5485 Clin Pathol 1995;48:548-552 CD 19 and immunophenotype of bone marrow plasma cells in of undetermined significance

M Zandecki, T Facon, F Bernardi, V Izydorczyk, L Dupond, M Fran9ois, R Reade, T Iaru, F Bauters, A Cosson

Abstract myeloma has been studied extensively, showing Aims-To determine whether a particular that early B lineage associated antigens, such as phenotype or antigen is preferentially re- CD10 and CD19,'-' as well as myelomonocytic lated to monoclonal gammopathies ofun- cell surface markers (CD33, CD13)'-7 are ex- determined significance (MGUS). pressed on neoplastic plasma cells. These data Methods-Bone marrow specimens from suggested either lineage infidelity or a primary 56 patients with MGUS were stained im- neoplastic event early in haematopoiesis.7 munocytochemically (ABC peroxidase) With regard to plasma cells, most information for CD38, CD56, CD9, CD1O, CD19, about their phenotype has resulted, as there CD20, CD22, and MB2. Specimens from are very few present in bone marrow and lymph patients recently diagnosed with multiple nodes, from extrapolation from malignant myeloma and reactive bone marrow myelomas31"'3 or myeloma cell lines6"3'5 and samples were studied in parallel. rarely from in situ characterisation.1016 Using Results-CD38 was expressed on all multiparameter flow cytometry, Terstappen et plasma cells from all MGUS samples al'7 recently showed that myeloid and early B tested, while 36% were positive for cell markers (CD10, CD 19, CD20, and CD22) CD56. CD9 and MB2 were both expressed are expressed within the normal plasma cell strongly; CD20 was moderately expressed, population. Harada et al, 8 using two colour and staining for CD10 and CD22 was un- flow cytometry to distinguish normal plasma common. For these five B cell antigens cells from mature myeloma cells, found that there was no clear difference between their among the various early B antigens tested, expression in MGUS and in multiple CD19 was consistently expressed on normal myeloma. A great difference was found for plasma cells and not on myeloma cells, whereas CD19: in MGUS this antigen was ex- CD10 and CD20 were not expressed on normal pressed on 2-91% of plasma cells (mean plasma cells. 35%/6) and 77% patients had >10% positive Monoclonal gammopathy of undetermined plasma cells; in its ex- significance (MGUS) is observed in up to 3% pression was low and only 12% patients had of patients aged over 70 years and in 1-1 6% >10% positive plasma cells. When these of those aged over 50 years.9-22 Some patients results were converted to numbers ofCD19 with MGUS will develop true multiple my- postive plasma cells per 100 nucleatedbone eloma or amyloidosis after five to 35 years, marrow cells, reactive bone marrow and whereas others will remain stable and finally MGUS specimens had a similar number die without evidence of multiple myeloma or ofpositive plasma cells. There was no cor- a related disorder.22 MGUS is associated with relation between expression of any of the a monoclonal immunoglobulin peak in serum antigens tested. Laboratoire and a slight elevation of monotypic bone mar- d'Hematologie, Conclusions-Many of the so-called pre- row plasma cells. CD56 expression on plasma Hopital Calmette, B, B or activation antigens are present on cells from monoclonal gammopathies has 59000 Lille, France plasma cells from MGUS specimens, and been studied extensively and its expression is M Zandecki of F Bemardi expression CD9, CD1O, CD20, CD22, high in multiple myeloma and low or absent V Izydorczyk MB2, and CD38 in MGUS was very similar in MGUS,182324 but may be expressed in L Dupond to that in multiple myeloma. CD56 was some.2527 By examining paraffin wax em- M Franmois frequently expressed in MGUS. In this T Iaru bedded bone marrow sections, Dehou et a128 A Cosson series CD19 was highly expressed in showed that the MB2 monoclonal MGUS but not in multiple myeloma. was strongly expressed by neoplastic plasma Service des Maladies Plasma cells bearing this antigen could du Sang, H6pital cells, whereas expression was low or absent in Huriez, Lille represent the non-neoplastic process and plasma cells of patients with MGUS. Other- T Facon determination of its expression could be wise, information about the immunophenotype F Bauters useful for the diagnosis