Myeloma Stem Cell Phenotype

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h4LJLTIPLE MYELOMA

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MYELOMA STEM CELL PHENOTYPE Implications For Treatment Joshua Epstein, DSc

The differentiation distance between the recognizable myeloma cell,

of plasma cell morphology and function, and its proliferative progenitor, the myeloma stem cell, is a topic of continuing controversy. Myeloma cells are plasma cells, the most mature cell in the Blymphocyte differentiation pathway, by morphology, phenotype, and function. Products of these cells are responsible for disease manifestations. Treatment is designed to decrease tumor burden; treatment success is judged by reduction in the number of recognizable tumor cells, their products, and manifestations. With recent innovations in the treatment of myeloma (discussed elsewhere in this issue), a considerable number of patients are induced into complete remissions. Still, cures have not been reported. Myeloma plasma cells are phenotypically heterogeneous. In individual patients, myeloma cells expressing lineage- and differentiation-associated surface molecules that indicate heterogeneity in their stages of maturation coexist. These myeloma cell populations are also functionally heterogeneous, lending support to the notion that the recognizable myeloma cells are the end product of a differentiation process, whereby myeloma progenitor cells, heretofore unrecognized, give rise to the

This work was supported in part by Grant CA-55819 from the National Cancer Institute, United States Public Health Services. ~

From the Division of Hematology/Oncology, Arkansas Cancer Research Center, University of Arkansas for Medical Sciences, Little Rock, Arkansas

HEMATOLOGY/ONCOLOGY CLINICS OF NORTH AMERICA VOLUME 11 NUMBER 1 * FEBRUARY 1997

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terminally differentiated, nonproliferative myeloma plasma cells. Such preplasmacytic myeloma progenitor cells are B lymphocytes that are morphologically indistinguishable from normal B cells. This article discusses the relevant phenotypic, functional, and molecular genetic data. PHENOTYPE OF PLASMA CELL THAT SUPPORTS DIFFERENTIATION

Myeloma plasma cells display a rather heterogeneous phenotype. Not only are there between-patient differences in the molecules expressed on the surface of tumor cells, but also among the myeloma cells of individual patients different populations can be defined, on the basis of expression of lineage- and differentiation-associated antigens. Thus, whereas all myeloma plasma cells express high levels of CD38, expresand CD56,26,30 and CD49E (VLA-5)26, 27 delinsion of CD10,I6 CD45,22-24 eates distinct subpopulations of myeloma plasma cells with a less differentiated phenotype, and identify maturation heterogeneity among plasma cells. Coexistence of these phenotypically mature and immature myeloma plasma cells indicates that the mature myeloma tumor cells are the end product of a continuous differentiation process. This is further supported by reports that the phenotypically diverse myeloma cell populations are functionally distinct. Whereas immature (CD38+ CD45 or CD38+ +CD49E-) myeloma plasma cells express interleukin-6 (IL-6) and its receptor, the mature (CD38' 'CD45- or CD38+CD49E+) cells do not express IL-6. Also, in most cases IL-6 receptor expression cannot be detected in mature myeloma cells, even by the sensitive reverse transcription polymerase chain reaction (RT-PCR).' Furthermore, this difference in IL-6 receptor expression translates to differences in response to 1L-6. Whereas the immature myeloma cells respond to IL-6 by increased proliferationz6and are protected by the cytokine from dexamethasone-induced apoptosis, IL-6 does not affect mature myeloma cells in a similar fashion.19 The proliferative activity (BUdR labeling index) of myeloma plasma cells decreases with the level of maturation, in inverse correlation with CD45 expression.25These functional differences between phenotypically distinct populations of myeloma plasma cells, joined by morphologic observations,3l lend strong support to the notion that myeloma plasma cells are the product of a continuous differentiation process of an earlier proliferative progenitor. +

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PHENOTYPIC SUGGESTION OF B-CELL INVOLVEMENT

The notion that the differentiation process that produces the myeloma plasma cell starts from a B lymphocyte is supported by experimental data. Anti-idiotype antibodies generated against IgA myeloma proteins were used to trace the myeloma clone down the B-cell differen-

