Document rBa3BKe2oq5oZOJGgwLbNg0bE

Phenotypic Heterogeneity in Aneuploid Multiple Indicates Pre-B Cell Involvement Myeloma By Joshua Epstein, Bart Barlogie, Jerry Katzmann, and Raymond Alexanian The expression of early and mature B cell markers, surface fl2-microglobulin (B2M) and cytoplasmic immunoglobulin (clg) by aneuploid tumor cells in bone marrow aspirates from 44 patients with multiple myeloma was evaluated by correlated DNA immunofluorescence flow cytometry. Myeloma tumor cells of almost 90% of the patients con- tamed monoclonal clg and expressed the mature plasma cell antigen Ri -3 as well as surface B2M; common acute lymphoblastic leukemia antigen (CAiiA) was present in 55%, B2 in 1 7%, and 84 in 23% of samples studied. M ULTIPLE MYELOMA is a B cell malignancy typi- cally associated with mature plasma cell morphology and monoclonal immunoglobulin secretion . Patient survival is inversely related to the tumor mass at diagnosis, which can be estimated from a number of laboratory parameters' or, as reported more recently, from the serum level of fl2-micro- globulin (B2M).2 The neoplastic involvement of earlier B cells has been suggested by the presence of idiotype- concordant and common acute lymphoblastic leukemia anti- gen (CALLA)-positive lymphoid cells in the blood of patients with multiple myeloma.35 CALLA+ lymphocytes from blood and bone marrow could be differentiated in vitro to mature plasma cells with concordant monoclonal immuno- globulin isotype expression and secretion.6 Recently, an aggressive clinical course has been described in myeloma with CALLA+ plasma cells.7 Our laboratory has investigated myeloma cellular features as a means of understanding the biology and the clinical course of the disease. Methods were devefoped for quantita- tive assessment of B cell and other cellular markers in relationship to DNA aneuploidy, which serves as an unequiv- ocal tumor cell marker present in about 80% of patients with plasma cell myeloma.8 We now report our studies on the expression of early and mature B cell markers by tumor cells in the bone marrow from 44 patients with aneuploid myelo- ma. MATERIALS AND METHODS Bone marrow aspirates were obtained from 44 patients with DNA-aneuploid myeloma in various stages of disease; ten of the patients were previously untreated. Informed consent was obtained from the patients before bone marrow aspiration. After Ficoll- Hypaque separation (density, 1,077 g/mL), interphase cells were washed in phosphate-buffered saline and reacted with a panel of monoclonal antibodies to surface antigens by using either direct or indirect immunofluorescence assays. After immunostaining, the cells were fixed in ice-cold 70% ethanol and counterstained with propidium iodide at 20 zg/mL for correlated DNA phenotypic analysis, which was carried out with an EPICS V flow cytometer (Coulter Electronics, Hialeah, FL).9 The investigated surface mark- ers included the pre-B cell antigen CALLA, which was studied by using the monoclonal antibody iS'#{17B6}2;, which is expressed by early to mature nonactivated B cells"; B4, which is expressed by all B cells but not by mature plasma cells'2"3 (Coulter); and the plasma cell marker RI-3, which was kindly provided by Dr iA. Katzmann (Mayo Clinic, Rochester, MN).5"4 In addition, cytoplasmic immu- noglobulin (clg) was also analyzed by using both anti-K and anti-A Coexpression of CALiA and clg in 46% of all patients identified a novel myeloma phenotype without known counterpart in the normal differentiation of B cells. CAiiA and clg were independently expressed