of MGUS. of plasma cells in MGUS is limited to small Service de (1 Clin Pathol 1995;48:548-552) series of patients.'8 27 Nephrologie, Hopital To characterise the immunophenotype of Calmette, Lille Keywords: Monoclonal gammopathy, immuno- R Reade phenotype, CD19, multiple myeloma. plasma cells in MGUS, bone marrow smears from 56 patients with MGUS were stained Correspondence to: immunocytochemically for CD38, CD56 and M Zandecki. various B cell CD Accepted for publication Over the past few years, the immunophenotype antigens including CD9, 10, 25 August 1994 of plasma cells from patients with multiple CD19, CD20, CD22, and MB2. Smears from Expression of CD19 in MGUS 549

107 patients presenting with multiple myeloma Antigenic expression was determined using were also studied for expression of the same an avidin-biotin peroxidase technique (Vecta- antigens. Our aim was to determine whether a stain ABC Elite, Vector Laboratories). Slides particular phenotype or antigen is preferentially were fixed for 10 minutes in cold acetone for related to MGUS. determination of CD19 and in acetone para- formaldehyde for all other antigens. Slides were incubated with successive layers of horse Methods serum, the relevant monoclonal at Fifty six patients fulfilling criteria for MGUS22 optimal dilution (see below) and biotinylated were studied. All patients had been followed antibodies. Endogenous peroxidase was for at least one year. Monoclonal peak was as quenched using methanol containing 0 3% follows: IgG K in 23 patients, IgG X in 19 perhydrol. Peroxidase conjugated avidin- patients, IgA K in four patients, IgA k in seven biotin complex was then layered on slides and patients, and biclonal in three patients (IgG K peroxidase activity revealed using diamino- and k for one patient, and IgG X and IgA k for benzidine and perhydrol. All slides were two patients). Patients presenting with IgM counterstained with diluted Giemsa. A negative gammopathy were excluded. control (no monoclonal antibody, or mouse The patients presenting with multiple my- IgG instead of monoclonal antibody) was in- eloma were classified according to the Durie cluded in some ofthe experiments. The optimal and Salmon staging system as follows: 39 dilution for each monoclonal antibody was that patients had stage I, 24 had stage II, and 44 used in our laboratory for the determination of had stage III disease. All patients were studied the immunophenotype of acute leukaemia or at diagnosis, before commencement of treat- peripheral lymphocytes with the ex- ment. ception of CD 10. For this latter antigen, the Eight patients with a moderate excess of monoclonal antibody was concentrated four- bone marrow plasma cells were also studied: fold and blood smears from a patient with two patients had drug agranulocytosis, two had common acute lymphoid leukaemia was used drug cytopenia, two had idiopathic thrombo- as a positive control. MB2 was used at a 1 cytopenia, and two had hepatitis A. None of in 20 dilution. For all monoclonal antibodies these eight patients had a monoclonal peak and tested (except CD10), the presence of positive their plasma cells were regarded as reactive. and negative lymphocytes was required on each Bone marrow smears were spread after sample for interpretation of reactivity. At least sternal or posterior iliac crest puncture. Slides 100 consecutive plasma cells were enumerated were stained with May-Grunwald-Giemsa. for reactive bone marrow and MGUS samples, For determination of the per cent of bone and at least 200 consecutive plasma cells were marrow plasma cells, 1000 consecutive cells enumerated for multiple myeloma samples. were enumerated for reactive bone marrow and Serum electrophoresis, immunoelectro- MGUS, and 500 consecutive cells for multiple phoresis and determination of the amount of myeloma. Remaining slides were allowed to each immunoglobulin class present were per- dry for 16-24 hours, were wrapped in alu- formed according to conventional methods. minium foil, and stored at -20'C until anal- Data analyses were performed using the X2 ysis. test and the Mann-Whitney U test. The following monoclonal antibodies were used: IOB 2 (CD9) and IOB 6 (CD38) (Im- munotech, Marseille, France); CD19 and Results CD20 (L26) (Dako, Glostrup, Denmark); B3 For each monoclonal