demonstrated that over 60% of the drug is excreted section will focus on the clinical utility in this setting of in the urine as a result of glomerular filtration and tubular three novel agents used in both denovo and relapsed MM: secretion [51]. Subsequently, Chen et al. carried out a Thalidomide, Bortezomib and Lenalidomide. If used in the multicentre, open label study to investigate the effect of setting of MM and AKI, these agents should probably be used renal impairment and dialysis on the pharmacokinetics in regimens that incorporated dexamethasone at cytotoxic of Lenalidomide. They enrolled 30 patients, whom they doses; this is because they act synergistically and early work stratified on the basis of renal function [52]. They confirmed comparing vincristine and doxorubicin alone or with high- that Lenalidomide is predominantly excreted by the kidneys dose dexamethasone suggested that most of the plasma cell (80% of total drug clearance) and that a creatinine clearance reduction was due to the high-dose dexamethasone [29, 30]. of <50 mls/min is the threshold at which dose modification Table 1 shows those chemotherapy studies that have reported is needed in respect of renal function [50]. For patients both the renal characteristics of the participants and their on dialysis there was a substantial decrease in trough renal outcomes [31, 32]. concentrations postdialysis, indicating that an extradose is needed following dialysis treatment [50]. 4.1. Thalidomide. Thalidomide was first developed in the Dimopoulos et al. reported a subgroup analysis of 1950s and was then used widely for numerous indications; the MM-009 and MM-010 trials; this analysis focused on however it was subsequently found to have devastating ter- the impact of renal impairment on safety and efficacy of atogenic effects and clinical use ceased. It was subsequently Lenalidomide and also on the renal outcomes of the cohort postulated that the teratogenic limb defects caused by the [40]. The degree of renal impairment was quantified by drug were caused by inhibition of angiogenesis, and this the calculation of creatinine clearance by the Cockcroft- prompted investigations into its potential as an antitumour Gault formula, and patients were divided into subgroups agent [41]. In 1965 the use of Thalidomide was first reported depending upon their renal function at baseline; these were in a patient with MM, with a subsequent delay in disease mild or no renal impairment (CrCl >60 mls/min), moderate progression. However, it was not until 1997 that patients renal impairment (CrCl 30–60 mls/min), or severe renal were first recruited into a clinical trial examining the effect impairment (CrCl <30 mls/min); however all patients had of Thalidomide in MM [42, 43]. Since then there have to have a baseline serum creatinine of <2.5 mg/dL [39]. been numerous studies using the drug that have established Renal recovery was defined as improvement in renal function its efficacy in MM, and it is now recommended for use as defined by transition to a subgroup with better renal in patients with MM in a range of clinical settings [44]. function. There was no difference in disease response rates Thalidomide is safe in renal impairment; it is not renally between subgroups, and response was not influenced by the excreted (the primary route of clearance appears to be degree of renal impairment. In moderate or severe renal hepatic), and there is no dose adjustment needed for renal impairment, 72% of the patients had an improvement in impairment or in patients undergoing dialysis [45, 46]. their renal function [40]. While individuals with very severe Tosi et al. considered the use of Thalidomide-containing renal failure were excluded and definitions for renal recovery regimens on the renal outcomes of patients. They enrolled are not precise, this work suggests that Lenalidomide is safe 20 patients (two who were dialysis dependent) with a serum and effective with a creatinine of up to 2.5 mg/dL and in this creatinine of ≥130 μmol/l and defined renal recovery as setting may improve renal function. Bone Marrow Research 5

Table 1: Renal characteristics of chemotherapy trials where both baseline renal characteristics and outcomes are reported.

Renal Inclusion Baseline renal Study Agents Renal outcomes criteria characteristics CrCl < 30 = 6% (B), 5% (control) Reversal = GFR >60 at end from San Miguel et al. CrCl 30–60 = 28% (B), <50 at start VMP versus MP No renal exclusions (VISTA) [31] 50% (control) VMP group = 44% reversal CrCl >60 = 46 (B), MP group = 34% 46% (control) All but 1 dialysis dependent, No clear defnof renal reversal Median sCr = 6.8 mg/dL B+varietyof Dialysis dependence 4 had “improved” renal function (1 pt Chanan-Khan et al. [33] (3.1–12.8)? before starting other agents mandatory never started dialysis, 2 came off dialysis dialysis after a CR and 1 off dialysis after a MR) No renal Bx VAD (or sCr >4mg/dL= 44% sCr <2 g/dL defined as renal reversal, 83% similar), M + sCr >2mg/dL sCr 2–4 mg/dL = 56% met this defn median TTR = 1.9 mo Kastritis et al. [34] hdD or D alone mandatory at 24% dialysis dependent 80% of those previously dialysed came off Group B: hdD + enrolment No renal Bx dialysis T+/ B Reversal = normalisation of CrCl, occurred CrCl 51–80 = 10.3% in 41% with no differences across V+DorV+ CrCl 30–50 = 19.7% Morabito et al. [35] No renal exclusions treatment subgroups D + other agents CrCl <30 = 70% 22% of those previously dialysed came off 12% dialysis dependent dialysis Mean sCr 9.05 mg/ml GFR <20 mls/min V, V + D or V, (5.2–12) Median sCr fell from 9.05 to 2.1 mg/dL Ludwig et al. [36] mandatory at D+A 63% dialysis dependent (0.8–2.4) enrolment No renal Bx Median GFR 20.5 mls/min No defined criteria for renal recovery, GFR <50 mls/min (3.7–49.9 mls/min) median GFR increased to 48.4 ml/min Ludwig et al. [37]VAD mandatory at 6renalBx(2showed (6.7–135.5 mls/min), improvement in GFR enrolment amyloid so patients correlated with tumour response excluded) sCr >130 μmol/l Initial sCr for all listed, Yes, Improvement defined as sCr Tosi et al. [38]T+othermandatory at Median = 155 μmol/l <130 μmol/l, 12/15 responding pts had enrolment Range 131–998 μmol/l renal recovery Recovery defined as either CrCl, 50 to >60 24 had baseline CrCl, 50 or movement from one subgroup to Dimopoulos et al. [39] R + D No renal exclusions (13–49), 2% dialysis another, 42% of those with renal dependent at baseline impairment had some improvement Improvement defined as movement from CrCl >60 = 71% one subgroup to a better one, 72% of those Dimopoulos et al. [40]R+DversusDsCr<2.5 mg/dL CrCl 30–60 = 24% who received R+D had some CrCl <30 = 5% improvement, data for D alone not quoted CrCl >80 = 56% CrCl 50–80 = 23.9% 26.6% had some change from one renal Dimopoulos et al. [40] R + D No renal exclusions CrCl 30–50 = 12.6% subgroup to another, 12.5% had CrCl <30 = 7.2% deterioration of renal function 3% dialysis dependent V: bortezomib, D: dexamethasone, M: Melphalan, P: prednisolone, A: doxorubicin, hD: high dose, T: Thalidomide, BMT: bone marrow transplant, R: Lenalidomide, CrCl: creatinine clearance (mls/min), Bx: biopsy, sCr: serum creatinine, CR: complete response, MR: minimal response, TTR: time to response, GFR: glomerular filtration rate.

Klein et al. reported a retrospective analysis of patients they report an improvement in renal function in 26%; with relapsed/refractory MM and renal impairment; the patients whose renal function stabilised or improved had cohort included patients who were dialysis dependent [32]. a higher frequency of PR than those whose renal function After treatment with Lenalidomide and dexamethasone deteriorated [41]. 6 Bone Marrow Research

