Adhesion Molecules on Human Myeloma Cells: Significant Changes in Expression Related to Malignancy, Tumor Spreading, and Immortalization1

[CANCER RESEARCH 55, 3647-3653, August 15, 1995] Adhesion Molecules on Human Myeloma Cells: Significant Changes in Expression Related to Malignancy, Tumor Spreading, and Immortalization1

Catherine Pellat-Deceunynck, Sophie Barille, Denis Puthier, Marie-JosÃ©Rapp, Jean-Luc Harousseau, RÃ©gisBataille, and Martine Amiot2

Laboratoire d'OneogÃ©nÃ¨seItnmiino-HÃ©matologiqiie [C. P-D., S. B., D. P., R. B.. M. A.], Inxertn i/2//. InstituÃ de Biologie IM. A.], 9 Quai Moncou\u and Departement il'HÃ©matologie IM-J. K.. J-L H.Â¡.HÃ´tel Dieu. 440ÃŒSNantes Cedex 01. France

ABSTRACT ment. Adhesion of HMCLs to BM stromal cells induced the secretion of IL-6 by BM stromal cells (11-13); IL-6 is the essential MM cell In order to evaluate putative changes of major adhesion molecule growth factor (14). The family of ÃŸland ÃŸ2integrin molecules expression on plasma cells (PCs) associated with malignant transforma contributed to these interactions, because mAbs against CD29, tion, tumor spreading, and immortalization, we have quantified and com CD49d, and CDlla inhibited the induction of IL-6 secretion in the pared the expression of CD56, CD44, CDlla, CD49e, and CD45 RO/RA on normal PCs, malignant PCs from multiple myeloma patients in chronic coculture of PCs with stromal cells (11, 12). The role of CD44 has phase, in accelerated phase with or without extramedullary progression, been established in different systems: in mice the growth of primary and from human myeloma cell lines. Plasma cell phenotype was defined plasmocytomas was shown to be dependent on CD44 physical contact with the use of two-color immunofluorescence in combination with li-li, with stromal cell feeder layers (15); and in humans the blockage of the or anti-CD38 antibodies. We found that all the adhesion antigens were CD44 interaction partly inhibited the adhesion of myeloma cells to expressed on normal PCs. Malignancy was characterized by an overex- stromal cells and the triggering of IL-6 by stromal cells (12). In the pression of CD56, whereas extramedullary spreading was associated with a dramatic down expression of CD56. Although ('1)44 remained un same context, we demonstrated recently that the interaction of my eloma cells with osteoblastic cells up-regulated the IL-6 secretion by changed, the subpopulation of PCs expressing CDlla, CD49e, and osteoblastic cells through cell-cell contact.4 In addition to the inter CD45RA/RO were significantly reduced during malignancy, and each of these negative subpopulations increased during disease acceleration. We actions through CD49d and CD44, we provided evidence for CD56 demonstrated that CDlla and CD49e expression were correlated and involvement in the cellular contact between osteoblasts and myeloma defined the same subpopulation of PCs. The phenotype of HMCLs was cells. This observation is of interest because it has frequently been similar to the expression profile of patients in accelerated phase with hypothesized that the lack of CD56 expression might play a role in the extramedullary spreading. In conclusion, we show that significant changes dissemination of the cells out of the BM (4, 16). All these data of PC phenotype were associated with malignancy, were correlated with emphasized the critical role of the adhesion molecules expressed on the disease evolution, and could be of diagnostic and prognostic value in myeloma cells for the cell-cell contact with the BM microenviron- individuals with monoclonal gammopathy and patients with multiple ment. Moreover, it is conceivable that changes in the density or in the myeloma. expression of these adhesion molecules could make possible the dissemination of PCs out of the BM. In that way, it appears important INTRODUCTION to quantify the changes in such major adhesion molecule expression in relation to malignant transformation, tumor spreading, and immortal MM3 is characterized by the proliferation of malignant PCs within ization. the BM. The mechanisms by which malignant PCs migrate and localize in the BM have not been yet elucidated. Various studies have documented the expression of adhesion molecules on myeloma cell MATERIALS AND METHODS surface (1-6). A high expression of CD44, CD29, CD49d (VLA-4), and CD54 has been reported on normal and malignant PCs, in contrast Patients. The expression of adhesion molecules has been studied in the BM to CD56 (NCAM) and CD58 (LFA-3), which were rather expressed of 49 patients with MM: they were 34 IgG, 11 IgA, and 4 pure Bence Jones only. The x:A ratio was 1.28. Twenty-seven patients have been studied either on malignant PCs than on normal PCs. The expression of CDlla at diagnosis (n = 23) or in the remission and plateau phase (;/ = 4). The (LFA-1) on malignant PCs was shown to be associated with high diagnostic criteria of MM were those of the Southwest Oncology Group (San tumor cell labeling index (7). On the other hand, Kawano et al. (8) Antonio, TX) (17). Sixteen patients have been studied at the time of relapse or have demonstrated that the expression of CD49e (VLA-5) distin guished two subpopulations, the CD49e+ cells, which were mature progressive disease due to primary treatment failures. Progressive disease was myeloma cells, and the CD49e~ cells, which were proliferative im defined as follows: (a) >50% increase of monoclonal immunoglohulin above previous levels; (h) worsening anemia; and (c) occurrence of new lytic bone mature myeloma cells. In addition, the expression of one or another lesions. In vivo, it has been shown that the growth fraction of patients at CD45 isoforms (CD45RA and CD45RO) on PCs was shown to define diagnosis or remission was low, generally <5% (18). On the other hand, the different stages of differentiation, CD45RO being expressed on the growth fraction of patients with relapse or progressive disease was signifi most differentiated PCs (9, 10). Among these adhesion molecules, cantly higher, generally >20%. Thus, to simplify the presentation of the results: (a) patients at diagnosis or in remission were pooled and termed as in chronic- recent studies have demonstrated the functional importance of some of them in the interactions of myeloma cells with the BM environ- phase; and (h) patients in relapse or with progressive disease were termed as in accelerated phase. Six patients in accelerated phase were in leukemic phase. They were termed as in the accelerated phase with extramedullary involvement. Received 2/24/95; accepted 6/8/95. The plasmocytc phenotype of MM patienls always established on BM cells The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with was compared to the plasmocytc phenotype of 12 healthy donors. Normal PCs 18 U.S.C. Section 1734 solely to indicate this fact. were analyzed in BM cell populations (9 cases) and in tonsil cell populations ' Supported by Association pour la Recherche sur le Cancer (6285) and the Ligue (3 cases). For all the markers tested, normal PCs presented an identical contre le Cancer de Loire-Atlantique grants. 2 To whom requests for reprints should be addressed. 1The abbreviations used are: MM, multiple myeloma; PC, plasma cell; HMCL. human 4 S. BarillÃ©.M.Collette, R. Bataille, and M. Amiot. Myeloma cells up-regulate IL6 Â¡n myeloma cell line; BM, bone marrow; PE, phycoerythrin; OR, quantum red; IL-6, osteoblastic cells through cell-to-cell contact hut down-regulate osteocalcin production, interleukin 6; mn. median of fluorescence intensity. submitted for publication. 3647

