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Cytoplasmic Immunoglobulin Content in Multiple Myeloma

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Downloded from on Mrch 2, 217. https://doi.org/1.1172/jci11233 Cytoplsmic Immunoglobulin Content in Multiple Myelom Brt Brlogie, Rymond lexnin, Mrk Pershouse, Leslie mllwood, nd Lon
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Downloded from on Mrch 2, 217. https://doi.org/1.1172/jci11233 Cytoplsmic Immunoglobulin Content in Multiple Myelom Brt Brlogie, Rymond lexnin, Mrk Pershouse, Leslie mllwood, nd Lon mith The University of Texs ystem Cncer Center, M. D. nderson Hospitl nd Tumor Institute, Texs Medicl Center, Houston, Texs 773 bstrct Bone mrrow cells of 82 ptients with multiple myelom were subjected to flow cytometric nlysis of DN nd cytoplsmic immunoglobulin (CIg) content using propidium iodide nd direct immunofluorescence ssys. Except for two ptients with nonsecretory myelom, there ws conformity in the immunoglobulin type derived from immunoelectrophoresis nd plsm cell CIg stining. One ptient with nonsecretory myelom exhibited monotypic CIg stining, while the second showed no rection. In eight ptients with IgG lmbd myelom, the sme tumor cells contined both lmbd nd kpp light chins, suggesting the productive rerrngement of both light chin genes. 14 ptients with previously unrecognized plsm cells of low RN content, ll of whom were resistnt to chemotherpy, were identified by CIg stining. By reveling previously unrecognized plsm cells with low RN content, CIg nlysis identified more ptients with tretment-refrctory myelom. Introduction The plsm cells of ptients with multiple myelom usully contin n bnorml DN nd n incresed RN content, permitting the quntittion of plsm cells by flow cytometry (1-3). Higher response rtes to both initil nd slvge therpy hve been noted with incresing RN content (4, 5), nd shorter survivl time hs been found in ptients with mrked bone mrrow plsmcytosis (4). typicl feture of plsm cells is the presence of cytoplsmic immunoglobulin (CIg).' We hve developed flow cytometric ssy for the concurrent nlysis of DN nd CIg content in order to quntify plsm cells when studies of DN nd RN content could not provide cler distinction from norml cells. Whether CIg stining might clrify the biology of plsm cells in ptients with nonsecretory disese or with other typicl clinicl fetures ws lso evluted. Methods tudies were performed in 82 ptients with dvnced stges of multiple myelom including 23 without prior therpy. The bone mrrow of ll ddress reprint requests to Dr. Brlogie, Deprtment of Hemtology, 6723 Bertner-Box 55, Houston, TX 773. Received for publiction 28 November 1984 nd in revised form Mrch bbrevitions used in this pper: CIg, cytoplsmic immunoglobulin; CV, coefficient of vrition. J. Clin. Invest. The mericn ociety for Clinicl Investigtion, Inc /85/8/765/5 $1. Volume 76, ugust 1985, ptients contined t lest 7% plsm cells on routine morphologicl exmintion. For flow cytometry studies, bone mrrow spirtes were seprted by Hypque-Ficoll grdient centrifugtion (6). Interphse cells were collected nd wshed once in phosphte-buffered sline. One liquot of cells ws lwys stined with the metchromtic dye cridine ornge for concomitnt DN nd RN nlysis using mercury rc flow cytometer (Phywe Co., Gottingen, Germny) (7). Routinely, 1, cells were nlyzed nd tumor cells gted on the bsis of bnorml DN nd RN content (1, 4). In this mnner, tumor cell DN nd RN index vlues were derived from the reltion to norml lymphocytes (specificlly, the rtio of medin fluorescence chnnel numbers of tumor to norml diploid cells) (1). The proportion of cells with bnorml DN nd/or RN content in the entire smple ws lso determined. second bone mrrow liquot ws processed for biprmetric DN-CIg nlysis with n EPIC V flow sorter (Coulter Electronics, Inc., Hileh, FL). Cells were fixed in 7% ice-cold ethnol for t lest 24 h. ingle cell suspensions were then exposed seprtely to ntilight-chin nd nti-hevy-chin regents (fluorescein-conjugted F(b')2 frgments; Cpell Lbortories, Westchester, P) t