4.3. Bortezomib. Bortezomib is a proteasome inhibitor that is Encouraging evidence with Bortezomib and dexametha- the first in class, and it was synthesised in 1995 and was first sone in patients with renal impairment has led to an Interna- used in humans until 1999. On the basis of a large phase II tional Myeloma Working Group recommendation that high- clinical trial it was recommended by the FDA for use in dose dexamethasone and Bortezomib are the recommended relapsed/refractory MM [52]. It undergoes hepatic clearance treatment for MM in patients with any degree of renal with no renal clearance, and therefore no dose adjustments impairment [30]. Given the previously poor prognosis for are recommended for patients with impaired renal function patients with renal impairment and MM it is logical to [53].Therehavenowbeenmanylargestudiesinvestigating focus on treatment with chemotherapy regimens that have the use of Bortezomib, initially in relapsed/refractory MM a high response rate and where that response is associated and more recently in de novo MM. A number of these with a rapid early decline in tumour (and therefore clonal have analysed the drug in impaired renal function, but few FLC) load. Where Bortezomib is not available, usually as a consider the renal outcomes of treated patients. consequence of cost, then Thalidomide should be used, again The largest study of renal outcomes is a subgroup analysis in combination with dexamethasone. of the VISTA study; this was a randomised phase III study In addition to an evolution in the chemotherapy options comparing melphalan and prednisolone (MP) to melpha- for patients with AKI and MM, there has been a recent lan, prednisolone, and Bortezomib (VMP). However the reevaluation of the role of extracorporeal removal of sFLC. study excluded patients with a serum creatinine >2mg/dL Whilst this may seem an attractive addition to therapies that (176 μmol/l) so was not applicable to patients with severe are available, it is critical to understand that this adjunctive AKI [31]. Despite this there was some indication of a benefit therapy will only add benefit in the setting of effective in the Bortezomib group with better renal outcomes in chemotherapy. Furthermore, any proposed use must be the patients treated with the drug (44% renal recovery in validated by properly designed prospective randomised treatment arm; 34% in the control arm; P = .001 the time controlled trials. to renal recovery was also faster in the VMP arm [31]. Ludwig et al. also considered the renal outcomes of 5. Extracorporeal LC Removal Strategies patients treated with Bortezomib; in their study it was used as part of a BDD (Bortezomib, doxorubicin, and dexametha- Plasma exchange has been variably used in clinical practice sone) regimen; they enrolled 68 patients with light-chain- for the removal of FLC; however, there is no convincing induced renal failure (defining acute renal failure as a recent evidence of benefit compared to treatment regimens that do decline in glomerular filtration rate (GFR) <50 mLs/min not include plasma exchange. More recently, novel protein where other causes of renal failure had been clinically permeable dialysis membranes have been developed, and excluded) [37]. All patients received the same treatment their utility in FLC removal has attracted considerable regimen, though only 58 patients were available for analysis interest. (seven died before the 2nd cycle, one discontinued after the 2nd cycle because of toxicity, one had progressive disease, 5.1. Plasma Exchange. Plasma exchange (PE) involves ex- and one had incomplete data) [36]. They reported complete tracorporeal processing of a patient’s blood to remove response rates of 31% and an overall response rate of pathogenic substances from the plasma; dependent on the 62%, the renal outcomes of the cohort were also reported, clinical indication these can include autoantibodies, cryo- and the median GFR increased from 20.5 mls/min (3.7– globulins, or other abnormal plasma proteins or immune 49.9 mls/min) to 48.4 mls/min (6.7–135.5 mls/min) [37]. It complexes [54]. A single plasma exchange removes approx- is important to note that the number of patients with imately 75% of the patient’s own plasma and the abnormal histological evidence of cast nephropathy was small; only constituent in the plasma [54]. Therefore PE was considered six patients had had a renal biopsy (of whom two were not asfortheremovalofremoveFLCsinMMandwas enrolled because of a diagnosis of amyloid), and the numbers investigated in a series of studies. The largest and most requiring dialysis at the outset and the end were also not robust study to date, reported by Clark et al., examined the reported. outcomes of 104 patients (of whom 97 completed 6-month Chanan-Khan et al. considered the use of Bortezomib followup) with acute renal failure at the time of presentation in dialysis-dependent renal impairment. They retrospectively of MM [55]. There were several design shortfalls in the examined the records of 24 patients who were treated for study; these included the absence of histological diagnosis MM and required dialysis at that time [33]. Of the 24 patients of cast nephropathy and the presence of dialysis dependency initially considered there was insufficient data available in at randomisation in only 28% of those recruited [55]. The six, so results on 18 patients were available for analysis [33]. patients were randomised to receive either conventional Of the remaining patients, 23% were independent of dialysis therapy with the addition of between five and seven plasma at analysis (one never actually needed dialysis, two became exchanges or conventional therapy alone. The results showed dialysis independent after a complete response (CR) and one that there was no benefit derived from plasma exchange by a after a minimal response (MR)) [33]. There was no increase composite end point of death, dialysis dependence, or renal in adverse events reported compared to patients without function at six months [55]. renal impairment [33]. As a result the authors concluded Other studies of PE have shown conflicting results, that Bortezomib was safe and effective in patients requiring but these are all limited by small sample sizes and other dialysis. methodological flaws [56, 57]. Collectively, these studies Bone Marrow Research 7 raised questions about why PE is ineffective. The most to have poor outcomes. A greater understanding of the important reason probably relates to the short duration pathogenesis of cast nephropathy may lead to successful of the treatment, which results in removal of FLC from prevention strategies and improvements in chemotherapy the intravascular space only. As 85% of the total FLC load and the emergence of an effective method of LC removal is in the extravascular compartment, this leads to rapid contribute to future improvements in clinical outcomes for redistribution following PE of FLC into the intravascular those affect. compartment [58]. 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Review Article Oncolytic Virotherapy for Multiple Myeloma: Past, Present, and Future

Chandini M. Thirukkumaran and Don G. Morris

Department of Oncology, Tom Baker Cancer Centre, 1331 29 Street NW, Calgary, AB, Canada T2N 4N1

Correspondence should be addressed to Don G. Morris, [email protected]

Received 21 December 2010; Accepted 1 March 2011

Academic Editor: Fausto Loberiza

Copyright © 2011 C. M. Thirukkumaran and D. G. Morris. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Multiple myeloma (MM) is a B-cell malignancy that is currently felt to be incurable. Despite recently approved novel targeted treatments such as lenalidomide and bortezomib, most MM patients’ relapse is emphasizing the need for effective and well- tolerated therapies for this deadly disease. The use of oncolytic viruses has garnered significant interest as cancer therapeutics in recent years, and are currently under intense clinical investigation. Both naturally occurring and engineered DNA and RNA viruses have been investigated preclinically as treatment modalities for several solid and hematological malignancies. Presently, only a genetically modified measles virus is in human clinical trials for MM. The information obtained from this and other future clinical trials will guide clinical application of oncolytic viruses as anticancer agents for MM. This paper provides a timely overview of the history of oncolytic viruses for the treatment of MM and future strategies for the optimization of viral therapy for this disease.

1. Introduction imperative to find novel, more effective treatment options for MM. Multiple myeloma (MM) is a clonal neoplasm of plasma cells derived from the B-lymphocyte lineage that is part of 1.1. Currently Available Therapies for Multiple Myeloma. a spectrum of diseases ranging from monoclonal gammopa- Disease management of MM has improved with the intro- thy of undetermined significance (MGUS) to plasma cell duction of several new agents such as bortezomib (Velcade, leukemia. It is the most common primary bone cancer and a proteasome inhibitor), thalidomide, and the thalidomide involves malignant plasma cells progressively infiltrating the analogue lenalidomide (Revlimid, immune modulator), and bone marrow and producing a monoclonal immunoglobulin (Ig) (M-protein) [1]. Overt myeloma (advanced disease) thus these drugs have now become current mainstays in is manifested by pathophysiological consequences such as MM treatment. These agents as monotherapies (bortezomib) osteolytic bone lesions, hypercalcemia, recurrent bacterial or in combination (thalidomide or lenalidomide) with dex- infections, anemia, and renal failure [2]. Over 70,000 amethasone have yielded improved patient outcome, yet people in North America are currently affected by MM long-term tolerance and toxicities associated with these with an annual incidence of greater than 15,000. Presently, drugs are limitations [6]. MM accounts for 10% of hematological malignancies and Stem cell rescue following high-dose chemotherapy with represent 1-2% of all cancer-related deaths [3]. The disease autologous (ASCT) transplantation has historically become remains incurable with current treatments with a median standard therapy for the subset of good-performance survival of 3–5 years [4, 5]. MM follows a relapsing course younger patients with MM. Yet minimal residual disease in the majority of patients, regardless of treatment regimen and/or contaminating tumour cells within the autograft or initial response to treatment. Accordingly, it has become leading to relapse is a concern with ASCT. 2 Bone Marrow Research

