- Email: [email protected]
Autologous Stem Cell Transplantation and Multiple Myeloma Cancer Stem Cells
SECTION V: MYELOMA CANCER STEM CELLS
Autologous Stem Cell Transplantation and Multiple Myeloma Cancer Stem Cells William Matsui,1 Ivan Borrello,1 Constantine Mitsiades2 It is well established that high-dose therapy (HDT) combined with autologous stem cell transplantation (ASCT) produces superior response rates and progression-free survival compared with conventional chemotherapy in patients with multiple myeloma (MM). Accordingly, MM currently represents the most common indication for ASCT. Despite these clinical improvements, the impact of ASCT on overall survival is unclear because the vast majority of patients eventually experience disease relapse and progression. The continual risk of relapse suggests that malignant cells resistant to HDT possess the clonogenic growth potential to mediate tumor regrowth, and in several diseases cancer stem cells (CSCs) have been identified that are both highly tumorigenic and resistant to standard anticancer approaches. Putative CSCs have been identified in MM, and their characterization may lead to the development of novel maintenance strategies that inhibit the production of new tumor cells, prevent disease relapse, and improve overall survival. Biol Blood Marrow Transplant 18: S27-S32 (2012) Ó 2012 American Society for Blood and Marrow Transplantation
AUTOLOGOUS STEM CELL TRANSPLANTATION (ASCT) IN MULTIPLE MYELOMA ASCT has been considered the standard approach for frontline therapy in eligible multiple myeloma (MM) patients based on several randomized trials comparing ASCT to conventional chemotherapy. Since 1996, 7 randomized clinical trials have been reported and clearly demonstrated the superiority of ASCT in inducing disease responses, especially complete remissions, and prolonging event-free survival. In contrast, the impact of ASCT on overall survival (OS) has remained unclear. The IFM90, MRC VII, and Italian MMSG phase III trials demonstrated significant prolongation of median OS in newly diagnosed patients undergoing ASCT [1-3]. However, these results are balanced by the PETHEMA, MAG91, MAG95, and US Intergroup S9321 trials
From the 1The Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, Division of Hematologic Malignancies, Johns Hopkins University School of Medicine, Baltimore, Maryland; and 2Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, Department of Medical Oncology, Harvard Medical School, Boston, Massachusetts. Financial disclosure: See Acknowledgment on page S30. Correspondence and reprint requests: William Matsui, MD, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Department of Oncology, CRB245, 1650 Orleans Street, Baltimore, MD 21287 (e-mail: [email protected]). Ó 2012 American Society for Blood and Marrow Transplantation 1083-8791/$36.00 doi:10.1016/j.bbmt.2011.10.036
that failed to detect a significant improvement in OS for patients undergoing ASCT compared with those receiving conventional chemotherapy [4-7]. Moreover, a recent meta-analysis of 2411 patients failed to identify a significant OS advantage for ASCT [8]. The uncertain impact of ASCT on OS is largely because of disease relapse and progression that affects virtually all MM patients regardless of whether or not they have undergone ASCT. Accordingly, the development of maintenance strategies to prolong responses and improve OS has been the subject of great interest.
MAINTENANCE THERAPY FOR MM IN THE POSTTRANSPLANT SETTING Several strategies have been examined in hopes of extending disease responses following ASCT. Initial studies focused on the use of interferon-alpha (IFN-a) based on its ability to improve OS when used as maintenance following conventional chemotherapy [9]. Patients with major reductions in tumor burden when initiating maintenance appeared to gain the greatest benefit. Therefore, the post-ASCT setting was thought to represent the optimal time to utilize maintenance IFN-a, and this concept was supported by a retrospective European Blood and Marrow Transplant analysis demonstrating improved progression-free survival (PFS) and OS in patients receiving IFN-a [10]. Two randomized studies were subsequently undertaken but failed to demonstrate improved OS in patients administered IFN-a following ASCT [7,11]. Although better tolerated post-ASCT than following conventional S27
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therapy, the relative toxicity of IFN-a combined with the unclear impact on survival has led to the abandonment of this approach. The introduction of novel agents including thalidomide, its analog lenalidomide, and the proteosome inhibitor bortezomib over the past decade have dramatically modified treatment approaches for MM [12,13]. All of these agents were initially tested and approved for use in patients with refractory and relapsed disease, but their relative efficacy has led to their examination and incorporation into both initial treatment paradigms as well as in the maintenance setting following ASCT. Maintenance thalidomide has been extensively studied, and to date, 5 randomized clinical trials have been reported [7,1417]. The IFM 99-02 study compared the use of thalidomide and pamidronate post-ASCT to pamidronate alone or no maintenance in patients with standard-risk myeloma [14]. Here, thalidomide demonstrated significantly greater response, PFS, and OS rates. These results were essentially replicated in a randomized study from Australia (ALLG MM6) that compared thalidomide and prednisone with prednisone alone [15]. In contrast, 3 other large studies have failed to demonstrate improvements in OS despite higher response rates and prolonged PFS and event-free survival. These studies have included Total Therapy 2, in which patients received thalidomide both during induction and as maintenance within a complex regimen that included induction chemotherapy, tandem ASCT, and IFN-a and dexamethasone post-ASCT [18], the HOVON 50 study utilizing thalidomide during induction therapy followed by thalidomide or interferon as maintenance [17], and the MRC Myeloma IX trial in which thalidomide was administered following either ASCT or conventional therapy [16]. Although an overview of these studies fails to provide definitive evidence for the use of thalidomide as maintenance following ASCT, distinct groups of MM patients appear to benefit. In the IFM 99-02 trial, thalidomide maintenance was found to be beneficial in patients failing to achieve less than a very good partial remission following ASCT, whereas a subsequent analysis of Total Therapy 2 at a median follow-up of 72 months demonstrated a significant improvement in OS in patients with detectable metaphase cytogenetic abnormalities who received thalidomide [19]. Therefore, it is likely that specific subgroups of MM patients may benefit from thalidomide maintenance, but this potential benefit must be weighed against adverse effects of the drug, most notably cumulative peripheral neuropathy that is dependent on the dose and duration of administration. Data concerning the use of bortezomib and lenalidomide as maintenance following ASCT are limited given their more recent approval. The incorporation
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of bortezomib into both induction and post-ASCT therapy has been the subject of 2 large randomized phase III studies in Europe [20,21]. Accrual has been completed on each of these trials and further followup will better define the utility of bortezomib in the maintenance setting. Posttransplantation lenalidomide is under study in several randomized studies, and preliminary findings have recently been reported. In the IFM 2005-02 trial, 614 patients were administered lenalidomide as consolidation following ASCT then randomized to receive maintenance lenalidomide or placebo [22]. PFS was significantly greater in patients in the lenalidomide maintenance arm. However, 5-year survival rates for each group was identical at 83%. In the CALGB 100104 trial, 460 patients were randomized to receive maintenance lenalidomide or placebo following ASCT [23]. Patients receiving lenalidomide experienced significantly prolonged time to progression, although the impact on OS is less clear given the low number of deaths. Lenalidomide was well tolerated in both trials, but an increased incidence in the rate of secondary malignancies has been observed in each. Therefore, the role of lenalidomide maintenance following ASCT remains unclear, and the balance between beneficial effects on MM relapse and potential toxicity must be further evaluated. CANCER STEM CELLS IN MM Disease relapse remains the major factor limiting OS; therefore, a better understanding of the processes responsible for tumor recurrence and regrowth may ultimately lead to improvements in long-term outcomes. Emerging data in a wide range of human malignancies, including MM, have demonstrated that cells within an individual tumor may be phenotypically and functionally heterogeneous despite their clonal origins [24]. The majority of cells appear to lack sufficient long-term replicative potential required for disease propagation. Instead, tumor growth arises from relatively rare populations of phenotypically distinct cancer stem cells (CSCs). In both hematologic malignancies and solid tumors, CSCs have been prospectively identified based on their ability to give rise to differentiated progeny that recapitulates the original tumor in the ectopic setting. Several studies have also found that CSCs are relatively resistant to standard anticancer agents compared with bulk tumor cells. Therefore, CSCs may play a central role in disease relapse because they have the ability to persist during treatment and give rise to new tumor cells. Several approaches have been undertaken to identify cancer stem cells [25]. Determination of specific cell surface antigens expressed by tumorigenic cells has been most commonly employed, and candidate markers have consisted of markers characteristic of
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normal stem cells or progenitors within the tissue of origin (eg, CD34 in myeloid leukemias), expressed by multiple normal stem cells (eg, CD133 in brain tumors), or associated with poor prognosis (CD44 in solid tumors). In addition, flow cytometric assays indicative of drug resistance, such as the Aldefluor assay that measures aldehyde dehydrogenase activity or detection of side population cells with increased drug efflux capabilities, have also been used to isolate tumor cells with relatively enhanced tumorigenic potential. In MM, each of these strategies has successfully identified candidate CSCs. Several studies have examined MM CSCs within the context of normal plasma cell differentiation. In a simplistic schema of normal B cell development, na€ıve B cells harboring immunoglobulin V(D)J gene rearrangements undergo somatic hypermutation in germinal center reactions to produce unique antibody idiotypes and improve antigen binding specificity and affinity. Germinal center B cells subsequently give rise to postgerminal center memory B cells that maintain long-term humoral immunity and differentiate into antibody secreting plasma cells upon antigen reexposure. The examination of immunoglobulin gene sequences in MM plasma cells have demonstrated that they are somatically hypermutated and remain constant throughout the entire clinical course, suggesting that the disease arises from postgerminal center memory B cells or plasmablasts [26]. The unique immunoglobulin gene rearrangement also provides a highly tumor-specific means of establishing clonal relationships among different tumor cell populations, and several studies over the past 2 decades have detected phenotypic B cells sharing the identical immunoglobulin gene rearrangements as MM plasma cells within the bone marrow and circulation of MM patients [27-30]. The clinical relevance of these clonotypic B cells has been unclear, but functional studies have demonstrated that these cells are tumorigenic and can give rise to MM plasma cells both in in vitro colony forming assays as well as in vivo in immunodeficient nonobese, diabetic, severe combined deficiency mice [31-33]. Moreover, clonotypic B cells isolated from the circulation of MM patients are capable of disease propagation during serial transplantation studies, demonstrating that they are capable of self-renewal [33]. Therefore, MM may be hierarchically organized and recapitulates normal plasma cell development. Studies have also demonstrated that clonotypic B cells are relatively resistant compared with plasma cells to several clinically utilized antimyeloma agents, including traditional cytotoxic chemotherapies and novel agents such as bortezomib and lenalidomide in vitro and in vivo [33,34]. In addition to cell surface antigen expression, functional flow cytometric assays have been used to identify