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. ADHESION MOLECULES ON HUMAN MALIGNANT PCS phenotype irrespective of their localization (tonsil or BM) except for CD44 as RESULTS indicated later in the text. Human Myeloma Cell Lines and Culture Conditions. Nine HMCLs To investigate the PC phenotype, we took advantage of the tech were studied: LP1, L363, and OPM2 were purchased from DSM (Braun nical approach that we described previously with the use of two-color schweig, Germany); U266 and RPMI 8226 were obtained from American Type immunofluorescence in combination with B-B4 or anti-CD38 antibod Culture Collection; the XG1-, XG2-, XG4-, XG6-, IL-6-dependent cell lines ies (16). A representative two-color staining of myeloma cells (Fig. were established in the laboratory (19). Cell lines were maintained in RPMI 1A) clearly demonstrated that all B-B4+ cells were located in the 1640 culture medium supplemented with 5% of PCS, 2 mM glutamine, anti CD38 bright fraction and vice versa (see also Fig. 4Â£).We further biotics (100 lU/ml penicillin and 100 fxg/ml streptomycin) and 10 /AM2-ÃŸ- confirmed that B-B4 mAb specifically stained PCs because all B-B4+ mercaptoethanol. For the XG cell lines, 1.5 ng/ml of rIL-6 was added to the cells were cytoplasmic K+ but A~ (the isotype of the monoclonal culture. Cell Preparations. On the occasion of a diagnostic iliac crest puncture, immunoglobulin of this MM patient was IgG/<; Fig. l, B and C). To 5-10 ml of BM aspirates were obtained, and BM mononuclear cells were compare the phenotype of malignant PCs at different stages of the dis isolated by Ficoll-Hypaque centrifugation. Tonsils were obtained from subjects ease, two parameters were analyzed for each patient: (a) the percent undergoing tonsillectomy. Adherent cells were then removed by allowing total age of positive cells in the PC population; and (b) the fluorescence mononuclear cells to adhere to a plastic flask in RPMI 1640-5% PCS for 90 intensity ratio for each specific marker. min at 37Â°Cin 5% CO2 humidified atmosphere; this short adherence did not CD56. Although CD56 has been used to distinguish malignant allow any PCs to adhere to plastic. In addition, BM and tonsil cells from from normal PCs, our recent study indicated that normal PCs ex healthy donors were depleted of T lymphocytes by sheep erythrocyte resetting. pressed CD56 (4, 16). In the present study, we first confirmed that 10 The percentage of PCs in the total cell population ranged from 0.5 to 3% and of 11 healthy donors presented a CD56+ plasma cell subpopulation from 3 to 85% for healthy donors and myeloma patients, respectively. ranging from 10 to 100%. We next compared the percentage of Antibodies. mAbs anti-CD44, anti-CD45RA, anti-CD38 directly conju CD56+-positive cells and the level of CD56 expression on normal and gated to FITC, control IgGl-FITC, and control IgGl-PE were obtained from Immunotech (Marseilles, France). Anti-CD45RO-FITC, anti-CD45RO-PE, malignant PCs. As shown in Fig. 2A, malignancy (chronic and accel and anti-CD lla-FITC were purchased from Dako (Glostrup, Denmark) and erated stage) led to a significant increase of the percentage of CD56+ anti-CD56-PE from Becton Dickinson (Heidelberg, Germany). B-B4-FITC and cells in the PC population. Moreover, the m^ was 12 for normal anti-CD lla-PE were obtained from Innotest (BesanÃ§on, France). Purified donors (n = 11), 52 for chronic MM (n = 25), and 44 for accelerated B-B4 antibody was a gift from Dr J. Wijdenes (Innotherapie, BesanÃ§on, MM (n = 16), indicating that the level of CD56 expression was France). The anti-CDlla mAbs used were