1.2. A Historical Perspective of Oncolytic Viruses. The concept in his IgA monoclonal protein levels [12]. The first formal of virotherapy in the treatment of cancer dates back to oncolytic viral MM clinical trial was conducted at the the early 20th century and more recently with a report of Mayo clinic with an attenuated oncolytic measles virus virally mediated tumour regression involving a patient with encoding thyroid sodium iodine symporter in order to cervical carcinoma that received an attenuated rabies vaccine improve sodium iodine uptake [13]. In addition, other [7]. Spontaneous remissions of heamatological malignancies naturally occurring viruses such as reovirus that is currently such as Burkitt’s lymphoma and Hodgkin’s disease have also undergoing phase III clinical trial testing for solid tumour been observed subsequent to clinical infections with measles histologies are anticipated to undergo a phase I clinical trial virus [8, 9]. Reports as early as the 1920s indicate that viral for MM in the near future. replication was responsible for consequent lysis of tumour in murine models [10]. Although anecdotal, these early 2.1. Measles Virus (MV). Measles virus is the most compre- observations provided the foundation to treat cancer patients hensively studied oncolytic virus for MM and is the first to with oncolytic viral therapy in the late 1940’s; however, undergo phase I clinical trial investigation for this disease. It results were disappointing likely owing in part to the rapid is a negative-strand RNA virus of the genus Morbillivirus that viral clearance by the resultant induced immune response causes the infectious measles syndrome. Its genome consists [11]. of 6 genes that encode 8 proteins: the nucleocapsid (N), Despite these setbacks over the last 15 years, there has phospho (P), matrix (M), fusion (F), hemagglutinin (H), been a revival of interest in developing oncolytic viruses and large (L) proteins in addition to C and V accessory as potential cancer therapeutics. An increasing number of proteins. The virus enters cells through the interaction of H- viruses have been shown to have oncolytic activity against glycoprotein with the CD46 receptor that is overexpressed both solid and haematological tumours in vitro and in vivo. in cancer cells including MM [14, 15] and the signaling The potential obstacles/limitations to a successful viable lymphocyte-activation molecules (SLAM) that are found in oncolytic therapeutic platform include: large-scale GMP B- and T-lymphocytes [16]. After receptor recognition by viral production, toxicity, immunogenicity and optimization the H-protein, the conformational changes that take place in of the schedule/route of administration. the F-protein lead to viral entry and cell fusion. Cytopathic Naturally occurring or engineered oncolytic viruses have effects of MV are mediated by massive cell fusion due to the theoretical therapeutic advantage over other cancer virus receptor recognition and the formation of syncytia therapies in that they can specifically infect, propagate, and (large mononuclear cell aggregates) [17]. The MV-Edm lyse cancer cells (including neighboring cancer cells) while (Edmonston vaccine strain) is a replicating measles strain sparing normal cells. Viral specificity depends on two major that was isolated from an 11-year-old pediatric patient and mechanisms to produce a productive viral infection and hitherto named after him. This virus has been attenuated subsequent cell lysis/death: (i) receptor-mediated uptake, after serial tissue culture passage and has been administered where cancer cells overexpress virus entry specific receptors, as a vaccine for over 50 years. Reversal of this strain to a and (ii) Utilization of aberrant cellular oncogenic signaling virulent form has never been reported, and thus it has been pathways for virus replication. With the use of recombi- exploited as cancer therapeutics based on its longstanding nant DNA technology, and our increasing understanding safety profile. of microRNA functions, the capacity for creating novel “designer viruses” appears to be limitless and therefore very 2.1.1. Preclinical Studies with MV. The success of MV-Edm promising. Viruses genetically engineered to express suicidal has been in part linked to the mutations of the H- genes, immune stimulatory products within the tumor glycoprotein that leads to improved interactions with the and/or tumor-specific inflammatory responses, and limit CD46 receptor [18], and it is of interest that over expression replicative potential in normal cells are attractive candidates of CD46 in cancer cells has been correlated with enhanced to be used as cancer therapeutics. cell death [19]. The earliest in vitro and in vivo work with MV-Edm 2. Viruses Used as Therapeutic and MM was carried out by Peng et al. in 2001 [20]. This Strategies for MM study demonstrated effective lysis of MM cell lines in vitro as well as patient MM tumor utilizing a GFP-tagged MV-Edm, Of the many viruses that are currently considered possi- with no adverse effects on normal blood lymphocytes. MM ble cancer therapeutics, four RNA viruses (measles virus, tumours implanted in a SCID/NOD murine xenograft model vesicular stomatitis virus, reovirus, coxsackievirus A21) and also showed complete tumour regression following intratu- two DNA viruses (adenovirus and vaccinia virus) have moural MV treatment and significant tumour response with been investigated as potential therapeutics for MM. These intravenous virus treatment. have been investigated preclinically as monotherapy, as A major drawback of standard viral vectors is the poor combination therapy in conjunction with chemotherapy delivery efficiency, especially in a clinical setting. Several and/or radiation therapy and as purging agents during ASCT. strategies have been adopted with MV for better delivery and Targeting multiple myeloma with virotherapy clinically to produce enhanced bystander effects that will facilitate the was first attempted in the late 1980s, involving intravenous killing of tumour cells that are shielded from virus. The MV- treatment of a Japanese male MM patient with an attenuated Edm derivatives utilizing human carcinoembryonic anti- AS vaccinia strain that demonstrated significant reductions gen (CEA; MV-CEA) or human sodium iodide symporter Bone Marrow Research 3

(NIS; MV-NIS) have successfully been utilized to produce humans [28]. Although, in newborn and severely immuno- significant local bystander effects through cell-cell fusion and compromised (SCID) mice, reovirus type 3 may cause syncytia formation [21–25]. encephalitis, myocarditis and death [29–32], immunocom- In addition to enhancing tumour targeting strategies, petent animals including humans have never exhibited any these engineered derivatives provide a means of tracking the of these toxicities [33]. Since reovirus is a common environ- in vivo spread of the virus and virus gene expression/kinetics mental virus, the vast majority of humans have neutralizing over time. These parameters provide insight for tailoring antibodies to this virus by the age of 8 [34]. Reovirus is viral delivery doses and frequencies of repeated cycles internalized into cells via the ubiquitous sialic acid receptor which could lead to a personalized approach of treatment. [35] and/or the junction adhesion molecule (JAM) [36]. It While both MV-NIS and MV-CEA allow expedient real- uses a strategy of cell infection and lysis through exploitation time monitoring of viral gene expression, MV-NIS has an of an already activated Ras/oncogenic signalling pathway in additional advantage in that it carries a membrane ion tumor cells [35]. Thus, reovirus specifically targets tumor channel (NIS) that can concentrate radioisotopes within cells cells for its replication and spares normal cells. thereby causing localization and the spread of the virus. This unique advantage of isotope trapping by NIS facilitates the 2.2.1. Preclinical Studies with Reovirus. The underlying non-invasive detection of the spread of the virus by γ camera, mechanism(s) behind the preferential cytotoxicity of re- PET, or SPECT/CT by using radioisotope tracers such as 123I, ovirus towards transformed cells has only recently been 124I, and 99MTc [26]. described and appears to be at the level of intracellular The oncolytic potential of MV-NIS against MM was signalling and not at cell surface attachment [35]. When investigated by Dingli et al. 2004 [22]. In vitro MM cell lines reovirus resistant murine NIH3T3 cells are transformed with and ex vivo MM tumour specimens showed marked sensi- oncogenes such as v-erbB2, sos and ras, reovirus susceptibility tivity, and the therapeutic efficacy of MV-NIS against MM is conferred [35, 37]. Reovirus likely exploits an activated xenografts was noteworthy as previously MV-NIS resistant Ras/oncogenic signalling pathway, taking advantage of the MM1 tumours showed complete regression of tumour with inhibition of the double-stranded RNA activated protein combination therapy with 123I. kinase (PKR) found in these cells [35]. Recent data has implicated the Ras/RalGEF/p38 pathway in an NIH 3T3 2.1.2. Clinical Studies with MV. The preclinical efficacy and model system of reovirus oncolysis [38]. the safety data generated for MV-NIS have translated these The oncolytic ability of reovirus against several neo- findings to a phase I clinical trial for recurrent or refractory plasms including breast, prostate, colorectal, brain, ovarian, MM that is ongoing at present [13]. This trial includes and hematological malignancies such as non-Hodgkin’s the intravenous administration of MV-NIS with or without lymphoma, chronic lymphocytic leukemia (CLL), and MM cyclophosphamide. In addition to being a chemotherapeutic has been shown by our group under in vitro, in vivo, and/or drug, cyclophosphamide is an immune suppressor, and thus ex vivo conditions [39–44]. prolonged viral dissemination and replication within these As in other tumor types, activating mutations in the ras immune-suppressed patients are expected as previously seen gene family are frequent in MM [45, 46]. The incidence of in animal models [27]. A 2-step protocol for the phase I activating M-ras and K-ras mutations appears to be common clinical trial was adapted where the maximum tolerated dose in MM and varies between 10 and 40% at presentation, but is to be evaluated in the first step where patients will be given may be as high as 70% at the time of relapse, indicating not 6 9 intravenous injections of MV-NIS ranging from 10 to 10 only a possible role in tumor progression but also potential TCID-50.ThesecondstepwillcommenceoncetheMTDis for reovirus sensitivity. Our laboratory exploited these reached where patients in groups of 3 will be pretreated with features of MM and investigated the potential of reovirus cyclophosphamide two days prior to MV-NIS injection. Pre as biological therapeutics against MM. Our initial studies and posttherapy hematological and biochemical parameters showed RPMI 8226 MM cell line and an ex vivo patient as well as antimeasles immunity will be determined in tumour to be sensitive to reovirus [44]. Expanding on these the patients. In addition to measurements of MV-NIS findings, we have tested 8 MM cell lines and 7 ex vivo patient levels in blood, urine, and gargle samples, patients would tumour samples in vitro and found 7 of 8 human MM cell 123 undergo serial imaging of virus biodistribution post- I lines and 5 of 7 ex vivo tumor specimens exposed to reovirus administration [13]. CD46 expression of patient myeloma to be exquisitely sensitive [47, manuscript submitted]. In cells isolated from bone marrow will be correlated with virus addition, we have shown that the potent antitumour efficacy infectivity in order to validate previous laboratory in vitro of reovirus is predominantly manifested through apoptotic studies. Interestingly, unpublished observations as reported cell death [48]. These results indicate the potential use of this 123 by Msaouel et al. [26] indicate the uptake of I in a localized virus as attractive therapeutic for MM. MM tumour of one patient as revealed by a SPECT/CT8 Most MM patients have symptomatic disease at diag- scanning 8 days after treatment. These encouraging results nosis, and autologous hematopoietic progenitor stem cell would hopefully lead to advance phase II/III clinical trials in transplantation (ASCT) is applicable for more than 50% of the future. patients with MM [49]. Stem cell rescue after high-dose ablative therapy has proven to be an effective and useful 2.2. Reovirus. Reovirus is a ubiquitous, nonenveloped treatment modality for a variety of hematologic malignancies double-stranded RNA virus with minimal pathogenicity in including MM as well as a few solid tumours [50–56]. 4 Bone Marrow Research