and gave rise to CAiiA+ /clg-, CAiiA+ /clg+, and CAiiA- /clg+ cells. The association of CALiA and mature plasma cell markers may define discrete stages of neoplastic plasma cell differ- entiation. a 1988 by Grune & Stratton, Inc. light chain antisera [F(ab')2 fragments; Cappel Laboratories, West Chester, PA].'5 We also studied surface expression of B2M (Dako, Santa Barbara, CA). which is secreted in proportion to tumor mass.2 Separate aliquots of all samples were also subjected to acridine orange staining for correlated analysis 01 DNA and RNA content as previously described.'6 Routinely, 10,000 cells were analyzed for each marker. Quantitative analysis was carried out with a Terak computer (Scottsdale, AZ) using cells stained with nonreactive antibodies as matching controls. RESULTS An example of DNA phenotype analysis in a patient with a hyperdiploid myeloma is presented in Fig 1 . 1-lyperdiploid tumor cells showed a high RNA content; reacted positively with anti-B2M, J5, and R1-3; and contained cIgA with monoclonal K light chain reactivity. Cells in the diploid cell compartment had a low RNA content, did not express clg, but were positive for B2M and iS. Because of scarcity of marrow samples, complete studies could be performed in 65% of the cases. Monoclonal clg and R I -3 were present in aneuploid tumor cells of 88% and 89% of the patients, respectively. The pre-B cell antigen CALLA was expressed by aneuploid myeloma tumor cells in 55% of 43 samples studied for this marker (Table 1). Phenotype coexpression by aneuploid tumor cells was observed in 80% of patients for monoclonal clg, Rl-3, and B2M, and in 45% of the patients, the pre-B cell antigen CALLA was present together with monoclonal clg and the mature plasma cell antigen Rl-3 (Fig I and Table 2). Only a few patients (13% and 15%, respectively) demonstrated coexpression of CALLA with B2 and B4 antigens. There was no correlation between the From the Department of Hematology. University of Texas Sys- tem Cancer Center, M.D. Anderson Hospital and Tumor Institute. Houston, and the Mayo Clinic, Rochester, MN. Submitted July 29. 1987; accepted November 9, 1987. Supported in part by Grants No. CA 37161. CA 28771. and CA 16672from the National Institutes ofHealth, Bethesda, MD. Address reprint requests to Joshua Epstein, DSc. M.D. Anderson Hospital and Tumor Institute. 15/5 Holcombe Blvd. Box 55. Houston, TX 77030. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact. 1988 by Grune & Stratton. Inc. 0006-4971/88/7104-0002$3.00/0 Blood, Vol 71, No 4 (April), 1988: pp 861-865 861 862 EPSTEIN ET AL r..'".'"..'." ( B2M 1"'' f1 a. C 0 R1-3 `S a "S K aI" 0 C C 1S SS S CIgA S. S. .( ,` a. I a. `S is CIgA 4#{149} a. "S S. . S. 4- S - -, S. S. IS `.S am a. Cellular DNA Content Fig 1 . Phenotypic heterogeneity in a patient with hyperdiploid multiple myeloma. Hyperdiploid cells show a high RNA content; react with anti-B2M. J5. and Ri -3; and show monoclonal clgA K expression. Diploid cells have low RNA contents without monoclonal clg reactivity but express B2M and CALLA. Abscissa. DNA content; ordinate, fluorescence intensity of cellular feature. proportions of tumor cells that stained positively for CALLA, R1-3, B2M, or clg in patients whose cells were positive for these markers, thus indicating their independent expression in plasma cell myeloma (Fig 2). Seven of the 44 patients displayed an additional diploid tumor DNA stem- line on clg analysis, a representative example of which is presented in Fig 3. Hyperdiploid cells with a high RNA content and monoclonal clg x were