MHM24 (FITC conjugated) and significantly enhanced during the malignant process [i.e., normal B-B,, (PE conjugated). The anti-CDlla F8.8 was a gift from Dr A. C. Bloem versus chronic (P < 0.01) or versus accelerated MM (P < 0.01); Fig. (University Hospital, Utrecht, Netherlands). Streptavidin conjugated to PE and to QR were obtained from Immunotech and Sigma, respectively. Purified B-B4 3/1]. No further significant differences could be observed between and CD49e were biotinylated with the use of biotinamidocaproate W-hydroxy- chronic and accelerated phases. However, the most striking finding succinomide ester (Sigma, St. Louis, MO) as described previously (16). was the loss of CD56 expression on BM myeloma cells from patients Phenotypic Analysis of PCs. The two-color immunofluorescence staining presenting an extramedullary involvement [mn = 1; Â«= 6 (P < 0.01); of PCs was done as described previously (16). Cells were analyzed on a Figs. 2A and 3/4]. These data confirmed that a strong association FACScan flow cytometer (Becton Dickinson). existed between the lack of CD56 expression and the spreading of For two-color immunofluorescence, FITC-anti-CD44, FITC-anti-CDlla, malignant PCs out of the BM. FITC-anti-CD45RO, and FITC-anti-CD45RA were associated with PE- CD44. CD44 is a proteoglycan, which was shown to be implicated streptavidin-biotinylated B-B4, whereas anti-CD56-PE and anti-CD49e-biotin with tumor metastasis and extracellular matrix attachment (20-22). A were associated with anti-CD38-FITC. When positive PCs represented >20% large number of related isoforms derived from a single gene has been of the total PC population, we determined the level of positivity for each marker; we divided the mean fluorescence intensity of each marker by the identified for CD44. In this study, to define the CD44 profile of expression on PCs we used an anti-CD44 mAb recognizing all CD44 control staining, thus, defining the fluorescence intensity ratio. For each group of patients the mn ratios for a given marker were determined. isoforms. On normal PCs, CD44 was always expressed on all BM PCs For B-B4, CD Ila and CD49e three-color staining, B-B4-FITC, and anti- (8 of 8 cases), whereas it was expressed on 70% of tonsil PCs (2 of CD lla-PE were associated with anti-CD49e-biotin-streptavidin-QR. For B-B4 3 cases). On myeloma patients, most of malignant PCs expressed and cytoplasmic immunoglobulin two-color staining, cells were first stained CD44 although about 20% of MM patients presented a large CD44- with B-B4-biotin-streptavidin-QR, fixed in PBS 1% paraformaldehyde, per- negative subpopulation (Fig. 2B). Also, 2 of 8 HMCLs were found meabilized in PBS-0.1% Tween 20, then stained with PE-anti-K or PE-anti-X negative for CD44. However, the level of CD44 expression during the in PBS-0.1% BSA-0.02% NaN3- 0.5% Tween, and fixed again in PBS-4% different phases of MM did not show any significant changes (Fig. 35; formaldehyde. mn were 36 and 45 for chronic and accelerated phases, respectively). Statistical Analysis. To determine whether significant modifications of the These results indicated that only 20-25% of MM patients presented a fluorescence intensity ratio or the percentage of positive cells for each given marker occurred during malignancy and during the different stages of MM, the negative PC population for CD44. values were compared with the Wilcoxon rank test. Linear correlations have CD49e. CD49e expression has been shown to distinguish prolifer- been established with the Pearson correlation coefficient. ative immature myeloma cells (i.e., CD49e negative) from mature