Because of its low treatment-related mortality rate (<3%) of cellular protein synthesis, abrogation of transport of and the absence of the need for a suitable donor (allo- cellular glycoproteins, proteolytic digestion of transcription transplantation), autologous transplantation has gained factors, and promotion of apoptosis [72]. widespread application, and globally the number of autol- ogous blood and marrow transplants now surpasses the 2.3.1. Preclinical Studies with CVA21. Work conducted by Au number of allotransplants [55, 57, 58]. et al. [69] showed that RPMI 8226, U266, and NCI-H929 Although ASCT following high-dose myeloablative cell lines were exquisitely sensitive to CVA21 producing 100– chemotherapy is considered standard therapy for many mul- 1000 fold increases in viral progeny at 24h after infection tiple myeloma (MM) patients, relapse post-ASCT still compared to controls. Normal peripheral blood cells in presents a major challenge in disease management. Gene- contrast were resistant to virus infection. Similarly, patient marking studies indicate that occult clonogenic tumour cells bone marrow biopsies exposing to CVA21 lead to purging within the autograft may be a partial contributor to relapse of CD138+ plasma cells up to 98.7% with minimal effects [59]. on progenitor function [69]. Since CVA 21 can cause severe Since MM currently remains the second most common myositis in suckling [73] and immunocompromised mice indication following lymphoma for autotransplantation [70], Kelly et al. [70] utilized a microRNA- (miRNA-) [60], we explored the possibility of using reovirus as a purg- based approach to decrease virus pathogenicity. In this study ing agent for MM. In significant contrast to its sensitivity virus tropism was modulated with the expression of tissue- towards MM tumour, we have shown that reovirus does (muscle-) specific miRNA within the engineered CVA21 not harm hematopoietic stem cells or their colony-forming thereby destabilizing viral replication in a tissue-specific abilities in vitro or in vivo. Furthermore, RPMI 8226 cells manner. SCID mice bearing subcutaneous Kas 6/1 MM admixed with human apheresis product (AP) cells showed tumours injected with miRT-CVA21showed complete tumor complete purging when treated with reovirus. To examine regression and sustained viremia but could not replicate in the potential use of this strategy under clinical conditions, we cells containing complementary miRNAs and therefore did recently utilized a murine model system that recapitulated not cause myositis. This study shows that naturally occurring the human course of MM and demonstrated that reovirus and differentially expressing miRNAs can be exploited to purged autografts do not abrogate human hematopoietic modulate viral replication cycles, thus providing a new stem cell repopulation in vivo.Further,wehaveshown paradigm of virotherapeutics. that reovirus purging leads to complete eradication of disease, prevents relapse, and leads to significant survival improvements in comparison to controls [47,manuscript 2.3.2. Clinical Studies with CVA21. Todate,noclinical submitted]. investigations have been initiated with CVA21 and MM. However, a phase I clinical study is currently underway for patients bearing melanoma, breast and prostate tumor 2.2.2. Clinical Studies with Reovirus. Upwards of 16 Reovirus that express cellular receptor ICAM-1 with or without DAF (REOLYSIN) phase I/II clinical trials in several cancers have expression [74]. The preclinical data reported to date suggest shown moderate efficacy, especially in combination with that CVA21 has the potential to purge MM during ASCT or radiotherapy and histology relevant cytotoxic chemotherapy be used as a systemic virotherapy agent for MM and warrants [61–66], and phase III trials are presently ongoing for further investigation. non-small cell lung carcinoma and head and neck cancers [66]. Reovirus’s extensive preclinical efficacy, replication competency, and low toxicity profile in humans have placed 2.4. Vesicular Stomatitis Virus (VSV). Vesicular stomatitis it as attractive anticancer therapeutics for further clinical virus (VSV) is a small negative-strand enveloped RNA virus testing for hematological malignancies. We anticipate that that belongs to the Rhabdoviridae family. With a wide host the preclinical data generated for MM will lead to a clinical range, its ability to cause vesicular lesions in farm animals is trial of reovirus in MM and possibly a reovirus purging trial common [75]; however, the incidence of human infections is in the near future. rare [76] and usually benign [77]. Naturally occurring VSV is highly sensitive to interferon (IFN) and exploits inherent IFN dysregulated pathways in tumour cells for its replication 2.3. Coxsackievirus A21 (CVA21). CVA21 is a nonenveloped, and eventual tumour destruction [78]. positive-sense single-stranded virus that belongs to the Picornaviridae family. Although it is known to cause res- piratory tract infections and myositis in humans [67, 68], 2.4.1. Preclinical Studies with VSV. Lichty et al. [79]have its oncolytic potential has been proven in several cancer shown several leukemic cell lines and multiple myeloma cell lines including MM [69, 70]. Intracellular adhesion ex vivo patient specimens to be exquisitely sensitive to molecule-1 (ICAM-1) and decay-accelerating factor are 2 VSV variants AV1, AV2 and heat resistant (HR) VSV [79]. receptors that are necessary for CVA21 infection and cell In addition, this group also demonstrated that leukemic lysis, and both these are reported to be upregulated in cell lines could be purged successfully with these VSV cancer cells including MM in comparison to normal cells variants with minimal effect on colony-forming ability of [69, 71]. CVA21-mediated cell death is manifested through hematopoietic stem cells suggesting potential use of these interruption of various cellular processes such as disruption VSV mutants as purging agents [79]. To date, in vivo testing Bone Marrow Research 5 of the ex vivo purging efficacy of these VSV variants has not vaccinia strain AS administered to this patient resulted in been undertaken. marked reductions in his IgA levels from 1,309 mg/dl in the Due to its small size, VSV is amenable to genetic early stages of treatment to 432 mg/dl on the 96th day of the manipulation, and recently VSV 51 has been engineered regimen. No adverse effects were noted. to express the human sodium iodide symporter (hNIS) for Since then, other clinical trials of VV mutants such combined imaging and radiotherapy of MM [80]. VSVΔ51- as JX-594 have been conducted in patients with refractory hNIS generated by Goel et al. [80] was oncolytic to MM cell primary or metastatic liver cancer with indications of efficacy lines, as well as primary patient tumours, and produced very [83]. JX-594 is a targeted, thymidine kinase(-) vaccinia high titers in MM cells under in vitro conditions. VSVΔ51- virus expressing human granulocyte-macrophage colony- hNIS administered to bg/nd/xid mice bearing subcutaneous stimulating factor (hGM-CSF) and is designed to selectively myeloma tumors demonstrated significant tumour regres- replicate in and destroy cancer cells with cell-cycle abnor- sion, and high intratumoral virus replication was noted [80]. malities and epidermal growth factor receptor- (EGFR-) Ras Utilizing a syngeneic 5TGM1 murine model of MM, aberrant signaling pathways. GM-CSF expression by JX-594 Goel et al. [80] further demonstrated the in vivo oncolytic stimulates shutdown of tumour vasculature and antitumoral ability of VSVΔ51-hNISwheresubcutaneousororthotopic immunity in addition to direct oncolysis. The encouraging tumours treated with VSVΔ51hNIS in combination of 131I clinical results seen with JX-594 will hopefully lead to future showed marked reduction of tumour and improved rates of clinical trials in hematological malignancies including MM. survival of mice. 2.6. Adenovirus. Adenoviruses (Ad) are non-enveloped 2.4.2. Clinical Studies with VSV. Since MM is a radiosensitive double-stranded DNA viruses, and wild-type Ad may cause tumour, in vivo work conducted with VSV 51hNIS suggests mild clinical infections of the upper respiratory tract; the potential usage of this virus in combination therapy for however, may cause significant morbidity and mortality clinical use in the future for MM. in immune-compromised patients. The majority of studies involving adenovirus as an oncolytic agent have utilized 2.5. Vaccinia Virus (VV). Vaccinia virus belongs to the family attenuated nonreplicative adenoviral vectors that have been poxviridae and is a close relative of the smallpox virus [81]. engineered either to deliver a prodrug activating enzyme It is a double stranded DNA virus with a large genome that such as thymidine kinase (TK)orexpresswild-typep53 is (of 190 KB) amenable to genetic manipulation. Due to selectively in tumour cells (reviewed in [86]). The tumour its strong immunogenic nature (that results in high T-cell cell selectivity of Ad is explained by the presence of the responses and circulating antibodies), VV has been utilized coxsackievirus and Ad receptor (CAR receptor) on tumour as a vaccine that is instrumental in eradicating smallpox cells in association with the expression of αvβ5 or αvβ3 [82]. Various VV strains have further been exploited in required for internalization of these virions into the immunotherapy of cancer and infectious diseases and as tumour cell [87]. Modification of the viral attachment fibre cancer therapeutics itself [82, 83]. knob [88] is an additional strategy that has been utilized for selective entry of Ad into tumour cells. 2.5.1. Preclinical Studies with Vaccinia Virus (VV). The thymidine kinase (TK) gene of VV serves as a site of DNA 2.6.1. Preclinical Studies with Adenovirus and MM. Teoh et al. insertion, and the first logical oncolytic VV was developed [89] showed that Ad vectors carrying the thymidine kinase by McCart et al. [84] which is highly attenuated. In this gene (TK) under the DF3 promoter could transduce OCI- double mutant VV, the TK and vaccinia growth factor (VGF) My5 and RPMI8226 MM cell lines efficiently. Treatment genes have been deleted and the gene for enhanced green of these cells in the presence of normal hematopoietic fluorescent protein (EGFP) has been inserted at the TK locus progenitor (HPC) cells could lead to a >6 log purging of resulting in a double-deleted GFP construct (vvDD-GFP). tumour cells leaving the HPC cells unharmed. Similarly, This attenuated virus’s tumour selectivity, safety profile, and Ad-mediated delivery of p53 to MM cell lines or patient ff oncolytic e ects have been evaluated in MM recently [85]. tumour resulted in substantial apoptosis if the cells were MM cell lines and ex vivo patient tumour exposed to vvDD- p53 mutant with low expression of bcl-2 [90]. In contrast, GFP showed sensitivity, whereas minimal viral infectivity HPC cells or normal lymphocytes were not permissive to was seen in normal peripheral blood mononuclear cells [85]. these Ad vectors [90]. Replicating Ad1337 with E1A and Systemic VV treatment of mice bearing My5 subcutaneous E1B deletions has also been shown to cause cytotoxicity to xenografts or RPMI 82226 disseminated tumour showed MM cell lines but not normal B cells [91]. More recently, significant reduction in tumour and improved survival over Fernandez et al. [92] examined growth inhibition of MM the controls suggesting the potential use of this virus as a cells potentiated by a conditionally replicating adenovirus clinical agent in the future. carrying a CD40 ligand transgene (AdEHCD40L). Their work demonstrated that MM cell lines were susceptible to 2.5.2. Clinical Studies with Vaccinia Virus. The first clinical AdEHCD40L-mediated apoptosis. RPMI 8226 xenografts in trial involving MM and VV virotherapy was a case study a SCID murine model were reduced by 50% by AdEHCD40L conducted with a 67-year-old Japanese male patient with treatment, whereas treatment with the vector alone showed IgA MM in the late 1980s [12]. Intravenous injections of only a 28% reduction in tumour. Since Ad5 serotype has been 6 Bone Marrow Research

