Document 80gRYMK7kLGq3XrZQJ3VN92k

CLIFT urnoy rb H , :laid I :705, i r 13, illlta- 19x7 WEVlEW ARTICLE Plasma Cell Myeloma- New Biological Insights and Advance- in Therapy By Bart Barlogie. Joshua Epstein, Peter Selvanayagam, and Raymond Alexa an 2e 4 ;'2&$$% 2 !. : 3._.~ PLASMA CELL myeloma has been the prototype of a monoclonal tumor cell proliferation that reveals a nionoclonal protein in the serum or urine in more than 90% of patients. The disease spans a spectrum of clinical entities, including localized and disseminated, as well as indolent and aggressive forms.'-' Patients typically sufler from painful fractures, anemia or renal failure, and from recurrent infeclions associated with immunodeficiency. With a median age of 60 years, current treatments have been palliative rather than curative.' Thus complete remissions from standard alkylating agent-glucocorticoid programs have been $3While the acquisition of new )NA stemlines on flow; L !!cytometry is rare,25 this techniq.: is insensitive for t h e ' s detection of structural aberrations r small numeric chromo- -4 ~ -j ~ % Isomal abnormalities.26 Indeed, tli : typical complexity o f t 3 ,w plasma cell karyotype suggests that common. With the prospect of betr clonal evolution must b e 3 :r in vitro culture meth- L.I . I1B" 9g i! (% -2ods, the authors expect that primar. and evolutionary karyo- typic anomalies will be identified regularly in myeloma d 3 progenitor cells. Such information n a y help define discrete disease entities and direct research into the molecular mech- six c anisms of abnormal growth and ditterentiation. achieved infrequently. and all patients succumb after a incdian of about 3 years, even though about 10%of current Cell Kinetics and In Vitro Growth I f Tumor Cells patients will survive 10 years.6 During the past 5 years, new insights have been gained of the cellular and molecular biology of myeloma, so that the previous notion of a terminally differentiated B-cell maligiiancy is giving way to the provocative concept of an early hciiiatopoietic stern-cell disorder manifesting itself mainly a t the mature stage of B-cell lineage. Therapeutically the growth-controlling effects of alpha-interferon7 and the feasi- bility of hematopoietic stem-cell-supported high-dose cytotoxic regimensn have raised hope for a fundamental change in prognosis that had been stagnant since the introduction of melphalan and prednisone some 25 years ago?." This review summarizes data that myeloma may be an early stem-cell disorder; describes features that predict clinical outcome; and reviews marrow-ablative programs for patients with newly diagnosed and refractory disease. Readers with interrst in traditional staging and treatments are referred to other recent reviews.''.'* Laboratory methods for cytokinetic studies have been refined, so that autoradiography with tritiated thymidine has been replaced by the more convenient bromodeoxyuridine (BUdR) immunofluorescence t e ~ l i n i q u e . ' ~A~s' ~expected in a tumor composed mainly of terminally differentiated B cells (ie, plasma cells), the labeling index after in vitro pulse exposure is usually low, with average values of I % a t diagnosis and higher values a t relapse." Both autoradio- graphic and BUdR techniques have identified even lower labeling indices in patients with monoclonal gammopathy of unknown significance (MGUS) and indolent myeloma.'&12A similar discrimination also resulted from the study of B lymphocytes in peripheral In patients with symp- tomatic myeloma, higher values of plasma-cell labeling index were associated with shorter survival time, independent of tumor Relative to values a t diagnosis, tumor cell-growth fraction (determined from tritiated thymidine administration in vivo) MYELOMA BIOLOGY &not ypic Studies Because of the low fraction of cells in D N A synthesis, few mitoses are usually available to define karyotype abnormali,ies.16.17 Unlike results in leukemia and lymphoma, diseasespecific anomalies have not yet been identified; the presence of complex numeric and structural rearrangements suggests a long disease course with many acquired genetic changes. Analysis of nuclear DNA content by How cytometry (that docs not depend on mitoses) has demonstrated aneuploidy in 80% of patients; hyperdiploidy was most frequent (70% of total, with a DNA excess of 10% to 20%); diploidy was present in 20%; and hypodiploidy in about 10%.'n-2A0lso was much higher during relapse," when in vitro colony growth is also more successful. The recent availability of antibodies that distinguish different DNA precursor molecules (BUdR and IUdR) permits a more expedient immunofluorescence-based analysis by flow cytometry of cell-cycle time parameters and growth fraction (J Gray, personal communication, June 1988).Several studies arc currenlly in progress in myeloma and other malignancies to deline the value of Ki-67 monoclonal antibody (MoAb) as a means of assessing the fraction of cells capable of cell proliferati~n.]'.'~ Consistent with the low proliferative activity of typical plasma-cell myeloma, attempts at establishing long-term confirmed by cytogenetic studies, DNA-hypodiploidy was associated frequently with only light-chain production and with resistance to standard therapies. (Table l).17~C*1hromosomal translocations, as in non-Hodgkin's lymphoma, were found in about 10%of cases, and t(8;14) abnormalities were arsociatd with an 1gA isotype." Deletions of the long arm of diroiiiosoinc 6 have recently been linked to lytic bone disease.'2 An adriamycin-resistant myeloma cell line (8226/ DOX) displaying the multidrug resistance (MDR) phenotype has a deletion of the long arm of chromosome 7," which is the site of the p-glycoprotein genc.I4 From the University of Texas M.D. Anderson Cancer Center, Department of Hematology. Houston. Submitted September 20. 1988: accepted November 18. 1988. Supported in part by Grants CA37161 and CA28771 from the Natiotrul Cancer Institute. National Institutes of Ileulth. Bethesda. MD. Address reprint requests to Bart Barlogie. MD. Department o/ tIematology. M.D. Anderson Hospital & Tumor Institute. University of Texas Box 30, 1515 Holcombe Blvd. Housfon, TX 77030. Q I989 by Grune & Sfratton.Inc. 0006-4971/(19/7304-00?1$3.OO/0 Sk~c:dV. ol 73,No 4 (March), 1989: pp 865-879 865 866 BARLOGIE ET AL Cytogenetic Abnumaliw Hypodiploidy t (8:141 (q24:q321 6q 7q- Trisomy 11 Table 1. Clinically Relevant Chromosomal Anomalies % Incidence Associations 10 Only Bence Jones protein Primary drug resistance c5 IgA 15 Lytic bone disease 7 Drug resistance, site of mdr gene 10 Decreased p2 1 (H-ras) levels Reference 17 21 17 22 23 111 cultures have met with limited success, so that only about a with progressively increasing labeling index and adverse dozen human myeloma cell lines are now available, often p r o g n ~ s i s . ' ~ ~A~s' a tumor perceived to be composed of with unusual phenotypic features typical of earlier stages of terminally direrentiated B cells with commitment to immu- maturation.40 Similar problems were met in sustaining short- noglobulin production and secretion, plasma-cell myeloma is term cultures for the study of in vitro drug s e n s i t i ~ i t y . " . ~ ~ composed mainly of cells that contain monoclonal cytoplas- Systematic laboratory efforts by Durie et al (BGM Durie, mic imm~noglobulin.~M' onoclonal clg expression by diploid personal communication, October 1987) have since led to the cells in about 20% of patients with DNA-aneuploidy indi- recognition that monocytes and CD4-positive T cclls provide cated the presence of DNA-biclonality that was also asso- important stimuli for in vitrogrowth of plasma cclls and their ciated with resistance to chemotherapy.*' Surprising was the precursors, while CD8-positive suppressor T cells seemed to coexpression of both kappa and lambda light chains in the inhibit growth. Employing a double-layer agar technique same DNA-aneuploid tumor cells in about 15% of patients. with irradiated HL-60 feeder cells, Millar et aL4' reported usually with IgG lambda isotype." The molecular basis of successful myeloma colony growth in almost 90% of these aberrations from the current dogma of exclusive kappa patients. or lambda light-chain expression and their association with Several cytokines are involved in the proliferation and gamma heavy chains remains to be ~ l a r i f i e d . " . ~ differentiation of normal murine and human B cell^.^^^^' Consistent with their major commitment to protein After B-cell activation by interleukin-4 (IL-4)46*4' and secretion, myeloma cells contain high levels of RNA, an expansion by interleukin-5 (IL-5),`8 B-cell stimulatory factor average six times more than unstimulated lymphocytes.6'6! 2 (BSF-2) induces the final maturation of B cells into Exploiting the metachromatic properties of acridine orange, immunoglobulin-secreting plasma cells.49Based on studies of cellular. DNA and R N A content can be measured simulta- D N A sequence homology, BSF-2, interferon-beta-2, and neously by two-parameter flow cytometry quantitating green hybridoma-plasmacytoma growth factor represent the same (double-stranded DNA) and red fluorescence (single- cytokine that is now termed interleukin-6 (IL-6).50+5P' ro- stranded RNA).6' The detection of aneuploidy also i n blwd duced by monocytes, fibroblasts, and T-cell lines, the domi- provided direct evidence for circulating tumor cells,?