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clonogenic MM cells. The expression of aldehyde dehydrogenase is characteristic of normal stem cells in a variety of adult tissues and can identify CSCs in both solid tumors and hematologic malignancies [25]. MM CSCs have been found to express relatively high levels of aldehyde dehydrogenase with tumorigenic potential both in vitro and in vivo [33]. The side population assay takes advantage of differential emission spectra following exposure to the DNA binding dye Hoechst 33342 and was initially developed to identify normal hematopoietic stem cells [35]. This assay has been used to study MM, and clonogenic tumor cells have been found to express the side population phenotype [33,36]. Further analysis of MM side population cells has also demonstrated increased expression of membrane bound drug transporters (ABCG2 and MDR/Pgp) suggestive of relative drug resistance [36]. Despite these findings, the existence and precise phenotype of MM CSCs remains unclear. Studies examining the B cell nature of tumorigenic MM cells have demonstrated that these cells lack expression of the characteristic cell surface antigen CD138 expressed by both normal and MM plasma cells [33,37]. On the other hand, studies examining MM side population cells have demonstrated that these cells are both CD138neg and CD1381 [33,36]. Moreover, the engraftment and growth of primary MM specimens directly injected into the ectopic human or rabbit bone fragments implanted into severe combined immunodeficiency mice (SCID-Hu, SCID-Rab) is restricted to CD1381 MM plasma cells [38,39]. The reasons for these discrepancies are unclear, but they likely represent the intrinsic differences between the animal models or patient specimens studied, and similar variations in CSC phenotypes have been observed in several other human malignancies. An important consideration, consistent with data emerging now from several lines of independent research in different malignant and normal models, is that some surface markers conventionally associated with the ‘‘stem cell’’ compartment of the tumor cell population may actually exhibit significant plasticity. Indeed, there is emerging evidence of substantial bidirectional interconversions between nonstem and stem-like compartments in a population of malignant or normal cells [36,40-42]. These results provide another possible explanation for some variations in the observations of different groups studying the phenotypic features of putative cancer stem cells. Importantly, the studies on bidirectional interconversions also indicate the critical significance of refocusing the investigation on putative cancer stem cell compartments toward functional assays, as this may provide a better approach to define the biologic and clinical implications of cancer cell populations with stem cell features.
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TARGETING MYELOMA STEM CELLS Given the unique functional attributes of CSCs, efforts to identify potential targeting strategies are currently under way. The impact of the tumor microenvironment on the proliferation and survival of MM plasma cells has been well documented, and these interactions have provided the scientific rationale for specific therapeutic strategies. The influence of the microenvironment on MM CSCs has recently been reported, and both the proliferation and relative proportion of side population MM cells can be enhanced by coincubation with bone marrow stromal cells [36]. Moreover, lenalidomide, but not thalidomide, has been found to significantly inhibit the number and colony-forming potential of MM side population cells associated with alterations in the phosphorylation of several important signaling molecules including Akt, GSK-3a/b, MEK1, c-JUN, p53, and p70S6K. These intriguing results suggest that recent clinical data demonstrating improved PFS and event-free survival in patients receiving post-ASCT lenalidomide is mediated by its impact on MM CSCs expressing the side population phenotype. Self-renewal appears to be a unique property required for the maintenance of both normal and cancer stem cells. The factors regulating MM CSCs are not fully understood, but the functional similarities between CSCs and normal stem cells suggest that shared cellular pathways may be involved. During development of the normal embryo, several highly conserved signaling pathways are required for cell fate specification, such as Notch, Wnt, and Hedgehog, and these have been increasingly implicated in the pathogenesis of a wide variety of human cancers. Hedgehog signaling has been studied in MM and may have multiple effects that depend on the precise cell type examined. In plasma cells, aberrant Hedgehog signaling primarily promotes cell survival, whereas it regulates cell fate decisions of MM CSCs [43,44]. In CD138neg cells, pathway activation by the Hedgehog ligand induces self-renewal and cell expansion, whereas the inhibition by antagonists of SMOOTHENED, a positive regulator of Hedgehog signaling, induces plasma cell differentiation and the loss of clonogenic potential. Telomerase may represent another potential means of targeting MM CSC self-renewal, and a recent study has reported that the inhibition of telomerase activity within CD138neg cells using specific inhibitors leads to decreased tumorigenic potential both in vitro and in vivo [45]. Therefore, shared self-renewal pathways may serve as novel therapeutic targets. The potential of immune-based therapies to target MM CSCs is evident through the allogeneic graftversus-tumor effect that may result in long-term remissions, and the tumor-specific nature of the M protein suggests that it may serve as an ideal antigenic