Table 1: Genetic composition, advantages, and disadvantages of oncolytic viruses presently evaluated for the treatment of MM.

Measles virus VSV Reovirus CVA21 Adenovirus Vaccinia virus Genetic composition ssRNA ssRNA dsRNA ssRNA dsDNA dsDNA Ability to genetically Easy Moderate Very difficult Moderate Easy Easy manipulate Titres achievable at >109 PFU/ml >109 PFU/ml >109 PFU/ml >1010 PFU/ml >1012 PFU/ml >109 PFU/ml clinical grade Ease of production Easy Difficult Easy ? Easy Easy Preclinical References References References References References References in vitro [20, 22][79, 80][44, 47][69, 70][89–93][85] ex vivo [20, 22][80][44, 47][69][92, 93][85] purging [79][44, 47][69][89] Preclinical in vivo [20, 22, 23][80][47][70][92, 93][85] Clinical studies Under Multiple myeloma Phase I (13) N/A N/A N/A Case study (12) discussion Recurrent glioblastoma Phase I- for Phase I/II for Phase I for Phase III for multiforme, melanoma, several solid primary or Other histologies N/A solid tumours recurrent breast, prostate tumours and metastatic liver (66) ovarian cancer (74) melanoma (106) cancer (26) Strategies for delivery of Intravenous Intravenous Intravenous Intravenous Intravenous Intravenous virus for MM VSV: vesicular stomatitis virus; CVA2: coxsackie virus A21; N/A: not available. approved for human use in solid tumours, Senac et al. [93] of virus from the blood vessels to the targeted tumour site. investigated the potential use of Ad5 for MM. Their work Various strategies have been developed by researchers to demonstrated that Ad5 could infect and kill the majority of overcome these barriers, and these are discussed in detail myeloma cell lines and ex vivo patient tumour as evidenced below. by reporter gene expression, viral DNA expression, viral titre, and cell death assays. When MM patient specimens were exposed to different adenovirus species, many showed the 3.1. Immune Modulation. A major drawback of virotherapy capability to kill MM tumour of patient origin and thus under clinical settings is that the majority of patients either suggested significant therapeutic potential. have baseline or generate neutralizing antibodies against the therapeutic virus of interest. Overcoming this immune Although the therapeutic advantages of adenoviruses ffi as an oncolytic agent for MM under clinical conditions responses would greatly enhance the therapeutic e cacy are yet to be investigated, the efficacy and safety profile of virotherapy. Although malignancy-reported immunosup- of many adenoviral vectors during phase I/II clinical trials pressioniscommoninMMpatients[94], it is imperative to demonstrated for solid tumours is of some concern, and develop strategies to overcome host immune viral responses whether or not this virus could be exploited for MM therapy if virotherapy is to be optimized. To this end, cyclophos- in the future is unknown. Table 1 depicts a comparison of phamide has been shown to be a suitable immunosuppresant oncolytic viruses that have been investigated for MM. in animal models as well as in early clinical trials with MV, herpesvirusaswellasreovirus[27, 95–99]. Preclinical data of MV-NIS in squirrel monkeys has shown prolonged viral 3. Optimization of MM Treatment with gene expression with cyclophosphamide administration to Oncolytic Virotherapy animals [27]. Similar administration of cyclophosphamide has shown to dampen the innate immune responses and In the clinical setting, oncolytic viral therapy needs to be increase the therapeutic efficacy of herpes virus with pro- ideally administered intravenously (IV) to MM patients due longed viral gene expression and enhance proliferation in to its hematological route of disease progression. The main tumor [95]. Clinical studies with reovirus in combination obstacles that prevent successful delivery of IV-injected virus with cyclophosphamide are presently being conducted in UK to tumour sites are recognition and irreversible clearance of for advanced malignancies of pancreatic, lung, and ovarian viral antigens by the immune system, nonspecific clearance cancer http://www.oncolyticsbiotech.com/clinical.html.The of virus by the liver and spleen, and minimal extravasation primary objective of this study is to determine the minimum Bone Marrow Research 7 effective immunomodulatory dose (MED) of cyclophos- of patients. Although the optimal strategy utilizing oncolytic phamide to obtain successful immune modulation. The viruses for the treatment of MM is unknown, it is anticipated secondary objective is the safety profile and obtaining any that the use of those viruses will not be used as monotherapy. evidence of antitumour activity. The combination of oncolytic viruses plus relevant cytotoxic or targeted drug may well prove to be synergistic. Further, one might envision that oncolytic virus treatment could be 3.2. Cell Carrier Based Methods. Cell-based viral carriers are used post- allo/-auto transplant once hematopoietic recovery another approach that has been developed to circumvent has been achieved. immune clearance of therapeutic viruses. Mesenchymal It appears from the advances made to date that the future progenitor cells, monocytes, and T cells have demonstrated of oncolytic virotherapy lies in combination therapies (versus the ability to shield virus from immune invasion and traffic monotherapy) for this malignancy, and a rationale for testing them to tumour sites leading to enhanced oncolysis [100– novel drugs for MM in combination with oncolytic viruses 102]. is justified. A major obstacle that hinders virotherapy is the Ong et al. [100] evaluated T cells as carriers for systemic host immune system that has evolved over the millennia in measles virotherapy for MM in the presence of antiviral to overcome infection. The many strategies that researchers antibodies. This study demonstrated that virus-infected T have undertaken to circumvent this problem such as immune cells expressing measles H/F fusogenic envelope glycopro- modulation, shielding the virus in cell carrier, and so forth. teins could efficiently transfer MV infection by heterofusion have provided insight into overcoming these barriers. Many with MM cells in vitro. These T cells were more efficient of the viruses mentioned in the aforementioned discussion in delivering virus to MM tumour sites than naked viruses are presently under phase II/III testing for solid tumors, and in mice that were passively immunized with low levels of phase I clinical trials are ongoing with measles virus for MM. antimeasles antibodies illustrating a similar strategy that may It is expected that the outcomes of these studies will facilitate be feasible with MM patients harbouring low immunity. to further testing of these viruses for MM in a clinical setting Another strategy that has been experimented is that the and be approved as cancer therapeutics in the near future. virus of choice could also multiply within the carrier cell, disperse and infect adjacent tumour cells. 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Review Article Multiple Myeloma: A Review of Imaging Features and Radiological Techniques