` lone nant action of IL-6 is the differentiation of activated B cells. suspected on the basis of monoclonal idiotype-concordant although effects on normal hematopoietic stem cells (in blood lymphocyte^"^^^ capable of plasma-cell dimerentiation concert with IL-3) have also been IL-6 probably in vitro.67Thus disease progression and spread may occur by accounts for the promotion of human myeloma growth by the hematogenous route, with specific tumor localization conditioned medium from adherent spleen cells when Balb/c enforced by marrow-derived monocytes and T cells providine mice were primed by intraperitoneal injections of pristane or growth-stimulating f a ~ t o r s . " - ~ ~ . ~ ' mineral oil." The recent demonstration that fresh myeloma In addition to facilitating the quantitation of marrow cells produced IL-6 constitutively and expressed IL-6 recep- plasmacytosis, flow cytometric studies of nucleic acids hate tor (IL-6R). with stimulation of D N A synthesis by exoge- provided useful prognostic information."RThus no patient nous IL-6 in some cases has suggested that IL-6 may with DNA-hypodiploidy has ever responded to standard function as an autocrine growth factor." This observation melphalan-prednisone or VAD; and higher response ratec has been questioned by Klein et who noted that IL-h have been observcd with increasing cellular R N A content. activity resided entirely in bone marrow-adherent cells and As with nuclear DNA, plasma-cell R N A index remained favored a paracrine growth mechanism. These authors had stable throughout a patient's disease and declined only rarely previously reported an autocrine function for BCGF I I with relapse. Therefore the undoubted clonal evolution that (IL-5) in human R P M I 8 2 2 6 ~ e l l s . 'In~vestigating the molec- produces drug resistance was not reflected in changes of ular basis of growth factor and receptor gene activation cellular D N A and R N A content. Occasional mixed clinical should clarify the pivotal mechanisms involved in the abnor- responses to therapy could be traced to direrent DNA mal proliferation and maturation of plasma-cell myeloma. stemlines present in different disease sites. Tumor Phenotype While the majority of tumor cells have plasma-cell features, self-renewal must be sustained by stem cells earlier in While a n individual patient has fairly uniform plasma cell the maturation s e q ~ e n c e . ~ ~S.e`v~e.ra~l~groups have searched morphology, there is moderate heterogeneity among dif- for the expression of early B, T and myelomonocytic mark- I ferent patients. The Mayo Clinic group has distinguished C ~ S . ' ' ~ I' n~ almost 50% of patients, the pre-B antigen common between plasmacytic, lymphoid, and plasmablastic variants acute lymphoblastic leukemia antigen (CALLA) WBS PLASMA CELL MYELOMA-INSIGHTS AND ADVANCES 867 Table 2. Differentiation and Lineage infidelity Phenotype Calla + Myelomonocytic T-antigen TCR-gamma rearranged LDH % Incidence 50 13 ? 40 50 Comment Coexpression with clg' and good prognosis Poor prognosis Poor prognosis when two myeloid markers present Coexpressionwith clg' and myeloid markers No clinical correlation High levels in terminal disease phase with lyrnphomalike features and poor prognosis Refarence 72 73 74 74 102 114 expressed in a large fraction of tumor cells (Table 2).72T h e cwxpression of CALLA and monoclonal clg by the same aneuploid tumor cells unmasked a novel tumor-cell phenotype without known counterpart in normal B-cell differentiation (Fig l). Such dilferentiation infidelity was also shared by the mature plasma-cell antigen RI-3, which was jointly cxpresscd with CALLA in about 40% of patients. T h e consistent presence, in all cases of DNA-aneuploid myeloma, of CALLA-positive cells with diploid D N A content (but without clg expression) raised the possibility that diploid precursor cells generated the aneuploid plasma cells. Subpopulations of cells with CALLA+/ cIg-, CALLA+/clg', and CALLA-/cIg+ may represent different maturation stages of plasma-cell myeloma. The authors have observed a more favorable clinical course in patients with CALLA expression, while others have concluded that this phenotype was harmful." Myelomonocytic antigen expression was reported recently in 13% of patients with myeloma (Table 2).74*7T5hose with dual myeloid antigen expression (M5and MY7) also showed T- and early B-cell features, high labeling index, and a poor prognosis. As expected from frequent elevations of serum beta-2-microglobulin (B2M) level, most patients showed tumor cell-surface expression of B2M." Both surface expression and serum levels of B2M can be augmented after treatment with interferon-alpha, although correlations with clinical response have not been detected (Epstein J. Barlogie B, Alexanian R, unpublished observations, June 1988). Thus myeloma is a disease in which the majority of tumor cells are aneuploid with a differentiated B-cell phenotype but with subpopulations that also express early B, possibly T, and even myelomonocytic features. The additional presence of diploid cells with an immature phenotype suggests that aneuploid myeloma originates from a stem cell with a different (ie, diploid) D N A complement.72 While differentiation and lineage infidelities may reflect merely malignancy-associated aberrations, they may indicate alternatively that transformation involved a pluripotent stem cell with a flexible pattern of gene expression. More specifically, the asynchrony of antigen expression in myeloma may reflect ! I- Fig 1. Phenotypic heterogeneity ina patient with hyperdiploid multiple myeloma. Hyperdiploid cells have high RNA content; react with anti-B,M. J5. and R l - 3 and show monoclonal clgA kappa expression. Diploid cells have low RNA content without monoclonal clg reactivity but express B,M and CALLA. Abscissa, DNA content; ordinate. Auoresceiice intensity of cellular featuru. C e h h v DNA Contnt 8 6 8 BARLOGIE ET AL Table 3. Factors Produced by Myeloma Cells Facta IL-1 beta TNF-beta TNF-alpha BCGFll (IL-51 IL-6 B,M Comment OAF, chromosome 24; autocrine growth factor? OAF, chromosome 6p2 1 Autocrine growth facta in CLL. HCL: not evaluatedin myeloma: on chromosome 6p2 1 Myeloma cell line (RPMI 82261-automine growth factor (7): chromosome 5q23.3-q32 Myeloma cells and marrow-adherent cells Increasedin most patients; poor prognosis with high Serum levels; chromosome 15q2 1-q22 Reference 78 79 77 80 56 54 55 138-141 abortive dimerentiaton during an ordered rather than stochastic hematopoietic development with sequential commitment to megakaryocytic, erythroid, myeloid, and, finally, B-cell lineage.76 The demonstration of erythroid or megakaryocytic antigens on myeloma cells would support such a model (Epstein J, Barlogie B, unpublished observations, Decembcr 1988). Consistent with the variety of disease features, myeloma cells produce many cytokines in addition to immunoglobulins. Osteoclast-activating factors have been identified as TNF-beta (lymphotoxin) and IL-I-beta (Table 3).77-'9While usually undetected in mature B cells, the continued expression of TNF-beta and IL-l -beta by myeloma cells7' suggests a role for those proteins in abnormal growth and differentiation, also invoked for IL-5'6 and 1L-6." Indeed, T N F c a n act as a tumor growth factor and can promote cell proliferation in two other related B-cell malignancies, namely hairy cell and chronic lymphocytic leukemia.'" Receptors for IL-5 ( B C G F 11) and IL-6 on myeloma cells may provide targets for therapy with immunotoxins or radioisotopes. Myeloma cells also express receptors for vitamin D,, glucocorticoids, and sex hormones (estrogens and progestins; Table 4).8f.84 Futurc studies should clarify the biological functions and implications of these receptors in the different B-cell tumors. In view of the clinical use of glucocorticoids" and alpha- interferon,"correlations of response with receptor expression are imp~rtant.".~' Imniunodeficiency mainly of B-cell, and less profoundly and frequently of T-cell, type is responsible for recurrent infections, usually of bacterial rigi in.^^"^ In murine plasma- cytoma, B-cell suppression has been traced to a plasma-cell factor that causes monocytes and macrophages to produce a suppressor of normal B-cell proliferation"; a similar factor has not yet been identified in humans. An alternative mechanism concerns the suppression of normal B cells by immunoglobulin-binding factor (IgBF), which may be identical to the Ig-Fc receptor shed by T cells." In the murine MOPC315 model, IgA-binding factor produced by IgA-induced T cells reacted with surface IgA on MOPC-315 cells: this resulted in the inhibition of both tumor growth and lg production by the suppression of myc and Ig gene transcription, re~pectively.~' The resistance to cytotoxic drugs poses a major obstacle to the successful management of myeloma and other neoplastic diseases. Refractoriness to multiple antitumor agents after exposure to one drug is referred to as MDR, a feature that eventually becomes dominant in cultured tumor cells.''