positive for CALLA (iS) and R 1-3; diploid cells also contained clg K, were CALLA- positive, but had a low RNA content and did not express R1-3. Interestingly, in all patients with CALLA+ aneuploid cells, the diploid cells also expressed CALLA. CALLA+ diploid cells were only seen in association with CALLA+ Table 1 . CAiLA Expression Number of patients studied Percentage of positive patients in Aneuploid Myeloma Tumor Cells B4 CALLA 35 43 23 55 B2 R1-3 82M clg 33 40 1 7 89 44 44 90 88 aneuploid cells. Further examination of CALLA/clg expres- sion revealed three different phenotypes, CALLA+/clg-, CALLA+/clg+, and CALLA-/clg+, that are present alone or in combination, at times with different ploidy levels (ie, diploid and hyperdiploid) (Figs 3 and 4). It has been reported that CALLA-positive myeloma has a particularly aggressive course with a short median survival of 6 months.7 The median survival from diagnosis of 2 1 patients with Table 2. Coexpression of Early and Late B Cell Markers Aneuploid Myeloma Plasma Cells (Percentage of Positive Patients) CALLA B2 R1-3 B2M B4 CALLA B2 R1-3 B2M 15 4 19 19 13 44 45 13 17 78 by clg 14 46 20 81 79 CALLA/cIG CO-EXPRESSION BY MYELOMA TUMOR CELLS 863 100 Med A, 80 60 40 20 100 11,2 A 80 60 A M,d ` . . . ... 20 A, Med 0 U Fig 2. Independent expression of cellular markers by myeloma tumor cells. The percentages of cells positive for each marker in bone marrow samples coexpressing these markers are plotted against each other. The correlation coefficient for each plot was < .5. 0 20 40 60 80 100 CALLA 100 M.d A, 80 60 40 S 20 A M.d 0 20 40 60 80 100 81-3 PERCENT POSITIVE 0 20 40 60 80 100 CALLA 100 M.d A, 80 60 40 .` .. . 20 A, Med 0 20 40 60 80 100 81-3 CALLA-positive mycloma was significantly longer than that of 23 patients with CALLA-negative disease (>80 v 24 months; P .004) (Fig 5). DISCUSSION Expression of several differentiation-associated B cell markers and surface B2M by tumor cells was evaluated in the bone marrow of 44 patients with DNA-aneuploid multiple myeloma by using correlated DNA and immunofluorescence flow cytometry. Tumor cells are unequivocally identified by DNA aneuploidy, which is present in more than 80% of all myeloma patients.8 The tumor cells of about 90% of the patients reacted with the mature plasma cell marker Rl-3, expressed surface B2M, and contained clg. Unexpectedly. however, tumor cells ofone halfofthe patients expressed the pre-B cell antigen CALLA together with the mature plasma cell features R I -3 and clg, recently also reported by Grogan et al.'7 Coexpression of CALLA and clg has no known counterpart in normal B cell differentiation and could repre- sent a unique tumor cell phenotype in myeloma. In casesofconcurrent CALLA and clgor Rl-3 reactivity, these markers were expressed independently of each other and represented CALLA +/clg-, tumor cell subpopulations that were either CALLA +/clg+ . or CALLA -/clg+. Although none of the CALLA-negative aneuploid cases showed iS reactivity in DNA-diploid cells, all cases with C 0 0, 0, 0, w 0, 0. 2 0, 60 40 Fig 3. Phenotypic heterogeneity expressed CALLA (J5). Hyperdiploid Cellular DNA Content in a biclonal myeloma with two DNA stemlines. Both diploid and hyperdiploid cells also reacted with Ri -3. whereas diploid cell expressed surface B2M. cells contained clg and 864 EPSTEIN ET AL CALLA --- .II r-'- r ` - -. a. -( - . - - - ` - -- , - -. - - -. - - a. .. 50 "S "S a. a. C,) as 55 z Ui S. z Ui --; o.-- -.:o---ai- 0 z Ui 0 Cig Ui es 0 1#{149} a.t:Ss a. S S --`:: r ` -- - 1- - - ` - Lai- -` I I. a. , as .::tr JF4SIfJ_! .. 