antibody tested, a positive (CD22) (Coulter, Florida, USA), MB2 reaction occurred when plasma cells stained (Clonab-Biotest, Dreieich, Germany), Leu 19 brown. Some lymphocytes were also stained (CD56) (Becton Dickinson, California, USA). brown and were easily distinguished from IOT 5 (Immunotech) was used to detect CD10 plasma cells by their morphology; their re- expression in all patients; J5 (Coulter) was used activity was used as a positive control (table). in 35 patients (20 with MGUS and 15 with A reaction was considered positive when at multiple myeloma) for comparison. All an- least 50% of plasma cells reacted. tigens could not be tested in all patients because CD38 was expressed on all plasma cells of of the limited number of bone marrow slides all patients with either reactive bone marrow, available. MGUS or multiple myeloma. No unreactive

Per cent ofpatients in whom most plasma cells (>50%) expressed the relevant antigen. For each antigen tested, the number ofpatients varied according to number of available smears Multiple myeloma Reactive bone marrow MGUS Stage I All patients Difference between MGUS (n = 4 to 7) (n = 20 to 52) (n = 16 to 34) (n = 31 to 96) and stage IMM (p value) CD38 100% 100% 100% 100% CD9 100% 80% 75% 70% NS MB2 100% 70% 68% 56% NS CD20 0% 20% 15% 16% NS CD1O 0% 0% 5% 1% NS CD22 0% 3% 6% 16% NS CD56 0% 36% 62% 55% p=0 01 MM= Multiple myeloma. 550 Zandecki, Facon, Bernardi, Izydorczyk, Dupond, Franfois, et al 100 r or extensive (>50%) staining, whereas in- termediate reactivity (11-50%) occurred more 90g- $ frequently (data not shown) in those with MGUS. 80 In seven of eight patients with reactive bone marrow 62 to 96% of their plasma cells ex- 70 pressed CD19. Only one patient with viral 60 hepatitis did not express this antigen. Ex- pression of CD19 in patients with MGUS var- ied (figure): plasma cells were unstained in a) 50 -8 o one patient and in all other patients reactivity 40 _- ranged from 2 to 91% (mean 38%); in 37 of 48 (77%) patients over 10% of plasma cells 30 were CD19 positive. Expression of CD19 in MGUS seemed to have a continuous spectrum. 20 8~~~~~~~~ Overall expression of CD 19 was low in patients with multiple myeloma: less than 10% of 10 plasma cells stained in 45 of 51 patients. CD 19 was not highly expressed in any of the patients 0 MGUS (all patients) (stage 1) (stages 11 and 111) with multiple myeloma and moderate staining Multiple myeloma (14-34%) was found in only six patients who all had stage I disease. A highly statistically Expression of CD19 on plasma cells from patients with MGUS and multiple myeloma. significant difference (p=0-0001) was found for expression of CD19 on plasma cells of patients with MGUS and multiple myeloma (overall, or in those with stage I disease only) plasma cells were observed in these patients. (figure). For CD9, plasma cells were positive in all four The per cent of bone marrow plasma cells patients with reactive bone marrow and in most was 09 to 3 0% in reactive bone marrow, 0 9 patients with MGUS and multiple myeloma. to 6-2% in MGUS, and 4 to 95% in multiple Plasma cells were also positive for MB2 in all myeloma. The actual number ofCDl9 positive four patients with reactive bone marrow, and plasma cells in bone marrow of individual 70% of patients with MGUS. Of the patients patients (% bone marrow plasma cells mul- with multiple myeloma, 68% of those with tiplied by % CD19 positive plasma cells) stage I and 56% overall (that is, all stages) expressed MB2. Plasma cells of 20%, 16% showed that the upper limit was 2-9% for multiple reactive bone marrow, 4-5% for MGUS and and 15% of patients with MGUS, 7-8% for multiple myeloma. However, in 45 of myeloma (all stages) and stage I multiple my- 48 patients with MGUS the actual number of eloma, respectively, were positive for CD20. CD1 9 positive plasma cells was within the same Expression of CD20 was low on reactive bone range as for reactive bone marrow; for multiple marrow samples, and only a few (1-25%) the actual number was lower than plasma cells stained positively. Both mono- myeloma, or clonal antibodies used to determine CD10 ex- that found in either reactive bone marrow MGUS samples. Therefore, an actual excess pression J5 and IOT 5) gave the same reactivity pattern (data not shown). Only one in 