.q' The MDR phenotype has been linked to the presence o r a p-glycoprotein (p170) that functions as an ellux pump for certain anticancer agents, such as anthracyclines and vinca a l k a l ~ i d s . U~ s~in. g~ ~the MoAb, C219. which recognizes the cytoplasmic domain of ~ 1 7 0 h.ig~h~levels of MDR expression have been observed i n myeloma cclls and have been linked to clinical resistance to the VAD regimen?' Such resistance has been overcome with the calcium channel blocker, verapamil,98presumably by retaining higher cellular levels of a d r i a m y ~ i n T. ~h~e frequency and degree to which V A D resistance can be overcome with verapamil is now under study@'. ' Molecrtlar Genetics Studies of immunoglobulin gene rearrangement (as the molecular hallmark of a B-cell tumor) may explain the phenotypic heterogeneity and differentiation infidelity of myeloma." As expected. .J,, rearrangement could be detectcd in proportion to the degree of marrow plasmacytosis. Millti- Table 4. Receptors Expressed on Myeloma Cells Receptor for Comment Reference BCGFll llL-5) Cell lines, autocrine mechanism ( 7 ) IL-6 Cell lines and fresh samples; me- diates autocrine or paracrine mechanism Interferon-alpha Active against low-mass disease: chromosome 2 1q2 1 Glucocorticoid Cell lines and fresh samples: correla- tion with clinical response to glu- cocorticoids not yet established: Estrogen, progesteron chromosome 5 q l l - q l 3 Biological effects (V:chromosome 6q24-q27 (ER) and 1 l q 1 3 (PA) Vit D 3 Mediates inhibitionof proliferation and induction of new phenotypes I bv Vit D 3 56 54 7 86 81 85 83 84 82 PLASMA CELL MYELOMA-INSIGHTS AND ADVANCES 869 plc rearranged bands were noted in only 5% of patients, myeloma showed rearrangement of bcl-2 (commonly indicating that clonal evolution was reflected rarely at the involved in follicular lymphoma), consistent with the rarity iiiiniunoglobulin gene level (Selvanayagam P, unpublished of t(14;18) translocation in myeloma. Five of 120 patients observations, September 1988). The concurrent rearrange- had rearrangement of bcl- 1 but without corresponding nient of Cu and J,, genes in some patients resulted either m R N A elevation.' lo from ditrerent tumor clones in different stages of B-cell Careful scrutiny of chromosomal breakpoints disclosed inaturation or from the coexpression of normally unasso- frequent involvement of the short arm of chromosomes 1 and ciated phenotypes. The authors' finding of clg kappa coex- 1I ,I' where Kirsten-ras and Harvey-ras genes a r e located. pression in IgG lainbda myeloma was confirmed at the Using pan-ras and H-ras antibodies with flow cytometry, 17 iiiolecular level, with Ck rearrangement present i n some of 23 patients with active myeloma had high p21 expres- patients with lambda light-chain production. sion."' An inverse relationship between p21 levels and Since early stem cells may be the source of myeloma, trisomy 11 suggested that suppressor genes were present on analysis was also carried out of T-cell receptor genes, which chromosome 1 I .'I2 The frequent elevation of p21 protein are part of the immunoglobulin supergene family. Surpris- indicated that H-ras oncogene is involved in the pathophysi- ingly, 35% of patients showed TcR-gamma rearrangement, ology of myeloma, as supported further by the shorter while TcK-beta and TcR-alpha genes were not aRected.lo2 survival time of patients with high p21 levels."' T h e mecha- Identical frequencies of rearranged bands with TcR-gamma nism of H-ras gene activation is currently under scrutiny, and J,, probes supported the neoplastic origin of TcR-gamma especially in regard to gene mutation as the commonest rsarrangemen t. mechanism of ras gene activation in other human tumors. In a survey of HLA-associated gene expression in human I)-cell oialiynancics. invariant chain messenger RNA (In- iriKNA) as well as 11LA DK-alpha and HLA DK-beta-chain Biology Summary and Future Projections inKNA were expressed in all patients with either acute or Cytogenetic and molecular data indicate the presence in chronic lymphocytic l e ~ k e m i a . " ~In contrast, only three of myeloma of D N A rearrangements similar to those observed I5 patients with myeloma demonstrated In-mRNA expres- in malignant lymphoma. Such chromosomal translocations sion. Thus the In gene was expressed in neoplastic cells early presumably result.from recombination errors during immu- in the maturation sequence, concurrent with the activation of noglobulin gene rearrangement (ie, B-cell commitment) with other genes encoding histocompatibility class 11 antigens, activation of juxtaposed oncogenes.'" Phenotypic evidence of and preceded the activation of immunoglobulin genes."" differentiation and even lineage infidelity, with coexpression While advances in chromosomal banding and molecular on terminally differentiated B cells of early B,' myeloid,74 techniques have clarified the role of cellular oncogenes in and T-cell markers" may reflect abortive differentiation in wine B-cell lymphomas and leukemias, the difficulty in the process of a n ordered sequential rather than a stochastic obtainirig adequate metaphase chromosomes and the dearth lineage commitment, thus placing the oncogenic events in of specific karyotypic aberrations have hindered similar myeloma possibly to an early stage in h e m a t ~ p o i e s i s . ~ ~ progress i n plasma cell myeloma. With the presumption of Disease evolution has not been studied systematically. pre- B-cell involvement in human myeloma72and the regular Preliminary data suggest disease deditrerentiation occasion- L w y c gene deregulation in murine plasmacytoma,lOJ-iM ally with cessation of myeloma protein secretion and produc- c-iiryc and other oncogenes implicated in malignant lym- tion instead of high levels of serum lactic dehydrogenase plionra have been studied also in human myeloma (Table 5). (LDtI) during the terminal phase of disease transforma- lligh lcvels of c-myc R N A expression were observed in 25% tion."' Similarly, the development of acute myeloid leuke- of patients, without apparent abnormalities of myc R N A mia in up to 25% of patients 10 years after diagnosis of transcript size.lo7 As with t(8;14) chromosomal transloca- multiple myeloma has occasionally been interpreted as a tions, myc gene activation was found more commonly in natural disease evolution rather than as a treatment-induced patients with IgA isotype but resulted only rarely from D N A secondary malignancy,ii5 thus supporting the notion of rearrangement and was not associated with DNA amplifica- myeloma as a tumor arising early during hematopoietic tion.IU"Analogous to recent observations in Burkitt's lym- differentiation (Fig 2). phoma, Meltzer et allDYreported mutations in the 3' region of Sequential investigations during the disease course of the first c-myc exon (Pvu 11 and Alu 1 endonuclease sites) in individual patients are likely to reveal a progressive expan- ten of 16 human myeloma samples and cell lines; we have not sion of myeloma progenitor cells with higher proliferative confirmed this observation. None of 60 patients with activity and earlier phenotypic characteristics. Such infor- Table 6. Oncogenes in Plasma-Cell Myeloma mation can then be used to probe for the presence of such features in rare cells earlier in the disease course. AtJnormJily ~ Rearrangement c-myc L-myc bcl-1 bcl-2 c-ti-ras 41120 0122 51120 0160 0140 Studies of human plasma-cell lines suggest that BCGF 11 (IL-5) may be an autocrine growth factor in some patients Amplification 01120 0122 01120 0160 0140 with m y e l ~ m a . ' ~Receptors for IL-6 (BSF-2) may mediate Deletion 01120 2/22 Ol120 0160 ND the biological activity of IL-6 produced mainly by bone HighmRNAexpression 9/37 ND 0140 0140 ND marrow-derived monocytes (paracrine m e ~ h a n i s m ) ~an' d/or Hiah orotein exoression ND ND ND 17/23 by the tumor cells themselves (autocrine mechanis~n).'~In ND. not done. view of the three-signal model advanced by Ki~hirnoto"~'f~or BARLOGIE ET AL Myeloma Cell Phenolype. Kinetics. and Drug Sensitivity ETIOLOGY In the mouse, an oil granuloma induces a preneoplastic chronic inflammation that progresses to plasmacytoma with myc gene deregulation'"; in contrast, the etiology of human myeloma remains elusive. Several oncogenes (c-niyc and H-ras) appear to be involved, but their link to the initiation and progression of myeloma is unclear. Radiation exposure. long known to induce myeloid leukemias and certain solid u tumors, has sometimes led to myeloma 15 to 20 years later 117-121 Other epidemiologic surveys have demonstrated an association with benzene"' and asbestos,"' as well as with certain agricultural o c c u p a t i ~ n s . ' ~S`e~ve~r~e ~immunoaiippression with organ transplantation, while inducing R-cell lymphoma, has rarely led to plasma-cell myeloma.'26Simi- larly, while B-cell lymphomas have been recognized as n Fig 2. Model of myeloma ontogeny. On the basis of available phenotype and molecular data (see text). e DNA-diploid myeloma stern cell early in hematopoiesis is postulated with sequential manifestation of the acquired immunodeficiency syndronic (AIDS), associations between human immunodeficicncy virus (HIV) infection and myeloma remain anecdotnl.tz'."`; abortive commitment to megekaryopoiesis. erythropoiesis, granulopoiesis, and. finally. B-cell lineage. Terminally differentiated B cells (plasma cellsl represent the dominant tumor phenotype, usually with DNA-aneuploidy and frequently asynchronous expression of earlier stages of differentiation. Proliferation is Only about 10% of persons with benign monoclonal gammopathy (one of the few benign disorders with DNAaneuploidy of plasma cells2') develop myeloma after at lcast 10 years of follow up? T h e persistence of a low-leve\ mono- highest at an early commitment stage with greater sensitivity to melphalan. whereas plasma cells (producinga variety of cytokines) are kinetically inactive and highly sensitive to glucocorticoids. clonal protein after marrow-ablative chemoradiotherapy for myeloma in remission suggests that a benign gamnlopitth! often precedes myeloma and is resistant to supralethal d o w of therapy because of the low proliferative activity of Rh,tfi. the activation, proliferation, and differentiation of normal B cells by 1L-4, IL-5 and IL-6, these lymphokines probably play an important role in sustaining the growth and differentiaton anomalies of human myeloma. For example, the production by tumor cells of IL-l,'a.79TNF,'7"aand IL-4 or I L - P may sustain both an autocrine growth-stimulatory loop and 1L-6 production by bone marrow monocytes," promoting the differentiation of earlier B cells into plasma cells. Abnormal differentiation with lineage infidelity (presence of myeloid and T-cell markers on plasma cells) suggests that receptors for B-cell growth and differentiation signals may also be expressed aberrantly. Comprehensive studies, at the single-cell level, of cytokine production by tumor cells, lymphokine receptor expression, proliferative activity, hema- associated plasma cells.'2q The presence of tumor cells in peripheral blood is relevnnl to an understanding of disease progression and has bccn suggested by several studies, including ( I ) isotype-con. cordant surface immunoglobulin expression by idiotypic lymphocyte^^'^"; (2) differentiation of CALLA-bearing pcripheral B cells to monotypic plasma cells in vitro": ( 3 ) circulating DNA-aneuploid cells20; and (4) presence of immunoglobulin gene rearrangement in blood.6' Wcrnc cl a113" examined the ratio of kappa- and lambda-positive lymphocytes in blood and noted the phenomenon of liphl. chain isotype suppression in stable phases of disease. con. trasting with isotype predominance during disease prb gression. topoietic lineage, and phenotype stage are required to clarify these questions. Multiparameter flow ~ytometry".'