C. F. Healy,1 J. G. Murray,1 S. J. Eustace,1 J. Madewell,2 P. J. O’G or ma n , 3 and P. O’Sullivan1

1 Department of Radiology, Mater Misericordiae University Hospital, Dublin 7, Ireland 2 Department of Radiology, University of Texas, MD Anderson Cancer Centre, Houston, TX 77030, USA 3 Department of Haematology, Mater Misericordiae University Hospital, Dublin 7, Ireland

CorrespondenceshouldbeaddressedtoC.F.Healy,[email protected]

Received 13 December 2010; Revised 6 April 2011; Accepted 30 May 2011

Academic Editor: Cesar´ O. Freytes

Copyright © 2011 C. F. Healy et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The recently updated Durie/Salmon PLUS staging system published in 2006 highlights the many advances that have been made in the imaging of multiple myeloma, a common malignancy of plasma cells. In this article, we shall focus primarily on the more sensitive and specific whole-body imaging techniques, including whole-body computed tomography, whole-body magnetic resonance imaging, and positron emission computed tomography. We shall also discuss new and emerging imaging techniques and future developments in the radiological assessment of multiple myeloma.

1. Introduction The role of radiological imaging in multiple myeloma is essentially three fold: in the initial staging of the disease, in Multiple myeloma is a neoplastic disorder of plasma B the detection and characterisation of complications, and in cells characterised by bone marrow infiltration and overpro- the evaluation of patient’s response to treatment. duction of monoclonal immunoglobulins. It accounts for approximately 10% of all haematological malignancies and 1% of all cancers with an increasing incidence, affecting 2. The Biology of Myeloma Bone Disease ff four in every 100,000 per year [1]. It predominantly a ects In the past decade, the role of the bone marrow microen- patients in the seventh decade and has high morbidity and vironment has been at the forefront of multiple myeloma mortality. Patient survival has improved over the past decade research. The “seed and soil” hypothesis was first introduced with the introduction of novel chemotherapeutic agents [2, in the late 1800s by an English surgeon, Dr. Stephen Paget, 3]. who proposed a neoplastic growth (the seed, e.g., the Standard investigations for multiple myeloma includes myeloma cell) will proliferate in an environment (the soil, a complete blood count, serum biochemistry, serum and e.g., bone marrow environment) that supports its’ replica- urine electrophoresis, and the gold standard for diagnosis: tion [6]. Since then, in particular in the early 21st century, a bone marrow aspirate and biopsy. The Durie/Salmon staging multitude of evidence has emerged demonstrating the role system introduced in 1975 used skeletal survey as its only of the myeloma cells local environment in augmenting its radiological criterion [4]. In an effort to standardize treat- survival. ment approaches and stage the disease as accurately as possi- The bony destructive lesions demonstrated by myeloma ble at the time of diagnosis, the Durie/Salmon PLUS staging imaging techniques are caused by myeloma cell-mediated system has been introduced, integrating the more sensitive promotion of osteoclast-mediated bony destruction and imaging techniques of magnetic resonance imaging (MRI), inhibition of osteoblast-mediated bone anabolism. Myeloma computed tomography (CT), and PET/CT information into cells attach to osteoclasts directly by numerous adhe- its classification system [5]. sion molecules, one example being vascular cell adhesion 2 Bone Marrow Research

R

Figure 1: Lateral radiograph skull: diffuse lytic lesions giving classical “pepper pot skull” appearance. molecule-1 (VCAM-1), with resultant stimulation of osteo- clastogenesis [7]. The effects of myeloma cells on attenuation of osteoblas- Figure 2: A-P radiograph right humerus: diffuse lytic lesions of tic activity can be explained, for the most part, by inhibition the right humerus (arrowed) with old pathological fracture distal of osteoblastic differentiation into mature osteoblasts. The diaphysis (arrow). main pathway involved in inhibition of osteoblastogenesis is by direct cell-to-cell contact between the mesenchymal stem cells (MSCs) and the myeloma cells. Adhesion of these two entities via VCAM-1 and very late antigen-4 (VLA-4) results in a reduction in Runt-related transcription factor 2 (RUNX2) expression, a critical factor involved in osteoblast transcription [8]. Secondly, myeloma cells secrete factors that inhibit differentiation of osteoblasts, such as Dickkopf 1 (DKK-1), tumour necrosis factor alpha (TNF-α), soluble frizzled-related protein-2 (SFRP-2), and Activin A. DKK-1 and SFRP-2 act by inhibiting the Wnt pathway, a pathway that plays a significant role in osteoblastic maturation [9].

3. Imaging Techniques 3.1. Plain Radiography. Afullskeletalsurveyincludesa frontal and lateral view of the skull, the cervical, thoracic and Figure 3: Axial CT pelvis: diffuse myeloma involving the sacrum lumbar spine, a coned-down frontal view of the dens axis, as and iliac bones bilaterally, with cortical destruction of the left iliac well as frontal views of the rib cage, humeri, femora, knees, bone (arrow). and pelvis. There is a clear association between the extent of disease, in terms of number of lytic lesions at presentation, ff and tumour load at diagnosis [4]. Almost 80% of patients 15]. Furthermore, di use osteopenia as a result of multiple with multiple myeloma will have radiological evidence of myeloma cannot be distinguished on plain radiographs from skeletal involvement on the skeletal survey most commonly more common causes of osteopenia, such as senile and effecting the following sites: vertebrae in 66%, ribs in 45%, postmenopausal osteoporosis [13]. A practical drawback skull in 40%, shoulder in 40%, pelvis in 30%, and long of plain radiography is that varied positions required for bones in 25% (Figures 1 and 2)[10]. Plain radiography has radiography films are painful for patients who are often the advantage over MRI in detecting cortical bone lesions. elderly and disabled due to previous pathological fractures. It also has the advantage of being universally available, and relatively inexpensive. 3.2. Computed Tomography (CT). CT is a sensitive imaging One of the major disadvantages of plain radiography is modality in detecting the osteolytic effects of multiple its high false-negative rate of 30–70%, leading to significant myeloma and has a higher sensitivity than plain radiography underestimation in diagnosis and staging of patients with at detecting small lytic lesions [16]. CT findings in multiple multiple myeloma [11–13]. Diffuse bone marrow involve- myeloma consist of punched-out lytic lesions, expansile ment, which may or may not be associated with cortical bone lesions with soft tissue masses, diffuse osteopenia, fractures, destruction, is not evaluable using conventional radiography. and, rarely, osteosclerosis (Figures 3 and 4)[16]. Mahnken et Lytic lesions become apparent on conventional radiography al. compared multidetector row CT with conventional radio- when 30–50% of the bone mineral density is already lost [14, graphy and MR imaging in patients with newly diagnosed Bone Marrow Research 3

S S

(a)

(b)

Figure 5: Low dose sagittal whole-body CT (a): note lytic lesion I posterior aspect of T10 vertebral body (arrow). Background of extensive osseous permeation from myeloma. Axial CT thorax in Figure 4: Volume rendering 3-dimensional reconstruction of the same patient at the level of T10 (b) identifying lytic infiltration lumbar spine and pelvis: multiple “punched-out” lytic lesions of vertebral body (arrow). throughout lumbar spine and pelvis (arrow).