dilutions of 1:2 (.1 mg/ml) nd counterstined for DN with propidium iodide (8). In three instnces of dul light chin rection, different ntiser were evluted in the sme mnner (Tgo Inc., Burlingme, C). uccessive flow cytometric nlyses were performed on 1, cells, ech stined for both light chins, nd, in subset of ptients, stined lso for two hevy chins (typiclly, IgG nd Ig). Except for eight instnces of dul light chin rection, mrked differences in stining intensity were observed between pirs of smples stined for light nd hevy chins (see Fig. 1). In the 8% of ptients with DN-bnorml stemlines, bright CIg fluorescence ws usully limited to neuploid cells, while residul diploid cells showed the sme dim fluorescence observed fter stining with the opposite light chin regent. The proportion of tumor cells ws redily determined by pplying n electronic gting procedure to the brightly stined cells with n bnorml DN content (Fig. 1). To obtin mesure of CIg content for ech ptient, CIg index ws computed from the rtio of medin CIg fluorescence intensities of neuploid nd diploid GI cells of the sme smple. For the remining DN-diploid smples, both the percentge of plsm cells nd their CIg index were determined in comprison with the nonspecific stining pttern of cells rected with the opposite light chin ntiserum. pecificlly, kpp nd lmbd distribution curves were superimposed electroniclly to identify the lower level of specific CIg fluorescence. The CIg index ws then computed from the rtio of medin intensities of specific nd nonspecific (opposite light chin) fluorescence. The degree ofheterogeneity of CIg content mong plsm cells from the sme ptient ws expressed s coefficient of vrition (CV) of the monotypic CIg distribution. The CV (percent) ws computed from the rtio of the stndrd devition nd the men of CIg fluorescence chnnel numbers times 1. There were eight ptients with dul cytoplsmic light chin expression. In the six neuploid cses, ll DN-bnorml cells demonstrted both kpp nd lmbd stining. This finding nd fluorescence microscopic evlution (fter double-stining with fluorescein- nd rhodmineconjugted ntiser) indicted tht the sme plsm cells rected with both light chin ntiser. s in the mjority of cses with monotypic CIg stining, the CIg index ws computed in reltion to dimly stined norml diploid cells. In the two cses of diploid myelom with dul light chin stining, ssessment of the degree of mrrow plsmcytosis Cytoplsmic Immunoglobulin Content in Multiple Myelom 765 Downloded from on Mrch 2, 217. https://doi.org/1.1172/jci11233 nd of the CIg index ws performed in comprison with norml bone mrrow smples seprtely stined with kpp nd lmbd light chin ntiser. Exmple of Norml Bone Mrrow ML nd Myolom It Results Fig. 1 shows typicl exmple of bivrite flow cytometric DN-CIg nlysis of bone mrrow cells from ptient with IgG kpp myelom. bright stining rection ws observed in hyperdiploid cells using nti-gmm nd nti-kpp ser, but not with nti-lph nd nti-lmbd regents. second exmple illustrtes ptient with concurrent multiple myelom nd cute myelogenous leukemi (Fig. 2). Compred with norml mrrow, two discrete DN stemlines (diploid nd hyperdiploid) with mrkedly incresed RN content were identified. Using light chin ntiser, only the hyperdiploid DN stemline contined monoclonl CIg of kpp type, nd hence, represented the myelom tumor popultion; the diploid DN stemline showed similr wek stining rections with both kpp nd lmbd regents nd represented the leukemic clone. Tble I summrizes the reltionship between CIg nd immunoelectrophoretic results. Concordnce of light chin phenotypes ws observed in 8 of 82 ptients, including 18 ptients on whom dditionl hevy chin nlyses were conducted. Two ptients lcked monoclonl protein by immunoelectrophoresis, but showed mrrow involvement microscopiclly (25 nd 42%) nd by DN-RN flow cytometry (18 nd 69%). One ptient hd kpp stining within the cytoplsm ( low secretor ), while the second showed no detectble nti-light-chin rection ( low producer ). third nfi- z, I1. I,14d DN16f in Diploid DN nti- eplj o -e D e E Diploid DN- o-.21 I I: Diloidneuploid Zs 