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target. A previous study found that the number of circulating clonotypic B cells was significantly diminished following vaccination of MM patients with purified M protein [46]. Interestingly, M protein levels were not significantly altered in these patients, suggesting that this strategy may preferentially target malignant precursors rather than differentiated plasma cells. Aberrant expression of the embryonic stem cell– associated transcription factor SOX2 was initially identified by screening reactive antibodies present in patients with monoclonal gammopathy of unknown significance and may represent another potential immune target preferentially expressed by MM CSCs [47]. In monoclonal gammopathy of unknown significance, the expression of SOX2 was restricted to CD138neg cells, and anti-SOX2 immunity limited the clonogenic growth of primary specimens in vitro. Tumor-infiltrating lymphocytes may exhibit antitumor activity, and the use of T cells derived from the bone marrow of MM patients is emerging. These marrow-infiltrating lymphocytes can be harvested from the bone marrow, efficiently activated and expanded ex vivo, and safely reinfused in patients with MM (I.B., personal observation) [48]. Moreover, functional analyses of marrow-infiltrating lymphocytes have demonstrated that they display greater specificity for tumor cells than normal peripheral blood lymphocytes and are capable of inhibiting both mature plasma cells and CD138neg CSCs. Thus, immune-based approaches hold promise as clinically effective MM CSC targeting strategies. CONCLUSIONS Evidence for both functional and phenotypic cellular heterogeneity in MM is emerging. However, the precise phenotype of tumorigenic MM cells has not been definitively established and controversy remains. Whatever their phenotype, it is likely that definitive proof of the existence of MM CSCs will be provided by actual improvements in long-term outcomes using clinical strategies based on CSC biology rather than further studies of various model systems. Increasing knowledge of the basic biology of MM CSCs should produce candidate approaches that can serve as the basis for novel therapies. The implementation of these strategies into clinical scenarios that allow MM CSC functions to be assessed, such as maintenance following tumor debulking by ASCT, may ultimately provide evidence for their clinical relevance. ACKNOWLEDGMENT Financial disclosure: The authors have nothing to disclose.
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17. Lokhorst HM, van der Holt B, Zweegman S, et al. A randomized phase 3 study on the effect of thalidomide combined with adriamycin, dexamethasone, and high-dose melphalan, followed by thalidomide maintenance in patients with multiple myeloma. Blood. 2010;115:1113-1120. 18. Barlogie B, Tricot G, Rasmussen E, et al. Total therapy 2 without thalidomide in comparison with total therapy 1: role of intensified induction and posttransplantation consolidation therapies. Blood. 2006;107:2633-2638. 19. Barlogie B, Pineda-Roman M, van Rhee F, et al. Thalidomide arm of total therapy 2 improves complete remission duration and survival in myeloma patients with metaphase cytogenetic abnormalities. Blood. 2008;112:3115-3121. 20. Cavo M, Tacchetti P, Patriarca F, et al. A phase III study of double autotransplantation incorporating bortezomib-thalidomidedexamethasone (VTD) or thalidomide-dexamethasone (TD) for multiple myeloma: superior clinical outcomes with VTD compared to TD. ASH Annu Meet Abstr. 2009;114:351. 21. Sonneveld P, Schmidt-Wolf I, van der Holt B, et al. HOVON-65/GMMG-HD4 randomized phase III trial comparing bortezomib, doxorubicin, dexamethasone (PAD) vs VAD followed by high-dose melphalan (HDM) and maintenance with bortezomib or thalidomide in patients with newly diagnosed multiple myeloma (MM). ASH Annu Meet Abstr. 2010;116:40. 22. Attal M, Lauwers Vc, Marit G, et al. Maintenance treatment with lenalidomide after transplantation for myeloma: final analysis of the IFM 2005-02. ASH Annu Meet Abstr. 2010; 116:310. 23. McCarthy PL, Owzar K, Anderson KC, et al. Phase III intergroup study of lenalidomide versus placebo maintenance therapy following single autologous hematopoietic stem cell transplantation (AHSCT) for multiple myeloma: CALGB 100104. ASH Annu Meet Abstr. 2010;116:37. 24. Rasheed ZA, Kowalski J, Smith BD, Matsui W. Concise review: emerging concepts in clinical targeting of cancer stem cells. Stem Cells. 2011;29:883-887. 25. Brennan S, Matsui W. Cancer stem cells: controversies in multiple myeloma. J Mol Med. 2009;87:1079-1085. 26. Bakkus MH, Heirman C, Van Riet I, Van Camp B, Thielemans K. Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation. Blood. 1992;80:2326-2335. 27. Bakkus MH, Van RI, Van Camp B, Thielemans K. Evidence that the clonogenic cell in multiple myeloma originates from a pre-switched but somatically mutated B cell. Br J Haematol. 1994;87:68-74. 28. Billadeau D, Ahmann G, Greipp P, Van Ness B. The bone marrow of multiple myeloma patients contains B cell populations at different stages of differentiation that are clonally related to the malignant plasma cell. J Exp Med. 1993;178: 1023-1031. 29. Bergsagel PL, Smith AM, Szczepek A, Mant MJ, Belch AR, Pilarski LM. In multiple myeloma, clonotypic B lymphocytes are detectable among CD191 peripheral blood cells expressing CD38, CD56, and monotypic Ig light chain. Blood. 1995;85: 436-447. 30. Rasmussen T, Jensen L, Johnsen HE. Levels of circulating CD191 cells in patients with multiple myeloma. Blood. 2000; 95:4020-4021. 31. Pilarski LM, Hipperson G, Seeberger K, Pruski E, Coupland RW, Belch AR. Myeloma progenitors in the blood of patients with aggressive or minimal disease: engraftment and self-renewal of primary human myeloma in the bone marrow of NOD SCID mice. Blood. 2000;95:1056-1065. 32. Pilarski LM, Seeberger K, Coupland RW, et al. Leukemic B cells clonally identical to myeloma plasma cells are myelomagenic in NOD/SCID mice. Exp Hematol. 2002;30:221-228. 33. Matsui W, Wang Q, Barber JP, et al. Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance. Cancer Res. 2008;68:190-197.