~and image analysis, together with in situ hybridization, are DIAGNOSIS AND STAGING Solitary and Indolent Myeloma suitable research tools for such investigations. The key trigger to self-sustained and ultimately relent- Solitary plasmacytoma of bone or soft tissue,'" indolent or smoldering m y e l ~ r n a , ' . ~ *a"n~d symptomatic generabed lessly progressive plasma-cell accumulation is likely to result from the abnormal expression of normal cellular genes by a variety of possible mechanisms, such as gene rearrangement. myeloma"' represent distinct clinical phases in the y w t r u m of plasma-cell myeloma (Table 6). The occurrence by chancr of a painful pathologic fracture from a single tumor mnv mutation, and/or lack of suppressor gene activity due to deletion of critical "antioncogenes." `Iz High myc expression, usually without DNA rearrangement, in about one quarter apparently allows the recognition of this disease about ? years earlier than otherwise possible. Provided strict criteria for staging a r e applied. local radiotherapy appears to be of patients suggests that point mutations may be the major cause, resulting in impaired nuclear protein binding to myc DNA sequences and consequently uncontrolled growth, as in Burkitt's l y m p h ~ m a . ' 'M~utational activation of H-ras unop- curative in about one half of the patients. Staging criteria include no signs elsewhere of monoclonal plasmacytosis (bcct by DNA-clg flow cytometry), lack of other bone Icsions or abnormalities on sensitive radiographic examinations (corn. posed by controlling sequences on chromosome 11 may be an puterized axial tomography [CAT1 and nuclear magnetic additional or separate mechanism of abnormal growth. resonance), and preservation of normal immunoglobulin I ..i PLASMA CELL MYELOMA-INSIGHTS AND ADVANCES 871 levels. Less rigorous criteria probably explain the divergent Table 6. Diagnosis and Staging conclusions reached in regard to the natural history of this ciitity. Following radiotherapy, abnormal globulin levels are _. c usually reduced markedly, indicating that the localized $ disease was encompassed by the radiation field. However, low monoclonal protein levels may persist indefinitely in wiiie patients, indicating residual plasma cells that remain Diagnosis MGUS Indolent myeloma oinwmmmg PWMIWIWS Normal bones, Hb. Ca. Ig: Marrow plasmacytosis <10% Labeling index 1% Few lytic bone lesions without fractures Normal Hb. Ca, Ig; dorniant in a manner analogous to BMG. Some localized Marrow plasmacytosls 4 0 % plasniacytomas are radioresistant (eg, those with DNA- Labeling index <1% hypodiploidy or with low R N A content), portending resis- Overt myeloma B,M t4.4-6. 2-6 m g k lance to later chemotherapy and a poor prognosis.68 The curability of localized plasmacytoma i n a substantial propor- Dune-Salmon StagingSystem Marrow plasmacytosis <20, 20-40, >40% lion of patients indicates that myeloma arises from a single focus; the persistence of monoclonal gammopathy without later evolution of generalized disease suggests that BMG is a prcneoplastic entity, requiring additional factors to progress IO iiiycloina. A siiiiilar explanation may pertain to the persistence of low levels of abnormal globulin after high-dose consolidation therapy for responsive multiple myeloma.'29 When a solitary lesion is not associated with bone destruc- tion (ie, perhaps because 1L-1 or TNF-beta production is survival of patients with plasmablastic myeloma may be related to higher proliferative activity,Mconfirmed in several clinical trials to shorten survival independent of tumor ~ass14,35.141 or B,M ln studies evaluating the role of high-dose alkylating agent therapy for VAD-refractory myeloma, the authors observed high-serum LDH levels of >300 U / L prior to or within 2 weeks after therapy in about one half of patient^."^ High low), generalized myeloma may develop and progress slowly without symptoms. Only a chance electrophoresis leads to h e diagnosis of such indolent or smoldering disease. Similar to patients with early phases of C L L or nodular lymphoma, chemotherapy is unnecessary until morbidity occurs or bscomes imminent (ie, myeloma protein >5 g / d L or new bone lesions). The similar prognosis of patients treated many riionths after diagnosis to that of the usual patient treated promptly indicates that drug-resistant tumor cells have not LDH conferred a rapidly progressive course with lymphomalike clinical features, in a manner similar to that seen in patients with the blast phase of chronic myelocytic leukemia or with transformed lymphoma. Thus high LDH seems to define a high-grade myeloma as a result of clonal evolution to a more undifferentiated tumor-cell phenotype and/or by progressive expansion of drug-resistant tumor cells.'j4 Quantitative studies of tumor-cell LDH content should distinguish these alternatives (Van NT, Epstein J, Barlogie B, unpub- expanded. A low plasma-cell labeling index can predict an indolent disease course in asymptomatic patient^,'^.^'.'' many of whom live longer than I O years after d i a g n o s i ~ . ~ . ~ ~ ~ lished observations, December 1988). Preliminary data in 100 previously untreated patients also showed that high LDH levels occurred more frequently with renal failure and Clinical Staging of Syntptoniatic Myeloma portended a poor prognosis.'42 Thus there are now a variety of quantitative and tumor Dilfcrent centers have defined the extent of myeloma from cell-derived parameters that correlate with prognosis (Table variations of a schema that considers the degree of anemia, 7). From a large retrospective analysis of previously hypercalcemia, and other disease f~atures."'~"B~ased origi- untreated patients who received similar chemotherapy, the i d l y on a system devised from direct measurements of total dominant variables associated with distinct clinical end- inyeloma mass,135increasing tumor burden has been asso- points were determined, eg, (1) absolute resistance with ciated with shorter survival time."` Although the application D N A hypodipoloidy and lower remission rates with low of different criteria for staging by different centers has plasma-cell R N A index and high BIM serum levels; (2) prevented a meaningful comparison of treatments between shorter remissions with high serum-LDH and low plasma- groups, the consistent use within a center or group has cell R N A index; and (3) short survival in patients with high allowed more reliable comparisons of new treatments with serum-LDH and B2M levels (Table 8). The importance of historicaI treatments. similar and mainly tumor-intrinsic parameters for both Recent studies have improved further the reliability of remission induction and long-term prognosis emphasizes the htagiiig systems. Thus the serum level of beta-2-microglobu- dominant role of marked cytoreduction from initial therapy lin (B,M), by reflecting both tumor mass and renal function, in each patient's ultimate outcome. ofers a single measurement that provides as clear a separa- The authors envision that, as in the leukemias and lympho- tion of risk groups as any staging ~ y s t e m . ' ~A"m~o~n~g 100 mas, cytogenetic and molecular disease entities will eventu- previously untreated patients, the authors found progressive ally be defined that are associated with unique clinical bhortening of survival time for patients with increasing B,M presentations and prognosis. Meanwhile, relationships Other studies have revealed that the extent of bone among the recognized prognostic factors should be clarified, lesions is an unreliable index of tumor load, that renal failure so that the adverse effects of high LDH,li4 low C A L L A occurs much more often with advanced disease, and that the expre~sion,~h'igh H-ras activity,"' myelomonmytic pheno- extent, morphology, and D N A / R N A content of marrow type,74and high proliferative activityI4` can be traced to few plasma cells are important factors. For example, the shorter critical biological features to be exploited therapeutically. 