MRI and PET/CT, it cannot assess continued activity of multiple myeloma [16]. Multi-detector CT was superior to myeloma in areas of prior bone destruction [5]. conventional radiography at defining lytic lesions and, in combination with MR imaging, aided in staging the extent of 3.3. Whole-Body MRI. Whole-body MR (WBMR) has the disease. CT allowed a more accurate evaluation of areas emerged as the most sensitive imaging modality to date at at risk of fracture than did MR imaging. CT is of use in iden- detecting diffuse and focal multiple myeloma in the spine, tifying bone destruction in cases where MR is negative, and as well as the extra-axial skeleton [19–21]. Due to its ability hence may provide complementary imaging information. CT to visualise large volumes of bone marrow without inducing has the advantage of accurately demonstrating the presence radiation exposure and in an acceptable amount of time, MR and extent of extraosseous lesions and is the tool of choice imaging has become a favoured imaging method for evaluat- utilised in image guided spinal or pelvic bone biopsy of MR ing disease within the bone marrow (Figure 6). MR also has imaging defined focal lesions [17]. prognostic significance; the number and pattern of lesions Traditionally, whole-body CT has not been used for detected on MRI correlates very well with treatment outcome screening purposes due to the high level of radiation and overall survival [5, 20]. The excellent correlation with exposure. Low-dose CT techniques are being developed as a survival outcomes is the primary reason for inclusion of MRI possible alternative to plain radiography (Figure 5)[18]. Due into the Durie/Salmon PLUS system [5]. It is important to to the high intrinsic contrast of bone, the tube current can note that MRI predominately reflects marrow infiltration, be lowered significantly (i.e., to 50–100 mA s, depending on which may or may not be associated with bone destruction. the weight of the patient), resulting in an effective equivalent ThetypeofMRsequenceappliedgreatlyaffects the dose in the same range as that of a skeletal survey (4–5 mS v). MR images. Multiple sequences have been proposed for CT also has the practical advantage of being quick, with use in identifying focal or diffuse disease of the bone mar- the patient lying comfortable on his or her back. Iodine- row. These include spin-echo (T1-weighted and T2-weight- containing contrast agents, which are contraindicated in ed), gradient-echo (T2∗-weighted), STIR (short time inver- patients with multiple myeloma due to the risk of cast sion recovery), and contrast-enhanced spin-echo (with and nephropathy and renal impairment, are not required for without fat suppression) sequences [22]. The term “dynamic skeletal CT making it an even more attractive screening contrast-enhanced MRI” denotes repeat scanning with high option. A drawback of CT is that it typically shows persistent temporal resolution before, during, and after intravenous bone lesions throughout the course of the disease and, unlike infusion of a gadolinium-containing contrast agent, using 4 Bone Marrow Research

Figure 6: Whole-body MRI coronal and selective axial STIR sequence. Left image: coronal STIR sequence demonstrating T2 bright myelomatous disease throughout the thoracic spine (rectangle). Centre image: coronal T1-weighted sequence demonstrating low signal marrow throughout the lumbar spine due the myelomatous infiltration (rectangle). Right image: 3 axial MRI images at the level of the vocal cords, lumbar spine, and ischium, T1-weighted sequence following administration of contrast.

depends on many factors, such as cell density or the presence of organised structures (e.g., fibres). Studies have shown that diffusion is impaired within tumours, and that a decrease in diffusion may indicate disease progression. Effective treatment may cause a transient decrease in diffusion, owing to toxic cell swelling, but thereafter, as the cellular load is reduced, diffusion increases significantly. Typical myeloma lesions are marrow based and have low signal intensity on T1-weighted images (Figure 7(a)), and a high signal intensity on T2-weighted sequences and STIR images (Figure 7(b)) and generally show enhancement on gadolinium-enhanced images. Four patterns of mar- row involvement have been identified. A normal marrow appearance is present at diagnosis in 50–70% of untreated Durie/Salmon stage 1 and in 20% of untreated Durie/Salmon stage 3 [23]. Other marrow appearances of untreated disease ff (a) (b) include a focal pattern, a di use pattern and a variegated appearance. Thus the main drawback of MR imaging is the Figure 7: (a) MRI sagittal T1-weighted sequence lumbar spine: dif- lack of specificity. Focal or diffusepatternsmayexistat fuse permeative low signal myelomatous marrow lesions through- diagnosis and may be a variation of the norm, or reflect an out the lumbar spine (arrow). (b) MRI sagittal T2-weighted STIR alternative pathological or physiological process. ff sequence (same patient): di use high signal myelomatous marrow Whole-body STIR imaging has gained wider acceptance lesions throughout the lumbar spine (arrow). in detecting occult malignant disease in the skeleton [24, 25]. With ongoing technical advances, such as the moving table, use of multicoil elements, and advances in image processing fast T1-weighted sequences [23]. The change in signal inten- technology, this technique is becoming more feasible and sity over time in a given region is a function of local perfu- quicker to perform. Increasing the number of sequences will sion, relative blood volume, capillary surface exchange area, improve specificity of images, the downside being an increase vessel permeability, and systemic elimination. Diffusion- in acquisition time. These newer techniques enable the inclu- weighted imaging (DWI) is increasingly being studied on sion of such sites as the sternum, skull, and ribs, which are patients with multiple myeloma [23]. DWI analyses the usually excluded from standard MR imaging protocols in freedom of movement of interstitial water molecules, which multiple myeloma. Bone Marrow Research 5

Figure 9: Axial fused PET/CT thorax at the level of the pulmonary bifurcation: massive right-sided chest wall plasmacytoma (arrow).