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Fig. 1. Specific identification of plasma cells with B-B4 mAb. Human BM cells were isolated from BM aspirates from one MM patient. Two-color analysis of BM cells with B-B4-biotin-streptavidin-QR and anti-CD38-FITC (A), anti-A-PE (B), and anti-K-PE (C) is shown.

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Fig. 2. Expression of adhesion molecules in terms of percentage of Â«T***4Â»ifCOI positive cells on normal and malignant PCs. Horizontal lines, median Ã¼ 60 - 60-oo. -*- of positive cells within each given specific marker and group of 40 - patients. M, medullary; EM, extramedullary. t oK 20-Â«t* Ã• (M) Â¡EM)

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myeloma cells (i.e., CD49e positive). Normal PCs (9 of 10 cases) profile for CDlla to patients in accelerated phases [i.e., a lack of were positive for CD49e on a subpopulation ranging from 35 to 85%. expression (mn = 1); Fig. 3D]. Malignancy led to a strong decrease of the CD49e-positive subpopu CDlla and CD49e Expressions Were Correlated on Malignant lation because the majority of MM patients in chronic or accelerated PCs. Because the proportion of the PC subpopulation expressing phases possessed <35% of positive PCs for CD49e (Fig. 2C). In terms CDlla and CD49e decreased on malignant PCs, we next investigated of the percentage of positive cells, significant decreases were ob whether there was a correlation between these two parameters. On a served when comparing PCs from patients in accelerated phase with large panel of 47 MM patients, a strong positive correlation was found an extramedullary involvement to normal PCs (P < 0.02) or to PCs of between the expression of CDlla and CD49e on malignant PCs in patients in chronic phase (P < 0.1; Fig. 2C). The level of CD49e terms of the percentage of positive cells. The correlation coefficient expression was not significantly different in healthy donors was r = 0.70 (P < 0.001; Fig. 5). (mn = 22), myeloma patients in chronic phase (mn = 16), or in To investigate more directly the coexpression of these antigens on accelerated phase (mn = 20) (Fig. 3C). HMCLs presented a CD49e individual malignant PCs, a three-color immunofluorescence analysis expression profile similar to the patients with an extramedullary was performed with the use of anti-CD 11a, anti-CD49e, and B-B4 involvement (mtl = 1). antibodies. For this purpose, CDlla and CD49e immunofluores- CD lia. As observed for CD49e, CDlla was always expressed in cences were analyzed on the restricted B-B4-positive population. A normal PCs: on a subpopulation of normal PCs (7 of 10 cases) or on healthy donor and three patients have been analyzed, and represent all the PCs (3 of 10 cases; Fig. 2D). Representative CD1 la expression ative cytograms of one of these patients are presented in Fig. 6. As profiles analyzed by two-color immunofluorescence in combination illustrated, most of the PCs (>92%) were either positive or negative with B-B4 antibody are presented in Fig. 4, C and F, for normal and for both CDlla and CD49e. In consequence, very small CDlla" CD49e+ and CDlla"1" CD49e~ subpopulations were observed. Sim malignant PCs, respectively. As shown in Fig. 2D, a significant decrease of the percentage of positive cells (P < 0.01) was correlated ilar results were obtained for the healthy donor and for the two other with malignancy (normal versus chronic P < 0.01). By consequence, patients (data not shown) demonstrating that a close correlation ex the mn decreased from 19 (healthy donors) to 4 (chronic MM) and isted between the CDlla and the CD49e expression on PCs. finally to 1 for accelerated MM (Fig. 2D). Analysis of the mn of CD45RO and CD45RA. Previous studies indicated that the dif CDlla expression during the different phases of the disease could not ferentiation of B cells toward PCs is characterized by the loss of show any significant modifications. HMCLs presented an identical CD45RA isoform expression and the simultaneous acquisition of 3649