71 ofCD19 positive plasma cells was an infrequent patients with multiple myeloma was positive finding in monoclonal gammopathies. for CD10; this patient had stage I disease. We failed to find any correlation between the Plasma cells from reactive bone marrow and expression of any of the antigens tested. For MGUS samples were negative for CD10 ex- CD19 and CD56, however, although no stat- pression although a few plasma cells (2-6%) istical correlation between high expression of stained positively in six patients. Plasma cells one antigen and low expression of another was from samples of reactive bone marrow were found, three groups could be identified: high negative for CD22, while only one in 30 CD19 (>50%) and low CD56 (<30%) ex- patients with MGUS was positive for this an- pression; high CD56 and low CD19 ex- tigen. Five of 31 (16%) patients with multiple pression; and low expression of both antigens. myeloma were positive for expression ofCD22; Interestingly, with the exception of one patient, one of these patients had stage I disease, and we failed to find a subgroup of patients with the other four had stages II or III disease. high expression of both CD19 and CD56. CD22 was expressed more frequently in stages In MGUS samples, the concentration ofIgG II and III multiple myeloma, but the number was 9 0 to 35 9 g/l (normal range 8-18 g/1) for of patients tested was too low to allow definite patients with an IgG peak and that of IgA was conclusions to be drawn. Some plasma cells (3 3 0 to 12 2 g/l (normal range 09-4.5 g/1) for to 28%) expressed CD56 in six of seven patients with an IgA peak. In seven patients patients with reactive bone marrow. CD56 the non-involved immunoglobulin (IgM, IgG (>50% plasma cells positive) was highly ex- or IgA) concentration was reduced. There was pressed in 36% of patients with MGUS, in no correlation between expression of CD19 on 55% of patients with multiple myeloma, and plasma cells and either the nature (IgG or IgA, in 62% when only patients with stage I disease K or k chains), the peak immunoglobulin were considered (p = 0 0 1). Most patients with concentration, or the reduction in non-involved multiple myeloma had either minimal (<10%) immunoglobulin concentrations. Expression of CD19 in MGUS 551 Discussion multiple myeloma."42735 In MGUS, using flow We used immunocytochemistry to determine cytometry, Leo et ai27 failed to find any reactivity the phenotype of plasma cells of patients with in the eight patients tested, whereas Harada et MGUS. CD1O and CD22 were expressed at all8 found variable positivity in all five patients low levels whereas positive staining occurred tested. Using immunocytochemistry, we also frequently for CD9, MB2 and CD20. While found that a variable but important percentage expression of CD19 and CD56 was not re- of plasma cells expressed CD19, and the stricted solely to either MGUS or multiple difference in expression between MGUS and myeloma, there was a statistically significant multiple myeloma was highly significant. difference in the staining pattern. Therefore it may be possible to use CD 19 CD 10 may be found on some normal expression to discriminate between MGUS and plasma cells,'7 but these data have not been stage I multiple myeloma. However, it is im- confirmed'8; conflicting results were also en- portant to note that, when CD 19 positive countered in multiple myeloma.'3182729-31 In plasma cells were expressed as a percentage of two small series'827 CD10 was not expressed all nucleated bone marrow cells and compared in MGUS, and our results confirm these data. with the value obtained for reactive bone mar- CD9 is commonly expressed on plasma cells row, no real excess of CD 19 positive plasma of patients with multiple myeloma,535 and in cells was found in most MGUS samples; in our series extensive expression was found in multiple myeloma these numbers were also low those with multiple myeloma, MGUS and re- and, in most cases, were even lower than those active bone marrow. CD10 and CD9 may be found in MGUS. In multiple myeloma the few related to the pre B nature of plasma cells CD 19 positive plasma cells may be residual and also to B cell activation32-34: in MGUS non-neoplastic cells and aberrant expression expression was low for CD10 and high for may be restricted to