Currently, PET/CT is being evaluated in patients with multi- (a) (b) plemyelomaandmaydetectearlybonemarrowinvolvement Figure 8: A-P radiograph right knee (a): 4 cm lucency medial in patients with apparently solitary plasmacytoma [28], to femoral condyle (arrow), radiographically difficult to visualise. MRI assess the extent of active disease, detect extramedullary coronal T2-weighted STIR sequence (b, same patient): high signal involvement or evaluate treatment response [29]. 4 cm plasmacytoma medial femoral condyle (arrow). The main limitation of PET imaging (without the CT component) is limited spatial resolution, which may result Dedicated focal MR imaging of the skull, spine, pelvis, in limitations in detecting subcentimetre lytic lesions seen on or of an extremity is also still a widely utilized technique. plain radiography. The advent of fusion scanning combining MR imaging is routinely required to evaluate the extent and both the PET component and CT component addresses this characteristics of a solitary lytic lesion identified at plain issue. In PET/CT fusion scanning, the patient receives an radiography (Figure 8).Thereisoftenawidedifferential for injection of FDG about 1 hour before image acquisition. An such a lesion, from benign lesions such as osteomyelitis and initial topogram is acquired to define the range of image fibrous dysplasia to malignant processes such as metastases acquisition. A spiral CT is then performed. The actual scan- and myeloma. ning time is shorter for PET/CT (approximately 30 minutes) than for a PET scan alone (approximately 1 hour) as CT data 3.4. Nuclear Medicine Imaging (Bone Scintigraphy, PET/CT, is used to perform attenuation correction (Figure 9). and MIBI Scanning) One of the most significant advantages of PET/CT imag- ing is its ability to distinguish between active myeloma (FDG 3.4.1. Bone Scintigraphy. Bone scintigraphy is of limited positive) and monoclonal gammopathy of undetermined sig- use in multiple myeloma. Detection of bone involvement nificance (MGUS) or smouldering disease. MGUS is usually using technetium 99-m (99mTc) labelled diphosphonates negative on PET/CT with neither diffuse marrow uptake relies on the osteoblastic response and activity of the skeletal nor focal disease in marrow sites [13]. Active myeloma is system for uptake. Multiple myeloma, however, is primarily FDG positive for focal and diffuse abnormities and FDG an osteolytic neoplasm. Lesions that are well defined on uptake decreases rapidly after effective therapy. Persistent isotope bone scans are the result of complications of multiple FDG positivity correlates with early relapse as is discussed myeloma, namely, osteoblastic response to a compression later. This rapid response to treatment is converse to that seen fracture of a vertebral body or pelvic insufficiency fracture. with MRI where there may be a time lag of 9–12 months in Bone scintigraphy may be helpful in evaluating areas not well the reversal of MRI abnormalities despite successful therapy demonstrated in plain radiography, such as ribs and sternum [20]. [26]. In a report comparing the skeletal survey with isotope How does PET/CT compare with other imaging modal- bone scans, uptake of the radioisotope in radiographically ities? In a series of 43 patients with multiple myeloma and abnormal regions occurred in 44% of cases, normal findings solitary plasmacytoma, Shirrmeister et al. [28]reportedfocal were seen in 48%, and diminished uptake was seen in 8% tracer uptake on PET/CT scans of 38 of 41 lesions (sensitivity, [27]. 93%) with known osteolytic pattern. PET/CT depicted 71 additional lesions missed on plain radiography in 14 patients, 3.4.2. PET/CT. Positron emission computed, tomography which resulted in a change in disease management in 14% (PET/CT) is a tomographic nuclear imaging technique of patients studied. Few studies exist directly comparing that uses a labelled radiopharmaceutical such as 18flouro- PET/CT with MRI. A recent study by Shortt et al. [30] deoxy-glucose (FDG) injected into the patient, followed by compared PET/CT with whole-body MRI in patients with tomographic scanning approximately 10–40 minutes later. bone biopsy proven multiple myeloma. Whole-body MRI Tumour cells can be imaged with this technique due to their performed better than PET/CT in assessing disease activity high metabolic rate and the resulting high glucose demand, having a higher sensitivity (68% versus 59%) and specificity allowing tumour cells to be distinguished from normal cells. (83% versus 75%) than PET/CT. When used in combination, 6 Bone Marrow Research

L 2 1 2

0.00 50% PET 3.3/

Figure 11: Axial fused PET/CT at the level of T2 vertebra: extensive FDG-avid biopsy-proven amyloid left deltoid muscle in a patient with multiple myeloma (arrow). Figure 10: Axial CT thorax postintravenous contrast at the level of T6: diffuse bilateral hilar lymphadenopathy from biopsy-proven multiple myeloma (arrows). producing masses in the surrounding soft tissues (Figures 9, 10,and11). CT is superior to MRI in depicting early cortical breaches [36]. PET/CT and whole-body MRI were found to have a positive predictive value of 100%. False-positive PET/CT scans may arise from inflamma- 5. Imaging of Response to Treatment tory changes due to active infection, chemotherapy within and Disease Progression the preceding 4 weeks, or radiation therapy within the The choice of imaging modality for assessing response preceding 2-3 months [31]. to treatment or disease progression generally depends on the findings from the initial workup and the patients’ 99 3.4.3. MIBI Scanning. Technitium sestamibi (MIBI) imag- specific treatment regime. Although new or enlarging lesions ing is an alternative nuclear imaging modality that uses generally signify disease progression, lytic bone lesions rarely Tc-99m-2-methoxy-isobutyl-isonitrile as a tumour-seeking show evidence of healing on plain radiographs, and routine tracer to identify areas of active disease in a variety of follow-up skeletal survey is of questionable benefit and not tumours including plasma cell dyscrasias. Using MIBI imag- routinely indicated in monitoring disease progression or ing, it is possible to scan both skeletal lesions and soft response to treatment. New vertebral compression fractures tissue lesions and the overall sensitivity is approximately on plain radiography do not always signify disease progres- 99 92% and specificity is 96% [32]. Tc-MIBI is superior sion and may occur even after effective treatment, due to the ff when compared with PET/CT for the visualisation of di use resolution of the tumour mass that was supporting the bony disease and despite its limited capacity in detecting focal cortex [37]. lesions, this technique may be an alternative option when CT in the followup of treated disease may demonstrate PET/CT is not available. In comparison to MRI, it has been the resolution of extramedullary disease, and the reappear- 99 shown that Tc-MIBI underestimates the extent of bone ance of a continuous cortical outline. Fatty marrow content marrow infiltration in the spine, especially in patients with may be seen in treated lytic disease in treated cases [38]. As 99 low disease stage [33]. Tc-MIBI may be useful in the mentioned earlier, one of the nuances of CT is that it cannot detection of indolent disease, as very low-level myeloma is assess continued activity of myeloma in areas of prior bone not detectable on PET/CT [5, 34]. An important nuance of destruction. 99 99 Tc-MIBI scanning is the enhanced uptake of Tc-MIBI by Although very accurate, MRI is not ideal for serial moni- drug-resistant myeloma cells versus enhanced uptake of FDG toring, as it takes 9–12 months for lesions evident on MRI to by metabolically active myeloma cells [34]. resolve and be clearly indicative of response [20]. There are a wide spectrum of treatment-induced changes seen on MRI 4. Imaging of Extramedullary following treatment. MR imaging may fail to demonstrate Multiple Myeloma evidence of regression of myeloma infiltration in the marrow. Focal lesions may shrink or remain unchanged in size. Extramedullary multiple myeloma is uncommon. In one A complete response to treatment may be evidenced by review of 432 patients with multiple myeloma, only 19 complete resolution of the preceding marrow abnormality, (4.4%) were identified as having extramedullary multiple and a partial response demonstrated by conversion of a myeloma [35]. Multiple myeloma often produces gross ster- diffuse to a variegated or focal pattern [22]. A good response nal expansion and distortion and vertebral body destruction. to treatment may also be evidenced by a reduction in signal The pathological tissue appears as soft tissue attenuation intensity on T2-weighted spin echo images and the absence similar to muscle at CT, with a degree of enhancement after of contrast-induced rim enhancement that was previously contrast. MRI typically reveals masses of uniform low signal present [19]. on T1-weighted image, and uniform high signal on T2- Diffuse or focal marrow changes may occur following weighted image. Later in the disease cortical breaches may treatment with synthetic growth factors including GCSF and occur, and further local spread from bone may be seen, erythropoietin that may simulate active disease on MR or Bone Marrow Research 7

PET/CT imaging [2]. Artificial alterations of FDG activity are potential tool for therapy monitoring and for evaluating the typically limited to a 1-month interval after discontinuation effectiveness of modern tumour treatment. of treatment [39]. New PET radiopharmaceutical tracers such as 3-flouro- PET/CT imaging has been shown to be useful in evalu- 3-deoxy-L-thymidine (18F-FLT) which is taken up into cells ating response to therapy, particularly when other imaging in relation to the rate of DNA synthesis are being studied techniques, such as MRI or CT have remained abnormal or [44]. They may visualise the higher cycling activity of inconclusive. PET/CT may identify new sites of disease as haemopoeitic cells in the bone marrow compartment and well as unsuspected extramedullary spread. Patients appear may be helpful in distinguishing separate haematological to have a particularly poor prognosis if abnormal FDG disorders. uptake is present following high-dose therapy or stem cell transplantation [40]. In a recent study by Bartel et al. in 2009, 7. Conclusion it was demonstrated that several imaging parameters related to tumour burden, such as focal lesion number assessed by It is clear that no one radiological technique in isolation MRI, and intensity of tumour metabolism on PET/CT, affect is perfect in accurately staging and monitoring patients survival outcomes [41]. 239 patients with newly diagnosed, with multiple myeloma, and that there are nuances and untreated symptomatic multiple myeloma enlisted in the pitfalls associated with even the most advanced techniques. Total Therapy 3 program and were evaluated using plain Whole-body MR, CT, and PET/CT can provide valuable radiographic survey, MRI and PET/CT. Complete FDG complimentary information when used in the correct setting. suppression in focal lesions and metastatic spread before As the availability of these techniques increase, so too transplantation conferred superior overall and event-free will their use. This is becoming increasingly important survival. At 30 months from first autotransplantation, 92% as clinicians strive to best assess the appropriateness and and 89%, respectively, were alive and event-free compared effectiveness of new and changing treatment regimes. Newer with 71% and 63% among those with less than 100% and more sensitive imaging techniques, including CT and suppression of FDG uptake in focal lesions or metastatic MR whole-body imaging and functional imaging modalities spread. 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