72n'o ntl-k DN Figure 2. Comprison of DN-RN nd DN-CIg nlysis in ptient with concurrent myelom nd cute myeloid leukemi (ML). Upper pnels represent DN-RN histogrms; lower pnels show DN-CIg histogrms. Compred with norml bone mrrow with prevlence of diploid cells with low RN content (upper left), both diploid nd hyperdiploid DN stemlines with mrkedly incresed RN content were found in ptient with both myelom nd ML (upper right). DN-CIg nlyses with nti-lmbd nd nti-kpp regents reveled monoclonl kpp expression in the hyperdiploid DN stemline, wheres diploid cells showed nonspecific stining. Hence, the hyperdiploid clone represented Myelom, nd the diploid cells with high RN content represented ML cells (see reference 7). Compred with the exmple in Fig. 1, nonspecific fluorescence (lower left) is shifted towrd higher chnnel numbers due to instrument setting. The CIg index of specific kpp fluorescence is reltively low (lower right). nti-x 2 nti- Figure. 1. Myelom phenotyping by CIg flow cytometry. Bone mrrow cells were stined with nti-hevy nd nti-light chin regents nd counterstined for DN with propidium iodide. Note the positive rection of hyperdiploid GI cells (window 1) only with ntigmm nd nti-kpp nd not with nti-lph nd nti-lmbd ntiser, confirming the presence of IgG kpp monoclonl protein. The CIg index s mesure of plsm cell CIg content ws computed in reltion to nonspecific fluorescence (window 2). Tble I. Immunoglobulin Typing by Immunoelectrophoresis nd CIg nlysis using Flow Cytometry CIg flow cytometry Immuhoelectrophoresis Light chin Kpp Lmbd No rection Kpp 45 1* Lmbd 35 No rection 11: Hevy chin G D No rection G 11 5 D 1 * Low secretor. i Low producer. ubset of 18 ptients in whom hevy chin nlyses were lso conducted. Ptient with lmbd light chin in cytoplsm nd urine; however, gmm hevy chin ws only detected in cytoplsm nd not in serum. 766 Brlogie, lexnin, Pershouse, mllwood, nd mith Downloded from on Mrch 2, 217. https://doi.org/1.1172/jci11233 ptient with positive rection of cells for lmbd light chin nd with Bence Jones proteinuri lso exhibited cytoplsmic IgG in the bsence of monoclonl serum pek. Fig. 3 illustrtes the correltions between the proportions of tumor cells identified by DN-RN or DN-CIg flow cytometry nd those enumerted by morphology for the 77 ptients in whom ll three ssys were vilble. Both cytometric ssys showed similr degrees of plsmcytosis in the 48 ptients with DN neuploidy, where bnorml cells were clerly seprted from norml cells (R =.83, P .1) (Fig. 3 ). mong the 29 ptients with diploid myelom, there were 8 ptients with DN-RN pttern indistinguishble from norml mrrow but monoclonl light chin rection ws evident in 5-69% of cells (medin, 26%). These cells corresponded to plsm cells on microscopic exmintion (Fig. 3 B). One ptient with nonsecretory myelom showed 6-7% plsm cells with high RN content but lcked monotypic CIg pttern ( low-producing myelom ). There were lso 6 ptients with neuploid myelom, in whom second diploid DN stemline with 5-2% plsm cells ws uncovered by monotypic CIg stining (four kpp nd two lmbd) (Fig. 4). ThUs, the combined nlysis of DN-CIg nd DN-RN permitted the identifiction of previously unrecognized cell popultions with low RN content in -2% of our ptients. Eight ptients with discrete DN-RN bnormlity hd plsm cells which expressed both kpp nd lmbd light chins. Immunoelectrophoresis reveled monoclonl IgG lmbd in ll instnces. n exmple of such double-stining is illustrted in Fig. 5 with three different DN stemlines in the diploid, the low degree hyperdiploid, nd the tetrploid rnge, ll of which expressed kpp, lmbd, nd IgG immunoglobulin (the ltter not shown). Fluorescence microscopy reveled concurrent kpp nd lmbd rections in the sme plsm cells. The dul light chin rection ws confirmed in three ptients using different source of nti-light-chin ntiser. There ws mrked vrition in the CIg stining per plsm cell in individul ptients, which ws expressed s CV rnging from 3 to 8% (medin, 5%). To compre the CIg content per cell mong different ptients, CIg index ws defined from the rtio of the medin