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Fig. 3. Expression of adhesion molecules in terms of fluorescence intensity ratio on normal and malignant PCs. Horizontal lines, median of fluorescence intensity ratios within each given specific marker and group of patients (mn). A/, medullary; EM, extramcdullary.

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Fig. 4. Immunofluorescen.ee profiles of CDl la Ã¯Ã¨'i'Ã¨2i"Ã¨HCONTROL-FITC Ã¯Ã¨3 ne0'"'Ã¯Ã´Â«'""Ã¯&2Ta3 Ta4 on normal and malignant PCs. Human BM cells Ã¯Ã´' were isolated from BM aspirates from one healthy CD38-FITC CD11a-FITC donor (A, B, and C) and one MM patient in chronic phase (Â£>,E, and F). Two-color analysis of BM cells with anti-CD38-FITC and B-B4-bioIin- streptavidin-PE (B and Â£)and wilh anti-CDlla- FITC and B-B4-biotin-streplavidin-PE (C and F) is shown. The corresponding controls with an irrele 0.-:Jl vant antibody are shown in A and D. *"*i2!O;O

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CD45RO (9, 10). On normal PCs, CD45RA and CD45RO were r=0.76 expressed on large subpopulations in most of the cases (illustrated in Fig. 2, E and F): the level of expression was 15 for CD45RA and 18 for CD45RO. On malignant PCs, both the level of expression and the percentage of positive cells for CD45RA and CD45RO were reduced significantly [chronic versus normal (P < 0.01)], but no additional significant differences could be observed during the subsequent stages of the disease (Figs. 2, E and F and 3, E and F). CD45RA and CD45RO Expressions Were Correlated on Ma 20 40 60 % of CD45RO positive cells lignant PCs. Because CD45RA and CD45RO showed similar mod ification of expression during the different stages of the disease, we Fig. 7. Correlation between CD45RO and CD45RA expression on malignant PCs. The CD45RO-positive population was plotted against the CD45RA-positive population for then analyzed the correlation between these two markers in terms of each of the 28 MM patients, then a linear regression was performed, and the correlation percentage of positive cells on malignant PCs. Twenty-eight couples coefficient (r) was calculated. were analyzed, and the correlation coefficient was 0.76, indicating a very high correlation between the expression of CD45RA and demonstrated the existence of a PC subpopulation expressing CD45RO (P < 0.001; Fig. 7). To confirm the coexpression of CD45RA and CD45RO on a single PC, a three-color staining was CD45RA and CD45RO. performed with the use of anti-CD45RA, anti-CD45RO, and B-B4 mAbs. Fig. 8 shows representative cytograms of one MM patient for DISCUSSION the CD45RA and CD45RO expression on the gated B-B4-positive In MM, PCs remain located essentially within the BM and rarely population. The results demonstrated that a small proportion of ma disseminate outside the BM, except in the terminal stage of the lignant PCs for this patient expressed CD45RA (5%) or CD45RO disease. A better understanding of the expression of adhesion mole (7%) (Fig. 8, A and B). The cytogram C reveals the existence of all four subpopulations CD45RA+CD45RO , CD45RA CD45RO+, cules and of their quantitative changes during the different stages of CD45RA+CD45RO+, and CD45RA CD45RO . In conclusion, we the disease may be critical for defining the mechanisms by which the PCs adhere to or detach from the BM environment. With regard to the CD56 expression, we confirmed that CD56 was expressed on normal PCs, up-regulated on myeloma PCs, and down-