a few patients. One might CD9, suggesting that the mechanisms trig- hypothesise that in MGUS CD1 9 positive gering their presence could differ. plasma cells could also be related to the non- Low or no expression of CD22 seems to be neoplastic process. Our data confirm those of a common finding on plasma cells in multiple Harada et al'8 that plasma cells do not coexpress myeloma,'4 myeloma cell lines,'4 normal CD19 and CD56. However, it is not yet clear plasma cells,'7 or MGUS (our series). CD20 whether patients with MGUS who express may'7 or may not'8 be found on some normal CD56 are more likely to develop true multiple plasma cells, and a subset of patients with myeloma than those who do not express this multiple myeloma express this pan-B antigen. antigen.5142735 In our series expression ofCD20 In conclusion, we have shown that no specific was not an infrequent finding, and its dis- immunophenotypic profile was associated with tribution was comparable on plasma cells from plasma cells in MGUS. CD38 was the unique patients with either MGUS or multiple my- antigen consistently expressed on all plasma eloma. Although expression varied slightly cells from patients with MGUS. The various when expressed as per cent positive cells, there B cell antigens studied were expressed with was no difference in the expression of CD 10, comparable frequency in MGUS, multiple my- CD9, CD22, or CD20 in reactive bone mar- eloma, and reactive bone marrow, with the row, MGUS and multiple myeloma samples. exception of CD19. This latter antigen was Paraffin wax embedded bone marrow sec- rarely expressed in multiple myeloma, whereas tions show strong cytoplasmic positivity to most or many normal and MGUS plasma cells MB2 in most multiple myeloma cases,36 were positive. Do CD19 positive plasma cells whereas plasma cells from patients with MGUS represent the non-neoplastic mass? Will these show either weak or no reactivity." Bone mar- plasma cells disappear or gradually lose this row smears are widely used in the diagnosis of antigen in patients developing true multiple MGUS, and we tested this monoclonal anti- myeloma? Whatever the explanation con- body on bone marrow slides. We observed cerning the presence of CD19 on plasma strong reactivity in 70% ofpatients with MGUS cells, as it is highly expressed in MGUS and and comparable results (56%) were found for not in multiple myeloma, determination of patients with multiple myeloma. So, when this the expression of this antigen may be used, monoclonal antibody is used on bone marrow along with determination of the proliferative sections, its ability to discriminate between activity," CD56i expression18 23-27 and multi- MGUS and multiple myeloma disappears. drug resistance,24 to discriminate between CD56 (N-CAM) is an adhesion molecule these two conditions. expressed on most plasma cells in 62 to 82% of patients with multiple myeloma91823-25 but We thank Roselyne Coudenis, Corrine Fougere and Daniele is absent or expressed at a low level on normal Talandier for their expert technical assistance. This study was supported by Grants from Comite du Nord de and reactive plasma cells. 18 25-27 In MGUS, la Lgue de Recherche contre le Cancer and Centre Hospitalier CD56 is expressed weakly or not at all on Regional et Universitaire de Lille. plasma cells.2324 Other reports,23-27 however, showed that some plasma cells from patients 1 Durie BGM, Grogan TM. Calla-positive myeloma: an ag- with gressive subtype with poor survival. Blood 1985;66:229-32. MGUS could express this antigen (36 5% 2 Tazzari PL, Gobbi M, Dinota A, Bontadini A, Grassi G, in our series). Although this percentage was Cerato C, et al. Normal and neoplastic plasma cell mem- brane phenotype: studies with new monoclonal antibodies. high, there was a clear statistical difference with Clin Exp Immunol 1987;70:192-200. stage I multiple myeloma. 3 Epstein J, Barlogie B, Katzmann J, Alexanian R. Phenotypic 9 heterogeneity in aneuploid multiple myeloma indicates CD1 is present in almost all stages of B pre-B cell involvement. Blood 1988;71:861-5. cell ontogeny37 and is not highly expressed in 4 King MA, Nelson DS. Tumor cell heterogeneity in multiple 552 Zandecki, Facon, Bernardi, Izydorczyk, Dupond, Franfois, et al

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