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tiation stages. These experiments confirmed the involvement of B-lymphocyte precursors in the malignant process. The experiments also indicated the presence of expanded pools of cells of the malignant clone expressing all major heavy-chain immunoglobulin isotypes, suggesting that pre-switch lymphocytes are involved in the process.28Involvement of CD10-expressing pre-B cells was also proposed by other investiga2o Involvement of a diploid CD10-expressing component in aneutor~.'~, ploid myeloma indicated that the aneuploid myeloma clone arises from involved, CD10-expressing diploid progenitors."j The presence of a conSinuous differentiation process within the myeloma clone has also been mggested from the expression of CD45 isoforms and adhesion molecules on circulating B cells in myeloma patients." 24 The potential role of B cells in the myeloma disease process is further supported by functional studies demonstrating their ability to differentiate into plasma cells expressing the M protein isotype. Lymphoid precursors of malignant plasma cells were identified in the bone marrow of myeloma patients. These cells could be induced to differentiate into plasma cells by phorbol ester.'O Circulating B cells were induced to differentiate into monotypic plasma cells by cytokine6 33 or by the bone marrow stromal en~ironment.~ During this process of differentiation, the lymphocytes undergo phenotypic changes, assume the phenotype of mature B cells, and express CDllb. Upon co-culture with bone marrow stromal cells, these mature B cells transform into monotypic plasma cells.% Although not conclusive, these observations and similar reports by other investigators not cited here lend support to the notion that myeloma is a malignant process that begins in the earliest stages of Blineage commitment and ends with the recognizable myeloma plasma cell. MOLECULAR DATA IN SUPPORT OF B CELLS

Additional support for clonal involvement of early B lymphocytes in myeloma comes from several investigative groups who employed genetic tools to address this fundamental issue. Circulating B lymphocytes had the same mutated ras oncogene as did the myeloma plasma cells? Also, in patients whose myeloma cells had a deletion of the retinoblastoma (Rb) gene,'3 circulating B lymphocytes had only one Rb allele (Dao and Epstein, unpublished comments). The specific immunoglobulin heavy chain VDJ rearrangement (CDR3) is an unequivocal clonal marker for myeloma cells (and all other clones of the B lineage). The sequence of the CDR3 has been used to identify myeloma cells and to estimate their abundance by allele specific oligonucleotide-PCR,8 and to demonstrate the clonal involvement of circulating B cells (defined by CD19 expression) in myeloma." ASO-PCR was also used to identify the constant region of the immunoglobulin heavy chain associated with the clonal CDR3 in myeloma patients.

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Regardless of the myeloma protein isotype, the myeloma clone was traced down the differentiation ladder to pre-switch B cells.7,l2 These findings confirm earlier speculations that were based on isotypic diversity of idiotypically identical cells.28 Thus, sensitive genetic approaches prove unequivocally that cells that belong to the same B cell clone as do the myeloma plasma cells span the whole spectrum of B-cell differentiation. Whether the clonal pre-immunoglobulin switch B cells or any other B cells with the same clonality as the myeloma cells are tumor cells still needs to be determined. Whereas it is more than likely that the proliferative myeloma cell is less differentiated than the recognizable myeloma plasma cell, its phenotype is yet to be identified. IMPLICATIONS FOR TREATMENT

The phenotypic heterogeneity of the myeloma clone presents specific challenges to the clinician. Myeloma cells of different maturation levels differ in the expression of cytokines, cytokine receptors, and 22, 26 This functional heterogeneity has direct response to cytokine~.'~, consequences in the clinical arena. Dexamethasone is frequently used, alone or together with other drugs, to treat myeloma. When used alone in previously untreated patients, dexamethasone induces rapid reduction in tumor burden and correction of disease manifestations. Nevertheless, cures are not achieved.', We and others have reported that IL-6 can protect myeloma cells from dexamethasone toxicity.21,29 More recently we have demonstrated that the bone marrow environment protects dexamethasone-sensitive myeloma cells in the bone marrow from dexamethasone-induced apoptosis. This protection is restricted to the immature myeloma ~el1s.l~ These findings explain the inability of dexamethasone to produce cures. Whereas the mature, IL-6 nonresponsive myeloma cells are eliminated, resulting in reduction in tumor mass, the immature cells are rendered dexamethasone-resistant by cytokines in their environment. Similarly, it is likely that mature and immature myeloma cells respond differently to a variety of drugs used for the treatment of the disease. Studies need to define conditions and strategies for the design of treatment regimens that address the potential differences in drug sensitivity between mature myeloma cells and their immature progenitors. In the case of dexamethasone, possibly the addition of IL-6 antagonists14,32 or IL-6 neutralizing antibodies3 could provide the answer. These two modalities of neutralizing IL-6 activity could by themselves provide a tool for controlling progression of this IL-6dependent ne0plasia.3~More specific targeting of treatment to eliminate myeloma progenitors is hindered by the fact that their phenotype has not been identified. ASO-PCR is a highly sensitive assay for the detection of myeloma clonal cells. It can be used for the detection of minimal residual disease, so that the frequency and value of attaining complete remissions in