872 BAALOGIE ET AL Table 7. Prognostic Factors in Myeloma Endpoint Adverlcr PretreatmentVariable Response -DNA hypodiploidy -Low RNA index -Hightumor mass -High B,M . . .MDR expression Time to relapse -Low RNA index -High 8,M -Hightumor mass -High LDH Survival time -High 8,M -Hightumor mass -High LDH -High labeling index . . . Low RNA index . . .DNA hypodiploidy . .. CALLA-negative . ..Butyrate esterase- positive . ..H-ras expressed -. established: . . . ,suggested. Aeference 68 68 68 142 97.100 142 138-143 136 and many others 114 34,35,144 68 68 72 74 111 OBJECTIVES OF THERAPY myeloma has been overlooked as an important endpoint. Few published reports have included this feature in the comparison of different treatments. Now that effective treatment alternatives for remission induction and consolidation are available, remission time assumes more importance in comparing treatments. Disease relapse may be detected early in most patients from rising myeloma protein levels. The median tumor-doubling time for patients with IgG or Igh myeloma protein is about 2 months, with more rapid relapse in those who responded rapidly. In recent years. and espccially after VAD, more patients have been recognized with "phenotypic escape.""' Most commonly this is manifested by increasing bone marrow infiltration with progressive anemia. bone destruction, hypercalcemia, and rising B,hl and LDH levels, despite low or even absent levels of myelonia protein. Transformation to a more aggressive malignancy with rising LDH has been associated with disease in lymph nodes, liver, brain, or blood."4 Whether such patients arc more likely to develop karyotypic or phenotypic changes from those present at diagnosis is not clear. The role of new therapies, such as VAD and/or high-dose melphalan, in causing such changes or allowing them to become manifest because of survival prolongation needs to be assessed. T h e degree of cytoreduction achieved in most responsive patients rarely exceeds 99%.'" With most available treat- TREATMENT ments, disappcarance of myeloma protein is evident in only For the past 25 years, intcrmittcnt mclplialan-prednisi)nc about IO% of those with IgG or IgA peaks, but is evident in (MP)has been the therapy of choice for multiple myelonin in about 60%of those with only Bence Jones protein, so that the community practice. Extensive clinical trials with other drug overall frequency of "complete remission" is about 20%. combinations have not shown a major improvement in dic- Rigid criteria for disappearance of abnormal protein should e a ~ e c o u r s e . ' ~ ~Si.n' ~ce' the details of numerous clinical trialc include immunoelectrophoresis and immunofixation tech- have been summarized e l s e ~ h e r e . ' ~t.h' i~s review will focus niques. Most centers define a response either as a 50% on those studies promising substantial future gain. Since reduction in myeloma protein c ~ n c e n t r a t i o n o' ~r ~as a 75% these treatments were developed first for refractory disease. reduction in M-protein synthesis"'*''8 with disappearance of effective salvage regimens will be reviewed prior to discuss- Bence Jones protein (as in this review). The speed of cytore- ing their role as initial therapy. duction, calculated from changes in IgG or IgA myeloma protein, varies with different treatments. Thus in responding Eflective Salvage Reginiens patients, the median time required for a 50% reduction in IgG or IgA myeloma protein was much shorter with primary VAD (0.4 months) or W A D - V A D (0.9 months), than with VCAP using bolus vincristine-adriamycin (1.2 months) or standard melphalan-prednisone (2.2 months). High-dose melphalan with total body irradiation achieved the most rapid cytoreduction with protein levels declining in accordance with their expected plasma clearance. The remission time of responding patients with multiple V A D and dexanrethnsone. Not until the introduction of VAD (combining pulses of high doses of dexamethasone with continuous infusions of vincristine and adriamycin) were there frequent, marked, and durable tumor cytoreductionc in patients resistant to standard therapies.'" Dexamethasonc alone was also effe~tive,~b'ut VAD induced more frequent remissions in patients with relapsing disease or with primary drug resistance of less than 1 year duration, provided favorable D N A / R N A features of plasma cells were present (Table 9). A longer duration of primary resistance we* Table 8. Multivariate Analysis of Prognostic Factors Among 100 Patients Treated at MD Anderson Cancer Center associated with DNA-hypodiploidy or low plasma-cell RNA index in nearly one half of the patients, providing one Adverse Variable biochemical explanation for such resistance. The greetcr Endpoint In Order d Enny P efficacy of VAD than dexamethasone among relapint Response Low RNA (<4) High B,M b 6 mg/L) .oo1 patients was attributed to the greater sensitivity of proliferel. .oo1 ing tumor cells to the cycle-active drugs vincristine end Relapse-freesurvival LDH (>200 U/L) .05 adriamycin. Identical to studies in newly diagnosed patientc. Survival 1 RNA (c4) High LDH I2200 U/L) High Mass (Durie-Salmon) High B,M (26mg/L) .06 .oo 1 .09 .10 a high serum B,M level (> 6 mg/L) was the dominanl adverse feature for survival time.I5* High-dose nielphalan and total body irradiation wirh hone inarrow fransplarrfafion. To date, 65 patients with PLASMA CELL MYELOMA-INSIGHTS AND ADVANCES 873 Table 9. Response to Salvage Treatment Releted to Duration of Primary Resistance and to DNA-RNA Features _ _ Treatment _I DEX VAD HDM c90 mg/m' HDM 90-140mgjm' HDM 140 mg/m' + TBI `Not hypodiploid and RNA index 24. Resurfantto MP MP MP and VAD MP and VAD MP and VAD % Responckng(No. Treated) Unresponsivef a 5 12 months + FavorableONAIRNA. 32 (28) 56 132) 67 112) 47 1151 77 (13) other 17 (181 14 (141 0 (81 53 (15) 100 (2) VAD-refractory niyeloma have been treated with high dose iiiclplialan (HDM), either a l ~ n e ' ~ 'o~r" w~ ith total body irradiation' (TBI; 850 cCy in five fractions I 2 hours apart) a i d supported with autologous bone marrow. The latter was harvested, whenever possible, during an earlier remission phase and contained less than 30%plasma cells. The rationole for autologous marrow transplantation in this marrowderived malignancy was based on the predominantly terminal B-cell phenotype with a small proportion of clonogenic tumor cells capable of self-renewal?* With the addition of TBI, virtually all patients responded (Table 9 ) ,and although tbc patients were selected more carefully, the median remis- sion and survival times were longer than with the melphalan alone (Table 10). The previous adverse influence of a long duration of priiriary drug resistance (512 months) and of certain DNA/ RNA features was no longer observed when high melphalan doses (ie. >90 mg/m') were employed, indicating both the lack of cross-resistance to VAD and, more importantly, critirely different mechanisms of drug action (Table 9). With bcttcr patient performance (Zubrod t2) and normal renal function. early mortality was virtually eliminated. The longest remission and survival times were observed among patients whose LDH levels were less than 300 U / L both prior to and during the first 2 weeks after therapy (Table 10 urd Fig 3)."' The short survival times in patients with higher LDII levels were due lo rapid recurrence of a high-grade iiiyeloiiia with lymphomalike features. Among patients receiving bone marrow autografts, neither the extent of plasmacytosis nor the tumor-mass kinetics a t the time of marrow harvest influenced remission and survival durations. Since complete remissions with current marrow-ablative regimens occurred rarely and since the phenotype of the niycloma stein cell was unknown, autologous bone marrow purging by immunologic means appears unwarranted. Instead, therapeutic research should focus on the development of regimens that provide even greater cytoreduction. Potential alternatives to marrow autografts include hematopoietic growth factors15' and/or autologous blood stem cell^,"^*^^' particularly in patients with extensive marrow plasmacytosis. The combined use of hematopoietic growth factors and autologous stem cells may reduce the duration of severe neutropenia and hence the risk of sepsis with ablative treatments. Allogeneic bone marrow transplanation. Because of the higher mortality from graft-v-host disease (GVHD) among older patients, the use of allogeneic bone marrow transplantation (BMT) has not been explored systematically. Cyclo- No TBI II 11 e I%LOW-LOW 1 I 17 L M b W -8.Nom-FYT. p .01 . . 0 6 16 24 32 40 0 8 16 24 32 40 Yonlhe Alter Treatment Fig 3. Survival of VAD-refractory myeloma after treatment with HDM with or without TBI (for details. aee textl. Longest disease control and survival were achieved in patients whose serum LDH level. did not exceed 300 U/L within 2 weeks of therapy, whereas patients with higher levels rarely derived clinical benefit. The prognostic importance of LDH applied whether or not TBI was added. Table 10. Proanostic Factors with HDMfor VAD-Refractory Myeloma ~~~ ~ ~ ~~~ ~~~~-~ Median RelapseFree Swvival (months) MedtanSurvival (months1 % TBI N RespOIlSl LOH. Low-Low. Othert P LDH, Low-Cow Othac No . 