6.5 -J LA. I.-...-.- `C `C - -- i: a. .. - a. a. _,#1.#{149}$ - -. a. as -C.- I. - 45 . as is i;. is a. S S #{243}s is ;S CS DNA CONTENT Fig 4. Heterogeneity of CALLA expression in aneuploid myeloma. CALLA + + /clg - + / (c) CALLA + + /clg + 4- . + . indicates the presence hyperdiploid cells. respectively. Three phenotypes of cellular marker; were observed: (a) CALLA - - /clg - + ; (b) - . the absence of marker for the diploid and CALLA expression by aneuploid tumor cells also showed expression of this pre-B cell marker by DNA-diploid cells, but usually without clg or RI -3. This phenotypic heterogene- ity with the coexistence of CALLA+/clg-, CALLA+/ clg+, and CALLA-/clg+ aneuploid tumor cell compart- ments and the additional presence of a CALLA+/clg- diploid compartment may reflect discrete stages in the development ofmultiple myeloma: CALLA+/clg- precur- sor cells mature progressively to CALLA+/clg+ and then to the expected mature plasma cell phenotype CALLA-/ clg+. The presence ofa diploid CALLA+/clg- cell popu- C 0 0 0. 0 0. 0 10 20 30 40 50 60 70 80 90 Months Fig 5. Superior survival of CALLA + myeloma pared with CALLA - patients. patients com- lation in all and only in aneuploid CALLA+ myelomas suggests the existence of a diploid tumor progenitor cell compartment. DNA aneuploidy may therefore be distal to more subtle genetic changes associated with malignant transformation. Such a model could explain the emergence, in some patients, of new ploidy levels (eg, hypodiploidy in a hyperdiploid myeloma) as the disease advances'8 and would imply that clonal changes during the course of the disease result from adaptation in response to selective pressures exerted by chemotherapy given at subcurative dose. The concept of pre-B cell involvement in the development of myeloma is supported by cytogenetic as well as molecular genetic studies: lymphomalike translocations [t(8;14) and t(l l;14)] were found in patients with multiple myeloma'9; and c-myc and Bc/I involvement have also been recently reported.#{176}2' These findings suggest that the malignant transformation in multiple myeloma occurs at an early stage of B cell maturation. The recently reported finding of T cell receptor `y gene rearrangements in three patients with plasma cell myeloma22 also raises the possibility that the malignant transformation in multiple myeloma occurs even proximal to the commitment to lymphoid lineage differentia- tion. CALLA expression by myeloma cells has been noted in association with an aggressive course and short survival.7 Even though obtained from patients at different stages of their diseases, our results are at variance with this report and, in fact, seem to indicate a more favorable clinical course in CALLA + myeloma, perhaps as a result of different chemo- therapy (vincristine, Adriamycin, dexamethasone), with marked efficacy also in CALLA-positive acute lymphoblas- tic leukemia in adults.23 CALLA/cIG CO-EXPRESSION BY MYELOMA TUMOR CELLS 865 ACKNOWLEDGMENT The authors wish to express their most sincere thanks to Kim Teague for her excellent technical assistance and to Eva Menefee for her secretarial assistance. REFERENCES .I Dune BGM, Salmon 5: A clinical staging system for multiple myeloma. Cancer 36:842, 1975 2. Alexanian R, Barlogie B, Fritsche H: Beta-2-microglobulin in multiple myeloma. Am J Hematol 20:345, 1985 3. Mellstedt H, MoIm G, Peterson D, Peest D: Idiotype-bearing lymphoid cells in plasma cell neoplasia. Clin Haematol I 1:65, 1982 4. Pilarski LM, Mant Mi, Ruether BA: Pre-B cells in peripheral blood ofmultiple myeloma