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sensitive molecular terms can be evaluated. The findings that myeloma clonal expansion can be traced to pre-immunoglobulin switch B cells present a special dilemma, however. The significance of these cells to the myeloma disease process is far from clear. Therefore, their detection by ASO-PCR could present a problem in interpreting the results from studies of minimal residual disease. The notion that myeloma arises from early B-cell progenitors posts rpecial dilemmas in the areas of autologous bone marrow transplantation. The potential of returning a significant quantity of myeloma stem ells has been recognized. The advent of autologous peripheral stem cell transplantation is subject to the same concerns.37,38 It has been demonstrated that during peripheral stem cell mobilization, myeloma dona1 cells, defined by ASO-PCR, also are mobilized.18 Although the kinetics of mobilization of CD34 cells appears to differ from that of myeloma clonal cells, this difference is not qualitative. The possibility that the clonal cells among the harvested stem cells are myeloma cell progenitors mandates complicated purification steps for elimination of contaminating clonal cells.17The complexity of the issue is amplified by the controversy over whether the immature clonal cells express CD34.4,36

SUMMARY The phenotypic heterogeneity of myeloma cells in fact delineates a differentiation process that appears to be an integral part of the disease process. Immature myeloma cells interact with their microenvironment differently than do the more mature cells. As a result of this interaction, the immature cells display different responses to chemotherapy than do the mature cells. Addressing this issue by tailoring treatment to target immature as well as mature myeloma cells may change dramatically the outcome of treatment. The ability to define the myeloma clone by molecular genetic techniques has markedly increased the ability to detect clonal cells. This technique provides a most sensitive tool for monitoring elimination of tumor cells; however, the role of the early clonal B cells identified through the use of ASO-PCR in the disease process needs to be clarified. Currently, a great deal of effort is directed towards development of treatment protocol that will eliminate all clonal cells, and a method for purging clonal cells from harvested mobilized peripheral stem cells. Understanding the biologic significance of early clonal B cells in myeloma will allow for a more rational approach to curative treatment.

References 1. Barlogie B, Epstein J: Multiple myeloma: Biology and therapy. J Cancer Res Clin Oncol 116109, 1990

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2. Barlogie B, Epstein J, Selvanayagam P, Alexanian R Plasma cell myeloma-new biological insights and advances in therapy [review]. Blood 73865,1989 3. Bataille R, Barlogie B, Lu ZY, et al: Biologic effects of anti-interleukin-6 murine monoclonal antibody in advanced multiple myeloma. Blood 86685,1995 4. Belch AR, Bergsagel PL, Szczepek A, et al: CD34+ B-cells in the blood of patients with multiple myeloma express clonotypic IgH sequences. Blood 84:A 385, 1994 5. Bergsagel PL, Belch AR, Pilarski LM: The blood B cells and bone marrow plasma cells in a patient with multiple myeloma include cells with the same N-ras mutation. Blood 84(suppl 1):524a, 1994 6. Bergui L, Schena M, Gaidano G, et al: Interleukin 3 and interleukin 6 synergistically promote the proliferation and differentiation of malignant plasma cell precursors in multiple myeloma. J Exp Med 170:613, 1989 7. Billadeau D, Ahmann G, Greipp P, Van Ness 8: 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 178:1023, 1993 8. Billadeau D, Blackstadt M, Greipp P, et al: Analysis of B-lymphoid malignancies using allele-specific polymerase chain reaction: A technique for sequential quantitation of residual disease. Blood 78:3021, 1991 9. Caligaris-Cappio F, Bergui L, Gregoretti MG, et a 1 Role of bone marrow stromal cells in the growth of human multiple myeloma. Blood 77:2688, 1991 10. Caligaris-Cappio F, Bergui L, Tesio L, et al: Identification of malignant plasma cell precursors in the bone marrow of multiple myeloma. J Clin Invest 76:1243, 1985 11. Chen BJ, Epstein J: Circulating clonal lymphocytes in myeloma constitute a minor subpopulation of B cells. Blood 871972, 1996 12. Corradini P, Boccadoro M, Voena C, Pileri A: Evidence for a bone marrow B cell transcribing malignant plasma cell VDJ joined to C mu sequence in immunoglobulin (1gG)- and IgA-secreting multiple myelomas. J Exp Med 178:1091, 1993 13. Dao DD, Sawyer JR, Epstein J, et al: Deletion of the retinoblastoma gene in multiple myeloma. Leukemia 8:1280, 1994 14. de Hon FD, Elders M, Rose-John S, et al: Development of an interleukin (IL) 6 receptor antagonist that inhibits IL-Mependent growth of human myeloma cells. J Exp Med 1802395, 1994 15. Durie BG, Grogan TM: CALLA-positive myeloma: An aggressive subtype with poor survival. Blood 66:229, 1985 16. Epstein J, Barlogie 8, Katzmann J, Alexanian R Phenotypic heterogeneity in aneuploid multiple myeloma indicates pre-B cell involvement. Blood 71:861, 1988 17. Gazitt Y, Reading CC, Hoffman R, et al: Purified CD34+ Lin- Thy+ stem cells do not contain clonal myeloma cells. Blood 86:381, 1995 18. Gazitt Y, Tian E, Barlogie €3, et al: Differential mobilization of myeloma cells and normal hematopoietic stem cells in multiple myeloma after treatment with cyclophosphamide and granulocyte-macrophage colony-stimulating factor. Blood 87805, 1996 19. Grigorieva I, Woodliff J, MacLeod SL, et al: Interleukin-6 negates the cytotoxicity of dexamethasone in myeloma [meeting abstract]. Proc Annu Meet Am Assoc Cancer Res 36:A128, 1995 20. Grogan TM, Durie BG, Lomen C, et al: Delineation of a novel pre-B cell component in plasma cell myeloma: Immunochemical, immunophenotypic, genotypic, cytologic, cell culture, and kinetic features. Blood 70:932, 1987 21. Hardin J, MacLeod S, Grigorieva I, et al: Interleukin-6 prevents dexamethasoneinduced myeloma cell death. Blood 84:3063, 1994 22. Hata H, Xiao H, Petrucci MT, et al: Interleukin-6 gene expression in multiple myeloma: A characteristic of immature tumor cells. Blood 81:3357, 1993 23. Jensen GS, Mant MJ, Belch AJ, et al: Selective expression of CD45 isoforms defines CALLA + monoclonal B-lineage cells in peripheral blood from myeloma patients as late stage B cells. Blood 78:711, 1991 24. Jensen GS, Mant MJ, Pilarski L M Sequential maturation stages of monoclonal B lineage cells from blood, spleen, lymph node, and bone marrow from a terminal myeloma patient. Am J Hematol41:199, 1992 25. Joshua D, Petersen A, Brown R, et al: The labelling index of primitive plasma cells