fluorescence intensities of specific vs. nonspecific nti-light-chin rections. mong 23 ptients studied t dignosis, the CIg index rnged from 2 to 27, with medin vlue of 1. There ws no correltion between the RN nd the CIg index, but the CIg index decresed with incresing mrrow tumor infiltrte (R = -.25, P .1). We lso exmined the clinicl implictions of CIg nlysis. mong the 23 previously untreted ptients, neither CIg index nor CIg dispersion (CV) correlted with response to chemotherpy. By CIg nlysis, we hd identified 14 ptients with low RN index (less thn four times the RN content of norml lymphocytes) in diploid plsm cells, either s their sole tumor cell popultion (8 ptients) or in ddition to n neuploid DN stemline (6 ptients). Five individuls in ech ctegory were evluted fter 1-3 mo of resistnce to initil chemotherpy (primry unresponsive disese); the remining ptients were studied t dignosis, nd none responded to stndrd chemotherpy. Thus, CIg nlysis unmsked diploid plsm cells with low RN content, which ws ssocited with complete resistnce to preceeding or subsequent chemotherpy. 1 8 z 6 zo *. * * ~*: - & & * * % bnorml Cells (DN-GIg) 81 1 Figure 3. Correltions between the proportion of tumor cells in the bone mrrow identified by flow cytometry of DN-RN nd DN- CIg s well s by microscopy. () Close concordnce between DN- RN nd DN-GIg cytometric ssys in neuploid myelom (v). mong the 29 diploid cses (., o), there were 8 with undetectble plsm cells on DN-RN histogrms () but with monotypic CIg stining (o), consistent with monoclonl plsm cells (B). *, o, o; C) t B. *. ~~~~ * * * * * * % bnorml Cells (DN-Cg) Diploid, n = 29, r =.4, P .1. &; neuploid, n = 48, r =.83, P .1. (B) Correltion between mrrow plsmcytosis by microscopy nd by DN-CIg cytometry. One ptient lcked monotypic CIg rection, but hd high RN content with typicl plsm cell morphology (o). o,., o; Diploid, n = 29, r =.75, P .1., neuploid, n = 48, r =.62, P .1. Cytoplsmic Immunoglobulin Content in Multiple Myelom 767 48 '. 44'h' 4 Hyperdiploid 36 G, 32 _ ' Diploid DN Diploid Hyperdiploid 'B r (nti-k) Figure 4. Biclonl myelom DN stemlines identified by DN-CIg flow cytometry. () DN-RN nlysis demonstrtes only one cell popultion with incresed RN nd DN content, s typiclly found in myelom (4). (B) Monoclonl light chin rection with nti-kpp serum in bob diploid nd hyperdiploid DN stemlines. (C) Nonspecific stining with nti-lmbd serum. 2,2 3. ~ _.._.)L ' Downloded from on Mrch 2, 217. https://doi.org/1.1172/jci11233 z 4 -.., C (nti-x).... s. W., _ B 1 2 4j 4 e 3 Discussion Plsm cells represent the lst phse of B lymphocyte differentition nd re redily identified by the presence of monotypic immunoglobulin in the cytoplsm (CIg) nd, more recently, by monoclonl ntibodies recting specificlly with the surfce membrne of plsm cells (9, 1). While flow cytometry hs been used to probe the lymphocyte differentition pthwy, this method hs not been pplied to ssess the biologic nd clinicl relevnce of CIg in ptients with multiple myelom (1 1). Using direct immunofluorescence technique, there ws excellent greement between immunoelectrophoresis nd CIg flow cytometry in immunoglobulin phenotyping. In conjunction with morphology nd DN-RN flow cytometry, DN-CIg nlysis helped define specil ctegories of low-secretory nd low-producing myelom. There were eight instnces of concurrent kpp nd lmbd rection in the sme neuploid cells. This observtion must be interpreted with cution, s ll eight ptients produced lmbd light chins. The dul stining rection, however, ws confirmed in three ptients using different regent; in one of those exmined further, we only eliminted the lmbd, but not the kpp stining of the doubly stined cells by preincubtion with free lmbd light chins. Thus, our findings seem consistent with those rre reports of coexpression of kpp - -c _ _... _.....,,... ~~~~~~~~~~~~~ I;! DN DN D FITC Figure 5. Dul light chin expression in myelom. () DN-RN nlysis revels three discrete popultions with diploid (1), low degree hyperdiploid (2), nd ner tetrploid DN stemlines (3), with typiclly incresed RN content in popultions (2) nd (3). (B), (C) DN-CIg nlysis demonstrtes positive rections in ll three DN RITC stemlines with both kpp (B) nd lmbd (C) regents. (D), (E) On microscopic inspection of doubly stined cells (kpp-fluorescein isothiocynte, D; lmbd-rhodmine isothiocynte; E), the sme plsm cells rect with both light chin regents. 