r=0.70 regulated on BM PCs from patients with an extramedullary involve ment. The PCs from patients with an extramedullary involvement I eoH were also analyzed in the extramedullary site (blood), and we ob served that they shared the same CD56 phenotype as the BM PCs 60 (results not shown). These findings extend and confirm our recent 40- report indicating that the loss of CD56 occurred in the BM and was 20- correlated with the capacity of cells to disseminate out of the BM. Because we demonstrated recently that osteoblastic cell lines interact 0 0 20 40 60 80 100 120 with myeloma cells through CD56, it is conceivable that the overex- % of CD49e positive cells pression of CD56 on myeloma cells favors the capacity of adherence of myeloma cells to the osteoblastic cells.4 Among the hematopoietic Fig. 5. Correlation between CDlla and CD49e expression on malignant PCs. The CD1 la-positive population was plotted against the CD49e-positive population for each of disorders, two presented a very particular CD56 expression profile on the 47 MM patients, then a linear regression was performed, and the correlation coeffi cient (r) was calculated. malignant cells (23, 24). Indeed, an abnormal expression of CD56 has

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Fig. 6. Coexpression of CDlla and CD49e on the same subpopulation of malignant PCs. The expression of CD1 la and CD49e was analyzed on the restricted plasma iÃ¨2 Ã¯Ã¨3 Ã•â€¢ cell population by three-color staining with the use of CONTROL-FITC B-B4-FITC anti-CDlla-FITC, B-B4-PE, and CD49e-biotin-streptavidin-QR as described in "Materials and Methods." Two- color analysis of BM cells with control-FITC and B-B4- biotin-streptavidin-PE (A) and with anti-CD38-FITC and B-B4-biotin-streplavidin-PE (ÃŸ)is shown. Cytograms C and D, expression of CD49e and CD1 la, respectively, on the gated B-B4 + + * population. Cytogram E, expression *' of CD49e veraÂ«CD1 la in the gated B-B4* + ' population. 1â€¢je4ffv0"10Â°

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Fig. 8. Coexpression of CD45RO and CD45RA on malignant PCs. The expression of CD45RO and CD45RA was analyzed on the restricted plasma cell population by three-color staining with the use of anti-CD45RA-FITC, anti-CD45RO-PE, and B-B4-biotin-strepta- S, vidin-QR. Cytograms A and B, expression of CD45RA and CD45RO, respectively, on the gated B-B4 ++ + population. Cyto- gram C, expression of CD45RA versus CD45RO in the gated B-B4+ + + population. TÃ¨H CD45RA-FITC