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34. Rasmussen T, Jensen L, Honore L, Johnsen HE. Frequency and kinetics of polyclonal and clonal B cells in the peripheral blood of patients being treated for multiple myeloma. Blood. 2000;96: 4357-4359. 35. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med. 1996;183:1797-1806. 36. Jakubikova J, Adamia S, Kost-Alimova M, et al. Lenalidomide targets clonogenic side population in multiple myeloma: pathophysiologic and clinical implications. Blood. 2011;117:4409-4419. 37. Matsui W, Huff CA, Wang Q, et al. Characterization of clonogenic multiple myeloma cells. Blood. 2004;103:2332-2336. 38. Yaccoby S, Barlogie B, Epstein J. Primary myeloma cells growing in SCID-hu mice: a model for studying the biology and treatment of myeloma and its manifestations. Blood. 1998;92:2908-2913. 39. Yata K, Yaccoby S. The SCID-rab model: a novel in vivo system for primary human myeloma demonstrating growth of CD138expressing malignant cells. Leukemia. 2004;18:1891-1897. 40. Gupta PB, Fillmore CM, Jiang G, et al. Stochastic state transitions give rise to phenotypic equilibrium in populations of cancer cells. Cell. 2011;146:633-644. 41. Chaffer CL, Brueckmann I, Scheel C, et al. Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state. Proc Natl Acad Sci USA. 2011;108:7950-7955.
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42. He K, Xu T, Goldkorn A. Cancer cells cyclically lose and regain drug-resistant highly tumorigenic features characteristic of a cancer stem-like phenotype. Mol Cancer Ther. 2011;10: 938-948. 43. Dierks C, Grbic J, Zirlik K, et al. Essential role of stromally induced hedgehog signaling in B-cell malignancies. Nat Med. 2007;13:944-951. 44. Peacock CD, Wang Q, Gesell GS, et al. Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proc Natl Acad Sci USA. 2007;104:4048-4053. 45. Brennan SK, Wang Q, Tressler R, et al. Telomerase inhibition targets clonogenic multiple myeloma cells through telomere length-dependent and independent mechanisms. PLoS One. 2010;5:e12487. 46. Rasmussen T, Hansson L, Osterborg A, Johnsen HE, Mellstedt H. Idiotype vaccination in multiple myeloma induced a reduction of circulating clonal tumor B cells. Blood. 2003;101: 4607-4610. 47. Kukreja A, Hutchinson A, Dhodapkar K, et al. Enhancement of clonogenicity of human multiple myeloma by dendritic cells. J Exp Med. 2006;203:1859-1865. 48. Noonan K, Matsui W, Serafini P, et al. Activated marrowinfiltrating lymphocytes effectively target plasma cells and their clonogenic precursors. Cancer Res. 2005;65:2026-2034.
Autologous Stem Cell Transplantation and Multiple Myeloma Cancer Stem Cells William Matsui,1 Ivan Borrello,1 Constantine Mitsiades2 It is well established that high-dose therapy (HDT) combined with autologous stem cell transplantation (ASCT) produces superior response rates and progression-free survival compared with conventional chemotherapy in patients with multiple myeloma (MM). Accordingly, MM currently represents the most common indication for ASCT. Despite these clinical improvements, the impact of ASCT on overall survival is unclear because the vast majority of patients eventually experience disease relapse and progression. The continual risk of relapse suggests that malignant cells resistant to HDT possess the clonogenic growth potential to mediate tumor regrowth, and in several diseases cancer stem cells (CSCs) have been identified that are both highly tumorigenic and resistant to standard anticancer approaches. Putative CSCs have been identified in MM, and their characterization may lead to the development of novel maintenance strategies that inhibit the production of new tumor cells, prevent disease relapse, and improve overall survival. Biol Blood Marrow Transplant 18: S27-S32 (2012) Ó 2012 American Society for Blood and Marrow Transplantation
AUTOLOGOUS STEM CELL TRANSPLANTATION (ASCT) IN MULTIPLE MYELOMA ASCT has been considered the standard approach for frontline therapy in eligible multiple myeloma (MM) patients based on several randomized trials comparing ASCT to conventional chemotherapy. Since 1996, 7 randomized clinical trials have been reported and clearly demonstrated the superiority of ASCT in inducing disease responses, especially complete remissions, and prolonging event-free survival. In contrast, the impact of ASCT on overall survival (OS) has remained unclear. The IFM90, MRC VII, and Italian MMSG phase III trials demonstrated significant prolongation of median OS in newly diagnosed patients undergoing ASCT [1-3]. However, these results are balanced by the PETHEMA, MAG91, MAG95, and US Intergroup S9321 trials
From the 1The Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, Division of Hematologic Malignancies, Johns Hopkins University School of Medicine, Baltimore, Maryland; and 2Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, Department of Medical Oncology, Harvard Medical School, Boston, Massachusetts. Financial disclosure: See Acknowledgment on page S30. Correspondence and reprint requests: William Matsui, MD, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Department of Oncology, CRB245, 1650 Orleans Street, Baltimore, MD 21287 (e-mail: [email protected]). Ó 2012 American Society for Blood and Marrow Transplantation 1083-8791/$36.00 doi:10.1016/j.bbmt.2011.10.036
that failed to detect a significant improvement in OS for patients undergoing ASCT compared with those receiving conventional chemotherapy [4-7]. Moreover, a recent meta-analysis of 2411 patients failed to identify a significant OS advantage for ASCT [8]. The uncertain impact of ASCT on OS is largely because of disease relapse and progression that affects virtually all MM patients regardless of whether or not they have undergone ASCT. Accordingly, the development of maintenance strategies to prolong responses and improve OS has been the subject of great interest.