768 Brlogie, lexnin, Pershouse, mllwood, nd mith Downloded from on Mrch 2, 217. https://doi.org/1.1172/jci11233 nd lmbd light chins by the sme tumor cells (12-14). Interestingly the dul light chin expression ws only observed in lmbd-secreting myelom. This fits the concept of hierrchy of light chin ctivtion, where kpp gene rerrngement precedes tht of lmbd (15, 16). mong 18 B cell smples exmined from ptients with chronic lymphocytic leukemi nd long term Epstein-Brr virus-trnsformed norml humn B cell lines, lmbd constnt region genes remined in the germ line configurtion in ll eight smples producing kpp light chins (16). In contrst, 1 lymphocyte smples producing lmbd light chins showed deletion of both kpp constnt region lleles in 9 instnces nd productive rerrngement of 1 kpp llele in 1 B cell line. The ltter cse indictes the possibility of productive rerrngement of both light chin genes in lmbd-secreting cells. Our CIg flow cytometric dt suggest tht this event my occur in s mny s 1% of ptients with myelom. There ws close conformity in the degree of bone mrrow involvement by tumor cells using morphologic nd flow cytometric ssys. The ltter hve the dvntge of objectivity nd reproducibility. DN-CIg nlysis identified some ptients with pprently norml bone mrrow on DN-RN studies, whose plsm cells were now recognized with very low RN content. This ssy lso reveled diploid plsm cells with low RN content in ddition to neuploid cells previously demonstrted by DN-RN nlysis. Both of these observtions re of mjor importnce becuse of the estblished resistnce to chemotherpy of myelom ptients either with low RN index or multiple DN stemlines (4, 5, 17). s we hd noted with RN content, the CIg content per plsm cell vried mrkedly within nd mong ptients. In previously untreted ptients, incresing bone mrrow plsmcytosis ws ssocited with decrese in CIg index nd RN index (18), which might reflect less differentited nd perhps more ggressive fetures of myelom. The resistnce to chemotherpy of low RN index myelom hs been estblished (4, 5, 17) nd my be relted in prt to greter degree of RN heterogeneity with incresing tumor burden (18, 19). imilr inferences cnnot yet be mde for CIg index nd CIg dispersion in view of the smll number of ptients evluted t dignosis. In summry, DN-CIg flow cytometry ids in severl spects of myelom reserch nd clinicl mngement. We were ble to recognize ptients with low-secretory nd lowproducing disese. The biologicl significnce of CIg dispersion nd the coexpression by the sme tumor cell of both light chins requires further study. The importnce of objective nd quntittive ssessment of the degree of mrrow tumor infiltrtion hs been demonstrted (4, 17). DN-CIg nlysis is superior to DN-RN cytometry for ssessing mrrow plsmcytosis becuse monoclonl CIg is more specific for bnorml plsm cells. In conjunction with DN-RN flow cytometry, CIg nlysis permitted the identifiction of more ptients with poor prognosis, whose plsm cells either hd low RN content, biclonl DN bnormlities, or both. cknowledgments This work ws supported by Ntionl Cncer Institute grnts C 16672, C28771, nd C References 1. Brlogie, B., J. Ltreille, D. wrtzendruber, L. mllwood,. Mddox,. M. Rber, B. Drewinko, nd R. lexnin Quntittive cytology in myelom reserch. In Clinics in Hemtology. W. R. chmidt, editor. W. B. unders Co Bunn, P.,. Krsnow, R. Mkuch, M. chlm, nd G. chechter Flow cytometric nlysis of DN content of bone mrrow cells in ptients with plsm cell myelom. Clinicl implictions. Blood. 59: Brlogie, B., M. Rber, J. chumnn, T. Johnson, B. Drewinko, D. wrtzendruber, W. Gohde, M. ndreeff, nd E. Freireich Perspectives in cncer reserch: flow cytometry in clinicl cncer reserch. Cncer Res. 43: Brlogie
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