patients. Blood 66:416, 1985 5. Ruiz-Arguelles GJ, Katzmann JA, Greipp RR, Gonchoroff NJ, Garton JP, Kyle RA: Multiple myeloma: Circulating lympho- cytes that express plasma cell antigens. Blood 64:352, 1984 6. Caligaris-Cappi F, Janossy GK, Bergui L, Tesio L, Pizzolo G, Malavasi F, Chilosi M, Campana D, van Camp B, Gavosto F: Identification of malignant plasma cell precursors in the bone marrow of multiple myeloma. J Clin Invest 76:1243, 1985 7. Dune BGM, Grogan TM: CALLA-positive myeloma: An aggressive subtype with poor survival. Blood 66:229, I 985 8. Latreille J, Barlogie B, Dosik G, Johnston DA, Drewinko B, Alexanian R: Cellular DNA content as a marker of human multiple myeloma. Blood 55:403, 1980 9. Look AT, Melvin SC, Williams DL, Brodeur GM, Dahl GV, Kalwinski DK, Murphy SB, Maura M: Aneuploidy detected by flow cytometry correlates with cell phenotype in childhood acute leuke- mia. Blood 60:959, 1982 10. Greaves Ml-, Hariri G, Newman RA, Sutherland DR. Ritter MA, Ritz J: Selective expression of the common acute lymphoblastic leukemia (gp 100) antigen on immature lymphoid cells and their .malignant counterparts. Blood 61:628, 1983 1 1 Nadler LM, Stashenko P. Hardy P. Van Agthoven A, Ter- horst C, Schlossman SF: Characterization of human B cell-specific antigen (B2) distinct from BI. J Immunol 126:1941, 1981 12. Nadler LM, Anderson KC, Marti G, Bates M, Park E, Daley JF, Schlossman SF: B4, a human B lymphocyte-associated antigen expressed on normal mitogen-activated and malignant B lympho- cytes. J Immunol 131:244, 1983 13. Foon KA, Todd RF III: Immunologic classification of leuke- mia and lymphoma. Blood 68:1, 1986 14. Gonchoroff NJ, Katzmann JA, Ruiz-Arguelles GJ, Greipp PR, Kyle RA: Development of mouse monoclonal human plasma cells. Hybridoma 2:130, 1983 antibodies to 15. Barlogie B, Alexanian R, Pershouse M, Smallwood L, Smith L: Cytoplasmic immunoglobulin content in multiple myeloma. JNCI 76:765, 1985 16. Barlogie B, Alexanian R, Dixon D, Smith L, Smallwood L, Delasalle K: Prognostic implications of tumor cell DNA and RNA content in multiple myeloma. Blood 66:338, 1985 17. Grogan T, Dune B, Lomen C, Spier C, Wirt D, Nagle R, Wilson G, Richter L, Vela E, Maxey V. McDaniel K, Rangel C: Delineation ofa novel pre-B cell component in plasma cell myeloma: Immunochemical, immunophenotypic, genotypic, cytologic, cell cul- ture and kinetic features. Blood 70:932, 1987 18. Barlogie B, Alexanian R: Cellular aspects of myeloma: Bio- logic and clinical implications, in Delamore 1W (ed): Multiple Myeloma and Other Paraproteinemias. Edinburgh, Churchill Liv- ingstone, 1986, p 154 19. Gould J, Goodacre A, Pathak S. Alexanian R, Barlogie B: Karyotype correlations with clinical features in multiple myeloma. Blood 68:209, 1986 (suppl I) (abstr 716) 20. Selvanayagam P. Blick M, Narni F, van Tuinen P. Ledbetter D, Alexanian R, Saunders G, Barlogie B: Alteration and abnormal expression of the c-myc oncogene in human multiple myeloma. Blood7l:30, 1988 21. Selvanayagam P. Goodacre A, Strong L, Saunders GF, Barlogie B: Alterations of bcl- 1 oncogene in human multiple myelo- ma. Proc Am Assoc Cancer Res 28:19(1/76), 1987 22. Lee MS. Selvanayagam P. Alexanian R, Stass 5, Barlogie B: Rearrangements (REARR) of T-cell receptor (TcR) gamma chain gene in multiple myeloma. Proc Am Assoc Cancer Res 28:1 78, 1987 (abstr 706) 23. Walters R, Kantarjian H, Dixon D, Keating M, Estey E, Beran M, Anderson B, McCredie K, Freireich E, Barlogie B: M.D. Anderson experience with acute lymphocytic leukemia. Proc Am Soc Clin Oncol 6: 152, 1 987 (abstr 596)