been underlined on some chronic myeloid leukemia, and among the is in agreement with the observation that a close correlation existed T-large granular lymphocyte leukemia, those expressing CD56 iden between the expression of CD45RA and CD45RO on malignant PCs. tified aggressive variant of this leukemia. Although the function of Taken together, our results seem to exclude a putative transition in CD56 remains unknown, in all of these disorders CD56 appears to be CD45 isoform expression in relation with the stage of maturation of an important biological marker probably involved in the adhesive the PCs. The phenotype of HMCLs is in good agreement with this mechanisms and tumor cell dissemination. hypothesis because the large majority of these cell lines were CD45RA" and CD45RO . According to the changes observed for the As reported previously, we found that the normal PCs expressed CD49e on a large subpopulation (8). On malignant PCs, we observed individual adhesion molecules during the different stages of MM, an important decrease of positive subpopulation although the number general characteristics associated with malignant transformation and of CD49e molecules expressed on the cell surface was not signifi tumor spreading can be proposed. Malignancy was characterized by a cantly modified. This result is in favor of the disappearance of the strong overexpression of CD56 in terms of both number of PCs positive subpopulation. The absence of CD49e expression on most expressing CD56 and number of CD56 molecules present on the cell HMCLs and on PCs from patients with an extramedullary involve surface. This overexpression of CD56 was associated with a decrease ment suggested a relationship between the lack of CD49e and the of the proportion of PCs expressing CDlla, CD49e, and CD45 increase of the plasmablastic compartment in the PC population. RO/RA, whereas CD44 remained unchanged. Disease acceleration These observations find counterpart in the study of Kawano et al. (8), within the BM was not significantly different of the chronic phase. which demonstrated that CD49e~ myeloma cells were proliferative Extramedullary spreading was associated with a dramatic down-ex immature cells, whereas CD49e+ cells were mature myeloma cells. pression of CD56 in association with a decrease of CDlla+ CD49e+ In contrast to CD49e, CD49d was always expressed on all normal and CD45 RO+/RA+ subpopulations but no significant modification and malignant PCs (results not shown), and this finding is compatible of the level of CD44 expression. Normal PC population always with the importance of CD49d in the terminal B-cell differentiation presented negative subpopulations for the antigens CDlla, CD49e, and with its involvement in the immunoglobulin secretion of PCs (25). CD45RA, and CD45RO that could identify the same subpopula- In this study, we demonstrated that CDlla expression was detected tion. By consequence, we can propose that this compartment on a subpopulation of normal PCs, the percentage of this subpopula- increased in relation with the malignancy and could be a good tion ranging from 20 to 100%. This finding is in disagreement with indicator of disease aggressiveness. Furthemore, we can notice that one previous report and may be explained by the difference in the CD44 and VLA-4 expressions, which were not significantly modified sensitivity of the detection methods (microscopy versus fluorescence- during MM, were also the antigens that did not define subpopulation on activated cell sorting analysis; Ref. 7); the differences are not sup normal BM PCs. ported by differences in the staining due to the mAbs used because we The loss of several adhesion molecules may reduce the cell-cell obtained the same results with 2 mAbs (MHM24 and B-B]5). More interactions, and it is tempting to speculate that these cells escape over, the staining of HMCLs with the three anti-CDlla mAbs more easily to the immune surveillance and present a selective ad (MHM24, B-B15, and F8.8) was similar (data not shown). As ob vantage of proliferation. Finally, the variations of the adhesion profile served for CD49e, the CDlla+ subpopulation decreased during ma phenotype presently described are easily quantified and could be of lignancy but the level of the intensity per positive cell was not diagnostic and prognostic value in individuals with MM. significantly changed. In human lymphoma, it has been well docu mented that the absence of CDlla was related to the aggressiveness ACKNOWLEDGMENTS of the lymphoma (26-29). Our results on malignant PCs are consist ent with this finding. Moreover, we demonstrated that the percentage We thank N. Robillard for expert advice in cytometry, M. Etrillard for of CD Ila-positive PCs was well correlated with the percentage of technical assistance, B. Racape for secretarial assistance, and Dr. L. Zagury- CD49e-positive cells in the malignant cell population, and by three- Bataille for editing the English text. color immunofluorescence we confirmed that CDlla and CD49e were expressed on the same subpopulation of PCs. On the basis of our REFERENCES

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1995 American Association for Cancer Research. Adhesion Molecules on Human Myeloma Cells: Significant Changes in Expression Related to Malignancy, Tumor Spreading, and Immortalization

Catherine Pellat-Deceunynck, Sophie Barillé, Denis Puthier, et al.

Cancer Res 1995;55:3647-3653.

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