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determines the clinical behaviour of patients with myelomatosis. Br J Haematol94:76, 1996 26. Kawano MM, Huang N, Harada H, et al: Identification of immature and mature myeloma cells in the bone marrow of human myelomas. Blood 82:564, 1993 27. Kawano MM, Mahmoud MS, Huang N, et al: High proportions of VLA-5-immature myeloma cells correlated well with poor response to treatment in multiple myeloma. Br J Haematol91:860,1995 28. Kubagawa H, Vogler LB, Capra JD, et al: Studies on the clonal origin of multiple myeloma. Use of individually specific (idiotype) antibodies to trace the oncogenic event to its earliest point of expression in B-cell differentiation. J Exp Med 150:792,1979 29. Lichtenstein A, Tu Y, Fady C, et a1 Interleukin-6 inhibits apoptosis of malignant plasma cells. Cell Immunol 162248, 1995 30. Omede P, Boccadoro M, Fusaro A, et al: Multiple myeloma: ‘Early’ plasma cell phenotype identifies patients with aggressive biological and clinical characteristics. Br J Haematol 85:504, 1993 31. Sailer M, Vykoupil KF, Peest D, et al: Prognostic relevance of a histologic classification system applied in bone marrow biopsies from patients with multiple myeloma: A histopathological evaluation of biopsies from 153 untreated patients. Eur J Haematol 54:137,1995 32. Savino R, Ciapponi L, Lahm A, et al: Rational design of a receptor super-antagonist of human interleukin-6. EMBO J 13:5863, 1994 33. Sawamura M, Murakami H, Tamura J, et a 1 Tumour necrosis factor-alpha and interleukin 4 promote the differentiation of myeloma cell precursors in multiple myeloma. Br J Haematol 88:17, 1996 34. Thomas X, Xiao HQ, Chang R, Epstein J: Circulating B lymphocytes in multiple myeloma patients contain an autocrine IL-6 driven pre-myeloma cell population. Curr Top Microbiol Immunol 182201, 1992 35. van Oers MH, van Zaanen HC, Lokhorst H M Interleukin-6, a new target for therapy in multiple myeloma? [review]. Ann Hematol 66219, 1993 36. Vescio RA, Hong CH, Cao J, et al: The hematopoietic stem cell antigen, CD34, is not expressed on the malignant cells in multiple myeloma. Blood 843283, 1994 37. Vesole DH, Jagannath S, Tricot G, et al: Autologous bone marrow and peripheral blood stem cell transplantation in multiple myeloma. Cancer Invest 14:378, 1996 38. Vesole DH, Tricot G, Jagannath S, et a1 Autotransplants in multiple myeloma-what have we learned? Blood 88:838, 1996

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