MAINTENANCE THERAPY FOR MM IN THE POSTTRANSPLANT SETTING Several strategies have been examined in hopes of extending disease responses following ASCT. Initial studies focused on the use of interferon-alpha (IFN-a) based on its ability to improve OS when used as maintenance following conventional chemotherapy [9]. Patients with major reductions in tumor burden when initiating maintenance appeared to gain the greatest benefit. Therefore, the post-ASCT setting was thought to represent the optimal time to utilize maintenance IFN-a, and this concept was supported by a retrospective European Blood and Marrow Transplant analysis demonstrating improved progression-free survival (PFS) and OS in patients receiving IFN-a [10]. Two randomized studies were subsequently undertaken but failed to demonstrate improved OS in patients administered IFN-a following ASCT [7,11]. Although better tolerated post-ASCT than following conventional S27
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therapy, the relative toxicity of IFN-a combined with the unclear impact on survival has led to the abandonment of this approach. The introduction of novel agents including thalidomide, its analog lenalidomide, and the proteosome inhibitor bortezomib over the past decade have dramatically modified treatment approaches for MM [12,13]. All of these agents were initially tested and approved for use in patients with refractory and relapsed disease, but their relative efficacy has led to their examination and incorporation into both initial treatment paradigms as well as in the maintenance setting following ASCT. Maintenance thalidomide has been extensively studied, and to date, 5 randomized clinical trials have been reported [7,1417]. The IFM 99-02 study compared the use of thalidomide and pamidronate post-ASCT to pamidronate alone or no maintenance in patients with standard-risk myeloma [14]. Here, thalidomide demonstrated significantly greater response, PFS, and OS rates. These results were essentially replicated in a randomized study from Australia (ALLG MM6) that compared thalidomide and prednisone with prednisone alone [15]. In contrast, 3 other large studies have failed to demonstrate improvements in OS despite higher response rates and prolonged PFS and event-free survival. These studies have included Total Therapy 2, in which patients received thalidomide both during induction and as maintenance within a complex regimen that included induction chemotherapy, tandem ASCT, and IFN-a and dexamethasone post-ASCT [18], the HOVON 50 study utilizing thalidomide during induction therapy followed by thalidomide or interferon as maintenance [17], and the MRC Myeloma IX trial in which thalidomide was administered following either ASCT or conventional therapy [16]. Although an overview of these studies fails to provide definitive evidence for the use of thalidomide as maintenance following ASCT, distinct groups of MM patients appear to benefit. In the IFM 99-02 trial, thalidomide maintenance was found to be beneficial in patients failing to achieve less than a very good partial remission following ASCT, whereas a subsequent analysis of Total Therapy 2 at a median follow-up of 72 months demonstrated a significant improvement in OS in patients with detectable metaphase cytogenetic abnormalities who received thalidomide [19]. Therefore, it is likely that specific subgroups of MM patients may benefit from thalidomide maintenance, but this potential benefit must be weighed against adverse effects of the drug, most notably cumulative peripheral neuropathy that is dependent on the dose and duration of administration. Data concerning the use of bortezomib and lenalidomide as maintenance following ASCT are limited given their more recent approval. The incorporation
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of bortezomib into both induction and post-ASCT therapy has been the subject of 2 large randomized phase III studies in Europe [20,21]. Accrual has been completed on each of these trials and further followup will better define the utility of bortezomib in the maintenance setting. Posttransplantation lenalidomide is under study in several randomized studies, and preliminary findings have recently been reported. In the IFM 2005-02 trial, 614 patients were administered lenalidomide as consolidation following ASCT then randomized to receive maintenance lenalidomide or placebo [22]. PFS was significantly greater in patients in the lenalidomide maintenance arm. However, 5-year survival rates for each group was identical at 83%. In the CALGB 100104 trial, 460 patients were randomized to receive maintenance lenalidomide or placebo following ASCT [23]. Patients receiving lenalidomide experienced significantly prolonged time to progression, although the impact on OS is less clear given the low number of deaths. Lenalidomide was well tolerated in both trials, but an increased incidence in the rate of secondary malignancies has been observed in each. Therefore, the role of lenalidomide maintenance following ASCT remains unclear, and the balance between beneficial effects on MM relapse and potential toxicity must be further evaluated. CANCER STEM CELLS IN MM Disease relapse remains the major factor limiting OS; therefore, a better understanding of the processes responsible for tumor recurrence and regrowth may ultimately lead to improvements in long-term outcomes. Emerging data in a wide range of human malignancies, including MM, have demonstrated that cells within an individual tumor may be phenotypically and functionally heterogeneous despite their clonal origins [24]. The majority of cells appear to lack sufficient long-term replicative potential required for disease propagation. Instead, tumor growth arises from relatively rare populations of phenotypically distinct cancer stem cells (CSCs). In both hematologic malignancies and solid tumors, CSCs have been prospectively identified based on their ability to give rise to differentiated progeny that recapitulates the original tumor in the ectopic setting. Several studies have also found that CSCs are relatively resistant to standard anticancer agents compared with bulk tumor cells. Therefore, CSCs may play a central role in disease relapse because they have the ability to persist during treatment and give rise to new tumor cells. Several approaches have been undertaken to identify cancer stem cells [25]. Determination of specific cell surface antigens expressed by tumorigenic cells has been most commonly employed, and candidate markers have consisted of markers characteristic of