36 Yes 16 46 80 7 3 .04 10 4 +18 6 .03 35 8 P .04 .06 ,006 .18 .19 'LDH. Low-Low: LDH ~ 3 0 U0/L for a t least 14 days atter treatment. tother: LDH 2300 U/L before or within 14 days after treatment. P ,009 .07 , 874 EARLOGIE ET AI. phosphamide plus TBI and modifications, with syngeneic or with an additional 50% of patients who achieved partin1 allogeneic BMT, has reduced tumor mass markedly in about responses; 10% sumered early mortality.'6" one dozen patients, many being treated during a partial The authors are conducting similar studies in newly diag- remission.'s8-'6' M onoclonal protein persisted in many nosed patients, in which VAD is prescribed for those with patients with progressive or unresponsive myeloma. In a intermediate or high tumor mass: responding patients then recent European trial, about one half of the patients achieved receive H D M (I40 mg/m2) and TBI (total dose of 850 cGy complete remission. and few patients developed severe in five fractions) supported by autologous remission marrow. GVHD.16' With the efficacy of antLCD5 MoAb in glucocor- Only one of five patients treated to date has achieved n ticoid-resistant GVHD, allogeneic marrow transplantation complete remission. may be better tolerated by older patients.'63 A comparison of autologous with allogeneic BMT (in patients with comparable disease features who received identical cytoreductive therapy) may clarify the possibly adverse effect of autologous tumor-cell reinfusion and the potential gain from a graft-v-myeloma effect. Problems and Promises of Therapy Combinations of alkylating agents with or without adrinmycin have not proven superior to standard MP introduccd about 25 years ago. During the past 5 years three effectivc treatments have been discovered: ( I ) alpha-interferon for Primary Therapy VCAD-VAD. Because of its superior activity i n melphaIan-refractory disease, the VAD regimen has been assessed in previously untreated patients. W e have not seen any major gain in survival with VAD or VCAD-VAD that includes oral cyclophosphamide (VCAD) in comparison with preceding regimens, so that no improvement in the frequency of complete remission or survival time has resulted after a 15-year experience with many drug ~ombinations."~The lack of survival prolongation with a regimen that was superior for resistant disease is puzzling, particularly since tumor halving times have shortened progressively from a median of 2.2 months with M P to 1.2 months with VCAP and t o 0.4 months with VAD. T h e authors reason that diffcrent tumor cells may be affected by the different treatments. VAD with high-dose glucocorticoid may reduce preferentially more differentiated tumor cells so that such manifestations as myeloma protein production, anemia, and hypercalcemia are controlled rapidly. This effect contrasts with standard MP, which may be more erective against myeloma precursors, so that slower but equally sustained tumor control is achieved (Fig 2). Inrerferon. Interferon-alpha is a n active cytoreductive agent for many patients with low tumor load both at low tumor-mass disease; (2) high-dose glucocorticoid thernpyS5 with further benefit from continuous-infusion vincric. tine-adriamycin in proliferating myeloma'"; (3) IIDM with superior antitumor effect from added TRI.R.129 T h e infrequency of complete reniisGms ilftcr iii:irrint. ablative therapies with autologous I3M'I'sccnis unlikcly tohc due to tumor-cell reinfusion. This reasoning is based on thr slight further reduction of residual myeloma protein in responding patients consolidated during remission and thc low number of reinfused plasma cells in proportion to thc number persisting in vivo. Furthermore, the infusion of autologous marrow with obvious plasmacytosis has not short. ened the duration of remission in patients with refractor! myeloma. The "plateau phase" of myeloma during reniission may represent a cytokinetic sanctuary this is resistant lo high-dose therapy.'" Alternatively, a BMG-like condition w,ith resistant and long-lived plasma cells may be a common precursor phase of myeloma remaining after successful ern& ication of the neoplastic tumor-cell population.'29 In addition, one must consider the persistence of causal agents nnd mechanisms sustaining the monoclonal gammopathy. l h w the continued production of a monoclonal protein after marrow-ablative therapy is not incompatible with durable disease control. diagnosis and during first remission."J'J6' The combination of interferon with VBMCP as initial therapy achieved a SUMMARY AND CONCLUSIONS higher frequency of complete remission than seen previously.'" However, interferon rarely benefits patients with resis- Plasma cell myeloma is a more complex neoplasni thnn suggested by the relative uniformity of its dominant plasma tant or relapsing disease several years after diagnosis. An attractive biological feature of interferon concerns the frequent recovery of normal immunoglobulins in 'many responding patients, an uncommon event with standard chemotherapy.'M HDM 2 TBI. The Medical Research Council reported that high-dose intravenous (IV) melphalan produced a 78% response rate (with 27% complete remissions) in 41 newly diagnosed patients with advanced di~ease.'~'.'~A' subsequent cells, which represent the terminal stage of normal R-cell differentiation. Phenotypic, molecular, and cellular genetic data favor the presence of a myeloma stem cell early in hematopoietic development so t h a t , a s in chronic myelogenous leukemia (CML), a far distance exists bctwctn the primordial malignant cell that was the target of malit nant transformation and the dominant clinical phenotyr Traces of pre-B, myeloid, and T cells are coexpresscd with the mature B-cell phenotype, an occurrence unknown in regimen included initial V A M P (vincristine-adriamycin by normal B-cell differentiation. continuous infusion plus methylprednisone instead of dexamethasone in VAD) followed, a t maximum cytoreduction, Analogous to CML. disease progression is markcd by disease dedifferentiation, occasionally with cessation d by H D M (200 mg/m2) with autologous remission marrow. myeloma protein production and development instcad d Preliminary data indicated a complete remission rate of 30%, extramedullary lyrnphomalike features with high 1.1)Il rn I M A CELL MYELOMA-INSIGHTS AND ADVANCES 875 m)cldysplasia/acute iiiyelogenous leukemia (AM L) syndiuiiirs. The prognostic importance of serum LDH Icvels crcn in nsMIy diagnosed m)elomd jugguts the early prescnu of tuiiior cells Hith " LDH phenotype," Hhish, as a iauli of drug resistance and proliferative advantage, expand prdcrentially during discase progression. Further characteriuiiua of ihese cells iuay provide important clues about the ontogeny of multiple myeloma. hiyeloma cells express many receptors for different biological signals that might be exploitable for therapy with immunotoxins or radioisotopes. Plasma cells and their precursors also produce a variety of cytokines, some of which have putatively autostimulatory functions (eg, IL- 1, IL-5, IL-6) and/or are related to disease manifestations (eg, IL- I and TNF-beta as OAF). The wealth of cellular expression by plasma cells provides clues for understanding the mechanisms of gene activation and the nature of abnormal growth a d direrentiation. The accuracy of prognostically relevant staging systems hab been refined with the use of new quantitative parameters that reflect tumor mass (ie, serum B,M levels) and biology. Further studies of cellular and molecular biology (ie, CALLA, 11-ras) may reveal those tumor cell features that define clinical entities, response to therapy, and long-term prognosis. The lack of a major advance in prognosis despite the use of more drugs and more intensive reginlens justifies the continued use of standard melphalan-prednisone for pdtients ~ i t ah hiyhl\ fdvordble prognosis, for the very aged. utd ior those ~ i t ah short life cxpectanc) due IO other mdjor medical probleiiis.i7i However, a radical departure from standard practice is required to improve the prognosis for younger patients with poor risk features. Especially rational is the application, soon after diagnosis, of three active treatments that lack cross-resistance, namely, alpha-interferon. VAD, and HDM. Exploiting supportive care measures with marrow or blood stem-cell support and/or with hematopoietic growth factors. one should seek a marked tumor reduction with ablative treatments to achieve durable remissions in most patients. As G V H D becomes more preventable and manageable, allogeneic BMT will become more feasible, and with comparative trials, the risk of autologous tumor-cell reinfusion can be assessed. The recent notion of an autocrine growth mechanism, as in acute myeloid leukemia,i72suggests that future therapy can be designed to interfere more specifically with the abnormal expression of cellular genes, such as H-ras and c-myc. ACKNOWLEDGMENT The authors wish to express their appreciation to Mattie ScottThomas and Eva Menefee for their secretarialassistance. REFERENCES I. Bataille R: Localized plasmacytomas, in Salmon SE (ed): Clinics in Hematology. Philadelphia, Saunders. 1982. p 113 2. Kyle RA: Monoclonal gammopathy of undetermined significance (MGUS): Review, in Salmon SE (ed): Clinics in HaematoloBY. London, Saunders, 1982. p I 2 3 3. Alexanian R: Localized and indolent myeloma. Blood 56:521, I980 4. Kyle RA, Greipp R: Smoldering multiple myeloma. N Engl J Med 302:1347, I 9 8 0 5. Bergsagel DE: Controversies in the treatment of plasma cell myeloma. Postgrad Med J 61:109, 1985 6. Alexanian R: Ten year survival in multiple myeloma. Arch Intern Med 145:2073, 1985 7. Mellstedt H, Ahre A, Bjorkholm M, Holm G , Johansson B, Skander H: Interferon therapy in myelomatosis. Lancet 1:245, 1979 8. Barlogie B, Alexanian R. Dicke K, Zagers G. Spitzer G, Jrgannath S, Horwitz L: High dose chemoradiotherapy and autologous bone marrow transplantation for resistant multiple myeloma. B l d 70:869, 1987 9. Bergsagel DE, Sprague CC, Austin C. Griffith KM: Evalualion of new chemotherapeutic agents in the treatment of multiple myeloma IV: L-phenylalanine mustard. Cancer Chemother Rep 21:87, 1962 IO. Alcxanian R, tiaut A, Khan AU, Lane M, McKelvey E, Migliore P, Stuckey W, Wilson ti: Treatment for multiple myeloma. JAMA 208:1680, 1969 I I . Costa G. Engle RL Jr, Schilling A, Carbone P, Kochwa S, (ilidewell 0: Melphalan and prednisone: An elTcctive combination lor ihe ireiltiiient ol inultiplc inyeionla.Am J Mrd 54589, 1973 12. Uuric UG,Salinon SE:The current status and future pros- pects of treatment for mthtiple myeloma. Clin Haematol 11:181, 1982 13. Sporn JR, Mclntyre OR: Chemotherapy of previously untreated multiple myeloma patients: an analysis of recent treatment results. Semin Oncol 13:318, 1986 14. Kyle RA, Greipp PR, Gertz MA: Treatment of refractory multiple myeloma and considerations for future therapy. Semin Oncol 13:326, 1986 15. Buzaid AC. Durie BG: Management of refractory myeloma: A review. J Clin Oncol6:889, 1988 16. DeWald GW,Kyle RA, Hicks GA. Greipp PR: The clinical significance of cytogenetic studies in IO0 patients with multiple myeloma. plasma cell leukemia or amyloidosis. Blood 66:380, 1985 17. Gould J. Alexanian R, Goodacre A, Pathak S, Hecht B, Barlogie B: Plasma cell karyotype in multiple myeloma. Blood 71:453, 1988 18. Latreille J, Barlogie B, Gohde W, Johnston D. Drewinko B, Alexanian R: Cellular DNA content as a marker of human multiple myeloma. Blood 55:403. 1980 19. Bunn PA. Krasnow S, Makuch RW. Schlam M, Schechter G: Flow cytometric analysis of DNA content of bone marrow cells in patients with plasma cell myeloma: Clinical implications. Blood 59528. 1982 20. Barlogie B, Latreille J, Swartzendruber D, Smallwood L, Maddox A, Raber M, Drewinko B, Alexanian R: Quantitative cytology in myeloma research, in Salmon SE (ed): Clinics in Haematology. London, Saunders, 1982, p 19 21. Smith L, Barlogie B, Alexanian R: Biclonal and hypodiploid multiple myeloma. Am J Med 80:841. 1986 22. Durie B, Baum V, Vela E, Mundy G: Abnormalities of chroniosoine 6q and osteoclast activating factor production in multiple n~ycloniaB. l d 68:208A, 1986 876 BARLOGIE ET AL 23. Dalton WS, Durie BGM. Alberts DS, Gerlach JH, Cress AE: 42. Salmon SE: in virro cloning and chemosensitivity of marrow Characterization of a new drug-resistant human myeloma cell line myeloma stem cells, in: Salmon SE (ed): Clinics in Hematology. that expresses pglycoprotein. Cancer Res 46:5125. 1986 Saunders, 1982, 11:47 24. Bell DR, Trent JM, Willard HF. Riordan JR, Ling V: 43. Millar BC, Bell JBG, Lakhani A. Aylime MJ, Selby PI. Chromosomal location of human p-glycoprotein sequences. Cancer McElwain TJ: A simple method for culturing myeloma cells from Genet Cytogenet 25:I4 I , 1987 human bone marrow aspirates and peripheral blood in vitro. Br .I 25. Barlogie B. Alexanian R: Cellular aspects of mycloma: Bio- Hncmatol 69:197, 1988 logic and clinical implications. in Dclamore IW (ed): Multiple 44. Kishimoto T Factors affecting B-cell growth and differentia. Myeloma and Other Paraproteinemias. Edinburgh, Churchill Liv- tion. Ann Rev lmmunol 3:133, 1985 ingstone. 1986, p I54 45. Kishimoto T B-cell stimulating factors (BSFs): Molcculw 26. Barlogie 8, Hittelman W. Spitzer G, Trujillo J, Hart J. structure. biological function, and regulation of expression. J Clin Smallwood L. Drewinko B: Correlation of DNA distribution abnor- lmmunol7:343, 1987 malities with cytogenetic findings in human adult leukemia and 46. Yokota T, Otsuka T, Mosmann T. Banchereau J, DeFrance lymphoma. Cancer Res 37:4400. 1977 T, Blanchard D, Vries JED, Lee F. Arai K: Isolation and characler- 27. Gratzner H: Monoclonal antibody to 5-bromo- and ization of a human interleukin cDNA clone, homologous to mnuw 5-iododeoxyuridine: A new reagent for detection of DNA replica- B-cell stimulating activities. Proc Natl Acad Sci USA 83:5894. tion. Science 218:474. 1982 1986 28. Dolbeare F, Gratzner H. Pallavicini M. Gray JW: Flow 47. Rabin EM, Mond JJ. Ohara J, Paul WE: B cell stimulatory cytometric measurement of total DNA content and incorporated factor (BSF)-I prepares resting B cells to enter S phase in response bromodeoxyuridine. Proc Natl Acad Sci USA 80:5573, 1983 to anti-lgM and to lipopolysaccharide. J Exp Med 164517, 1986 29. Durie BGM, Salmon SE: Staging, kinetics and now cytome- 48. Shimizu K, Hirano T, Ishibashi K. Nakano N. Taga T. try of multiple myeloma, in Wicrnik P,Canellos G, Kyle R, Schill'cr Sugamura K, Yamamura Y. Kishimoto T: Immortali7ation of , C (eds): Neoplastic Diseases of the Blood. New York, Churchill BGDF (BCGF 11)-and BCDF-producing T cells by human T cell Livingstone. 1985, p 513 leukemia virus (HTLV) and characteriiation of human BGDI: 30. Greipp PR, Kyle RA: Clinical morphological and cell kinetic (BCGF 11). J Immunol 134:1728.1985 differences among multiple myeloma. monoclonal gammopathy of 49. Hirano T, Yasakuwa K, Hanada [I, Tagar T. Watanabe Y. undetermined significance, and smoldering multiple myeloma. Matsuda T, Kashiwamura S . Nakajima K, Koyama K. lwanatsu A. Blood 62:166. 1983 Tsumasawa S . Sakijama F, Matsui H, Takahara Y. Taniguchia T, 3I . Boccadoro M, Gavarotti P,Fossati G . Pileri A, Marmont F. Kishimoto T Complementary DNA lor a novel human interleukin Nerretto G, Gallamin A, Volta C. Tribalto M, Testa M. Amadori S , (BSF-2) that induces B lymphocytes to produce immunoglobulie. Mandelli F, Durie BGM: Low plasma cell '[HI thymidine incorpo- Nature 324:73, 1986 ration in MGUS. smoldering myeloma and remission phase myelo- 50. Van Damme J, Opdenakker G, Simpson RJ, Rubira M R . ma: A reliable indicator of patients not requiring therapy. Or J Cayphas S , Vink A, Billiau A. van Snick J: Identification of the Haematol 58:689, 1984 human 26 kd protein, interferon beta-2 (IFN-beta-2), as a 0-mil 32. Greipp PR. Witzig TE, GonchorolT NJ, Habermann TM, hybridoma/plasmacytoma growth factor induced by interleukin and Katzmann JA, OFallon WM. Kyle RA: Immunofluorescence label- tumor necrosis factor. J Exp Med 165:914, 1987 ing indices in myeloma and related monoclonal gammopathies. 51. Gauldie J, Richards C. Harnish D, Landsdorp P, Raumann Mayo Clin Proc 62969,1987 H: Interferon beta-2/B cell stimulatory factor type 2 shares mnn* 33. Boccadoro M, Gavarotti P, Fossati G, Massaia M.Pileri A, cyte-derived- hepatocyte stimulating factors and the major acute Durie BGM: Kinetics of circulating B lymphocytes in human phase protein response in liver cells. Proc Natl Acad Sci USA myeloma. Blood61:812, 1983 84:7241, 1987 34. Durie BGM, Salmon SE, Moon TE: Pretreatment tumor mass, cell kinetics and prognosis in multiple myeloma. Blood 55:364, 1980 52. Vink A, Coulie PG, Wauters P, Nordan RP, van Snick 1: B-cell growth and dimerentiation activity of interleukin I. IIP I a d related plasmacytoma growth factors. Synergy with Interleukin I. 35. Latreille J. Barlogie B, Dosik G. Johnston DA, Drewinko B, Eur J lmmunol 18507, 1988 Alexanian R: Ploidy and proliferative characteristics in monoclonal gammopathies. Blood 59:43.1982 36. Durie BGM, Young LA. Salmon SE: Human myeloma in vitro colony growth Interrelationship between drug sensitivity, cell kinetics, and patient survival duration. Blood 61:929. 1983 37. Drewinko B. Alexanian R, Boyer H. Barlogie B, Rubinow SI: The growth fraction of human myeloma cells. Blood 57:333, 1981 38. Gerdes J, Dallenbach F, Lennert K: Growth fraction in malignant non-Hodgkin's lymphomas (NHL) as determined in siru with the monoclonal antibody Ki-67. Hematol Oncol 2:365, 1984 39. Grogan TM. Lippman SM. Spier CM, Slyman DJ, Rybski JA. Range1 CS. Richter LC, Miller T P Independent prognostic significance of a nuclear proliferative antigen in diffuse large cell 53. lkebuchi K, Wong GG, Clark SC. lhle JN. Ilirai Y,0aa.a M: Interleukin 6 enhancement of interleukin 3-dependent prolifefa. tion of multipotential hemopoietic precursors. Proc Natl Acad %! USA 84:9035,1987 54. Kawano M, HiranoT. Matsuda T. Taga T, Horii Y,IwatoK. Asaoku H, Tang B. Tanabe 0,Tanaka 11, Kuramoto A. Kishimata T: Autocrine generation and requirement of DSF-2/11.-6 for humma multiple myelomas. Nature 332:83, 1988 55. Klein B. Zhang X-G. Jourdan M, Content J, lioussiau F. Aarden L, Piechaczyk M, Bataille R: Paracrine but not aut&n regulation of myeloma-cell growth and dimerentiation by interln kin-6. Blood (in press), 1989 lymphomas as determined by the monoclonal antibody Ki-67. Blood 71:1157. 1988 56. Klein R, Jourdan M, Vasquez A, Ihgas R. Rntaille R. Production of growth factors by human myeloma cells. Cancer Re 40. Matuzaki H, Hata H, Takeya M. Takatsuki K: Establish- 47:4856, 1987 ment and characterization of an amylase producing human myeloma 57. Greipp R, Raymond NM, Kyle R A , OFallon WM: MulIIfi cell line. Blood (September), 1988 myeloma: Significance of plasmablastic subtype in rnorpholnpid 41. Hamburger AW, Salmon SE Primary bioassay of human classification. Blood 65:305, 1985 I myeloma cell stem cells. J Clin Inv 60:848, 1977 58. Barlogie B, Alexanian R, Pershouse M, Smallwood I. Smith PLASMA CELL MYELOMA-INSIGHTSAND ADVANCES 877 I.: Cytophsniic immunoglobulin content in multiple myeloma. J Clin Invest 76:765, 1985 S9. Choi Y, Wong M: Double light chain production by leukemic cells of cummon clonal origin: A rare report with a review of pcrtinent literature. Am J Hematol I1:93. 1981 60. Hieter PA, Korsmeyer SJ, Waldemann TA, Leder P Human immunoglobulin kappa light chain genes are deleted or rearranged in lambda producing B cells. Nature 290:368. 1981 61. Barlogie B. Latreille J. Freireich E. Fu C, Mellard D, Msistrich M, Andreeff M: Characterization of hematologic malignancies by Bow cytometry. Blood Cells 6:719, 1980 b2. Barlogie B. Alexanian R, Gehan EA, Smallwood L, Smith T. Drswinko 8: Marrow cytometry and prognosis in myeloma. J Clin lnvcst 72:853. 1983 63. Traganos F, Darzynkiewicz 2,Sharpless T, Melamed MR: Siniultaneousstaining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system. J Histochem Cytochem 25:46, 1977 64. Mellstedt H. Hammorstrom S, Holm G: Monoclonal lymphocyte population in human plasma cell myeloma. Clin Exp lmmunol 11:371.1974 65. Bast E. van Camp B, Reynaert P, Wirniga G, Ballinx R: ldiotypic peripheral blood lymphocytes in monoclonal gammopathy. c'liii Exp lmmunol47:682. 