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normal stem cells or progenitors within the tissue of origin (eg, CD34 in myeloid leukemias), expressed by multiple normal stem cells (eg, CD133 in brain tumors), or associated with poor prognosis (CD44 in solid tumors). In addition, flow cytometric assays indicative of drug resistance, such as the Aldefluor assay that measures aldehyde dehydrogenase activity or detection of side population cells with increased drug efflux capabilities, have also been used to isolate tumor cells with relatively enhanced tumorigenic potential. In MM, each of these strategies has successfully identified candidate CSCs. Several studies have examined MM CSCs within the context of normal plasma cell differentiation. In a simplistic schema of normal B cell development, na€ıve B cells harboring immunoglobulin V(D)J gene rearrangements undergo somatic hypermutation in germinal center reactions to produce unique antibody idiotypes and improve antigen binding specificity and affinity. Germinal center B cells subsequently give rise to postgerminal center memory B cells that maintain long-term humoral immunity and differentiate into antibody secreting plasma cells upon antigen reexposure. The examination of immunoglobulin gene sequences in MM plasma cells have demonstrated that they are somatically hypermutated and remain constant throughout the entire clinical course, suggesting that the disease arises from postgerminal center memory B cells or plasmablasts [26]. The unique immunoglobulin gene rearrangement also provides a highly tumor-specific means of establishing clonal relationships among different tumor cell populations, and several studies over the past 2 decades have detected phenotypic B cells sharing the identical immunoglobulin gene rearrangements as MM plasma cells within the bone marrow and circulation of MM patients [27-30]. The clinical relevance of these clonotypic B cells has been unclear, but functional studies have demonstrated that these cells are tumorigenic and can give rise to MM plasma cells both in in vitro colony forming assays as well as in vivo in immunodeficient nonobese, diabetic, severe combined deficiency mice [31-33]. Moreover, clonotypic B cells isolated from the circulation of MM patients are capable of disease propagation during serial transplantation studies, demonstrating that they are capable of self-renewal [33]. Therefore, MM may be hierarchically organized and recapitulates normal plasma cell development. Studies have also demonstrated that clonotypic B cells are relatively resistant compared with plasma cells to several clinically utilized antimyeloma agents, including traditional cytotoxic chemotherapies and novel agents such as bortezomib and lenalidomide in vitro and in vivo [33,34]. In addition to cell surface antigen expression, functional flow cytometric assays have been used to identify
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clonogenic MM cells. The expression of aldehyde dehydrogenase is characteristic of normal stem cells in a variety of adult tissues and can identify CSCs in both solid tumors and hematologic malignancies [25]. MM CSCs have been found to express relatively high levels of aldehyde dehydrogenase with tumorigenic potential both in vitro and in vivo [33]. The side population assay takes advantage of differential emission spectra following exposure to the DNA binding dye Hoechst 33342 and was initially developed to identify normal hematopoietic stem cells [35]. This assay has been used to study MM, and clonogenic tumor cells have been found to express the side population phenotype [33,36]. Further analysis of MM side population cells has also demonstrated increased expression of membrane bound drug transporters (ABCG2 and MDR/Pgp) suggestive of relative drug resistance [36]. Despite these findings, the existence and precise phenotype of MM CSCs remains unclear. Studies examining the B cell nature of tumorigenic MM cells have demonstrated that these cells lack expression of the characteristic cell surface antigen CD138 expressed by both normal and MM plasma cells [33,37]. On the other hand, studies examining MM side population cells have demonstrated that these cells are both CD138neg and CD1381 [33,36]. Moreover, the engraftment and growth of primary MM specimens directly injected into the ectopic human or rabbit bone fragments implanted into severe combined immunodeficiency mice (SCID-Hu, SCID-Rab) is restricted to CD1381 MM plasma cells [38,39]. The reasons for these discrepancies are unclear, but they likely represent the intrinsic differences between the animal models or patient specimens studied, and similar variations in CSC phenotypes have been observed in several other human malignancies. An important consideration, consistent with data emerging now from several lines of independent research in different malignant and normal models, is that some surface markers conventionally associated with the ‘‘stem cell’’ compartment of the tumor cell population may actually exhibit significant plasticity. Indeed, there is emerging evidence of substantial bidirectional interconversions between nonstem and stem-like compartments in a population of malignant or normal cells [36,40-42]. These results provide another possible explanation for some variations in the observations of different groups studying the phenotypic features of putative cancer stem cells. Importantly, the studies on bidirectional interconversions also indicate the critical significance of refocusing the investigation on putative cancer stem cell compartments toward functional assays, as this may provide a better approach to define the biologic and clinical implications of cancer cell populations with stem cell features.
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TARGETING MYELOMA STEM CELLS Given the unique functional attributes of CSCs, efforts to identify potential targeting strategies are currently under way. The impact of the tumor microenvironment on the proliferation and survival of MM plasma cells has been well documented, and these interactions have provided the scientific rationale for specific therapeutic strategies. The influence of the microenvironment on MM CSCs has recently been reported, and both the proliferation and relative proportion of side population MM cells can be enhanced by coincubation with bone marrow stromal cells [36]. Moreover, lenalidomide, but not thalidomide, has been found to significantly inhibit the number and colony-forming potential of MM side population cells associated with alterations in the phosphorylation of several important signaling molecules including Akt, GSK-3a/b, MEK1, c-JUN, p53, and p70S6K. These intriguing results suggest that recent clinical data demonstrating improved PFS and event-free survival in patients receiving post-ASCT lenalidomide is mediated by its impact on MM CSCs expressing the side population phenotype. Self-renewal appears to be a unique property required for the maintenance of both normal and cancer stem cells. The factors regulating MM CSCs are not fully understood, but the functional similarities between CSCs and normal stem cells suggest that shared cellular pathways may be involved. During development of the normal embryo, several highly conserved signaling pathways are required for cell fate specification, such as Notch, Wnt, and Hedgehog, and these have been increasingly implicated in the pathogenesis of a wide variety of human cancers. Hedgehog signaling has been studied in MM and may have multiple effects that depend on the precise cell type examined. In plasma cells, aberrant Hedgehog signaling primarily promotes cell survival, whereas it regulates cell fate decisions of MM CSCs [43,44]. In CD138neg cells, pathway activation by the Hedgehog ligand induces self-renewal and cell expansion, whereas the inhibition by antagonists of SMOOTHENED, a positive regulator of Hedgehog signaling, induces plasma cell differentiation and the loss of clonogenic potential. Telomerase may represent another potential means of targeting MM CSC self-renewal, and a recent study has reported that the inhibition of telomerase activity within CD138neg cells using specific inhibitors leads to decreased tumorigenic potential both in vitro and in vivo [45]. Therefore, shared self-renewal pathways may serve as novel therapeutic targets. The potential of immune-based therapies to target MM CSCs is evident through the allogeneic graftversus-tumor effect that may result in long-term remissions, and the tumor-specific nature of the M protein suggests that it may serve as an ideal antigenic
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target. A previous study found that the number of circulating clonotypic B cells was significantly diminished following vaccination of MM patients with purified M protein [46]. Interestingly, M protein levels were not significantly altered in these patients, suggesting that this strategy may preferentially target malignant precursors rather than differentiated plasma cells. Aberrant expression of the embryonic stem cell– associated transcription factor SOX2 was initially identified by screening reactive antibodies present in patients with monoclonal gammopathy of unknown significance and may represent another potential immune target preferentially expressed by MM CSCs [47]. In monoclonal gammopathy of unknown significance, the expression of SOX2 was restricted to CD138neg cells, and anti-SOX2 immunity limited the clonogenic growth of primary specimens in vitro. Tumor-infiltrating lymphocytes may exhibit antitumor activity, and the use of T cells derived from the bone marrow of MM patients is emerging. These marrow-infiltrating lymphocytes can be harvested from the bone marrow, efficiently activated and expanded ex vivo, and safely reinfused in patients with MM (I.B., personal observation) [48]. Moreover, functional analyses of marrow-infiltrating lymphocytes have demonstrated that they display greater specificity for tumor cells than normal peripheral blood lymphocytes and are capable of inhibiting both mature plasma cells and CD138neg CSCs. Thus, immune-based approaches hold promise as clinically effective MM CSC targeting strategies. CONCLUSIONS Evidence for both functional and phenotypic cellular heterogeneity in MM is emerging. However, the precise phenotype of tumorigenic MM cells has not been definitively established and controversy remains. Whatever their phenotype, it is likely that definitive proof of the existence of MM CSCs will be provided by actual improvements in long-term outcomes using clinical strategies based on CSC biology rather than further studies of various model systems. Increasing knowledge of the basic biology of MM CSCs should produce candidate approaches that can serve as the basis for novel therapies. The implementation of these strategies into clinical scenarios that allow MM CSC functions to be assessed, such as maintenance following tumor debulking by ASCT, may ultimately provide evidence for their clinical relevance. ACKNOWLEDGMENT Financial disclosure: The authors have nothing to disclose.
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