1982 66. Berenson J. Wong R. Kim K. Brown N, Lichtenstein A: Evidence of peripheral blood B lymphocyte but not T lymphocyte involvement. Blood 701550, 1987 67. Caligaris-Cappio F, Bergui L, Tesio L. Pizzolo G . Malavasi F,Chilosi M.Campana D, van Camp B. Jonossy G: Identification of malignant plasma cell precursors in the bone marrow of multiple myeloma. J Clin Invest 76:1243. 1985 68. Barlogie B, Alexanian R, Smith L, Dixon D, Smallwood L. Drlasalle K: Prognostic implications of tumor cell DNA and RNA content in multiple myeloma. Blood 66338, 1985 69. Kubagawa 14, Vogler LB, Capra JD, Conrad ME, Lawton AK, Cooper MD: Studies on the clonal origin of multiple myeloma. Uie of individually specific (idiotype) antibodies to trace the oncogenic event to its earliest point of expressionin B-cell differentiation. J Exp Med I50:792.1979 70. Bast BJEG. Boom SE, Ballieux RE: Characterization of the mlony forming cell in monoclonal gammopathies. Blood 60~608, 1982 71. Grogan TM. Durie BG: Lomen C. Spier C, Wirt DP, Bagle R. Wilson GS,Richter L, Vela E. Maxey V, McDaniel K, Rangel C: Delineationof a novel pre-B cell component in plasma cell myeloma: Iiiiniunochcmical,immunophenotypic, genotypic,cytologic,cell culture and kinetic features. Blood 70:932, 1987 72. Epstein J. Barlogie B, Katzmann J, Alexanian R: Phenotypic heterogeneity in aneuploid multiple myeloma indicates pre-B cell involvement. Blwd 71:861, 1988 13. Durie BGM. Grogan TM: CALLA-positive myeloma: An aggressive subtypc with poor survival. U l d 66:229, 1985 74. Grogan TM, Durie BGM, Spier CM, Richter L, Vela E Myelomonocytic antigen positive multiple myeloma. Blood (in press), 1989 15. Durie BGM, Grogan TM, Spier C, Vela E, Baum V, Rodriqucz MA, Frutigcr Y: Myelomonocytic myeloma cell line (LB W l ) . U l d (in prcss), 1989 16. Brown G, Bunce CM, Howie AJ, Lord JM: Stochastic or orderd lincage commitment during hemopoiesis, editorial. Leukemia 1:1500155, 1987 77. Garrett LR, Durie BGM, Nedwin GE, GillespieA, Bringman T, Sabatini M, Uertolini DR, Mundy GR: Production of the bone resorbing cytokine lyinphotoxin by cultured human myeloma cells. N Engl J Mzd 317526. I987 78. Cozzolino F, Torcia M, Aldinucci D, Rubartelli A, Miliani A, Shaw R, Lansdorp AR. Di Guglielmo R: Production of interleukin-1 by bone marrow myeloma cells: Its role in the pathogenesis of lytic bone lesions. Blood (in press), 1989 79. Kawano M, Yamamoto I, lwato K, Tanaka H, Kideki A, Tanabe 0.lshikawa H, Nobuyoshi M, Ohmoto Y.Hirai Y, Kuramoto A: Myeloma cells produce IL-I, having bone-resorbing activity. Blood (in press), 1988 80. Cordingley IT. Hoffbrand AV, Heslop HE, Turner M, Bianchi A, Reittie JE. Vyakarnam A. Meager A Tumour necrosis factor as an autocrine tumour growth factor for chronic B-cell malignancies. Lancet 8592:969, 1988 8 I.Marchetti D, Van NT. Gametchu B, Thompson EB, Kobayashi Y, Watanabe F, Barlogie B: Flow cytometric analysis of glucocorticoid receptor using monoclonal antibody and Huoresceinated ligand probes. Cancer Res (in press). 1989 82. Rossi J-F, Durie BGM, Duperray C. Braich T, Marion SL, Pike JW. Haussler MR, Janbon C, Bataille R Phenotypic and functional analysis of 1,25-dihydroxyvitamin D3 receptor mediated modulation of the human myeloma cell line RPMI 8266. Cancer Res 48:1213, 1988 83. Danel L, Menouni M. Cohen JH. Magaud JP, Lenoid G, Revillard P, Saez S: Distribution of androgen and estrogen receptors among lymphoid and haemopoietic cell lines. Leuk Res 9:1337, 1985 84. Danel L. Vincent C. Rousset F. Klein B, Bataille R, Flacher M, Durie BGM, Revillard P Estrogen and progesterone receptors in some human myeloma cell lines and murine hybridomas. J Steroid Bioc'hem (in press), 1989 85. Alexanian R; Barlogie B. Dixon D: High-dose glucocorticoid treatment of resistant myeloma. Ann Intern Med 105:8, 1986 86. Alexanian R, Gutterman J, Levy H: Interferon treatment for multiple myeloma, in Clinics in Haematology. London, Saunders, 1982, p 21 I 87. Van NT. Raber MN, Barrows G, Barlogie B: Estrogen receptor analysis by flow cytometry. Science 224:876, 1984 88. Katzmann JA: Myeloma-induced immunosuppression: A multistep mechanism. J Immunol 121:1405. 1978 89. Jacobson DR, Zolla-Pazner S: Immunosuppression and infection in multiple myeloma. Semin Oncol8:282, 1986 90. Ullrich S , Zolla-Pamer S: lmmunoregulatory circuits in myeloma. Clin Hematol I1:87, 1982 91. PreudHomme JL, Brouet JC. Seligmann M: Membranebound IgD on human lymphoid cells, with special reference to immunodeficiency and immunoproliferative diseases. lmmunol Rev 37:127. 1977 92. Roman S , Moore S. Darby C, Muller S. Hoover RG: Modulation of Ig gene expression by I g binding factors. Suppression of alpha-H chain and lambda-2-L-chain mRNA accumulation of MOPC-315 by IgA-binding factor. J lmmunol40:3622, 1988 93. Ling V: Multi-drug-resistant mutants, in Gottesman MM (cd): Molecular Cell Genetics. New York, Wiley, 1985, p 773 94. lnaba M. Watanabe T, Sugiyama Y: Kinetic analysis of active efflux of.vincristine from multidrug-resistant P388 leukemia cells. Jpn J Cancer Res 78:397. 1987 95. Willingham MC. Cornwell C. Cardarelli C, Gottesman MM, Pastan 1: Single cell analysis of daunomycin uptake and efflux in multidrug-resistant and -sensitive KB cells: effects of verapamil and other drugs. Cancer Res 46594 I, I986 96. Kartner N, Everndcn-Porelle D, Bradley G, Ling V: Detection of p-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies. Nature 316:820, 1985 97. Epstein J, Barlogie B, Ling V, Alexanian R: P-glycoprotein expression in plasma cell myeloma is associated with resistance to VAD. Blood (in press), 1988 878 BARLOGIE ET AL 98. Tsuro T, lida H. Tsukagoshi S. Sakurai Y: Overcoming vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res 41:1969, 1981 99. Cornwell M, Pastan I, Gottesman MM: Certain calcium channel blockers bind specifically to multidrug-resistant human KB carcinoma membrane vesicles and inhibit drug binding to p-glycoprotein. J Biol Chem 262:2166,1987 100. Dalton WS, Durie BGM. Salmon SE, Grogan TM, Scheper RJ, Meltzer PS:Multidrug resistance in multiple myeloma. Detection of p-glycoprotein in clinical specimens and reversal of resistance with verapamil. Blood 70834A. 1987 101. Selvanayagam P, Barlogie B: Manuscript in preparation. 102. Lee M, Selvanayagam P, Alexanian R, Stass S,Barlogie B: Rearrangement (REARR) of T-cell receptor (TCR) gamma chain gene in multiple myeloma. AACR. Abstract X706. 1987 103. Narni F. Kudo J, Mars W. Calabretta B, Florine D, Barlogie B, Saunders G: HLA-DR-associated invariant chain is highly expressed in chronic lymphocytic leukemia. Blood 68:189. 1986 104. Narni F,Selvanayagam P. Barlogie B, Saunders G:HLA- associated invariant chain gene expression in human B cell neoplasia. Leukemia 1304. 1987 105. Fahrlender PD, Sumegi J, Yang J-Q.Wiener F, Marcu K. Klein G:Activation of the c-myc oncogene by the immunoglobulin gene enhancer after multiple switch region-mediated chromosomal rearrangements in a murine plasmacytoma. Proc Natl Acad Sci USA 82:3746,1985 106. Potter M: Plasmacytomas in mice. Semin Oncol 13:275, 1986 107. 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. Blood 71:30. 1988 108. Sumegi J. Hedberg T. Bjorkholm M, Godal T, Mellstedt H. Nilsson MG, Perlman C. Klein G: Amplification of the c-myc oncogene in human plasma cell leukemia. Int J Cancer 36:367. 1985 109. Meltzer P. Shadle K. Durie B: Somatic mutation alters a critical region of the c-myc gene in multiple myeloma. Blood 70:985A,1987 110. Selvanayagam P. Goodacre A, Strong L, Saunders G. Barlogie B: Alterations of bcl-I oncogene in human multiple myeloma. AACR, Abstract #76. 1987 I 1 I. Tsuchiya H,Eptein J. Selvanayagam P, Dedman J, Gallick G, Alexanian R,Barlogie B Correlated flow cytometric analysis of H-ras p21 and nuclear DNA in multiple myeloma. Blood 72:796, 1988 1 1 2. Harris H: The Analysis of malignancy by cell fusion: The position in 1988. Perspective in Cancer Research. Cancer Res 48:3302, 1988 113. Klein G,Klein E Myc/Ig juxtaposition by chromosomal translocation: Some new insights. puzzles and paradoxes. lmmunol Today 6:208.1985 114. Barlogie B. Smallwood L, Smith T, Alexanian R: High serum levels of lactic dehydrogenase identify a high grade lymphoma-like myeloma. Ann Intern Med (in press), 1989 1 IS. Bersagel DE, Bailey AJ, Langley GR, McDonald R, White D. Miller A: The chemotherapy of plasma cell myeloma and the incidence of acute leukemia. N Engl J Med 301:743. 1979 116. Zajac-Kaye M. Gelmann EP, Levens D: A point mutation in the c-myc locus of a Burkitt lymphoma abolishes binding of a nuclear protein. Science 2401776,1988 117. Najarian T, Castleman B: Is low dose radiation associated with myeloma? N Engl J Med 300:1278. 1979 118. lshimaru T. Finch SC: More on radiation exposure and multiple myeloma. N Engl J Med 301:439,1979 119. Cuzick J: Radiation-induced myelomatosis. N Engl J Med 304204.198I 120. lchimaru M. IshimaruT, Mikami M, Matsunaga M: Multi- ple myeloma among atomic bomb survivors in Hiroshima and Nagasaki. 1950-76 Relationship to radiation dose absorbed by marrow. JNCl69:323.1982 121. Smith RJ: Study of atomic veterans fuels controversy. Science 221:733.1983 122. Rinsky R, Smith H,Hornung R, Filloon T, Young R,Okitn A. Landrigan P: Benzene and leukemia. N Engl J Med 316:1044. I987 123. Kagan E, Jacobson RJ, Yeung KY, Haidak DJ, Nachnani GH: Asbestos-associated neoplasms of B cell lineage. Am J Med 67:325. 1979 124. Gallagher RP, Spinelli JJ, Elwood JM, Skippen DH: hller. gies and agricultural exposure as risk factors for multiple myeloma. Br J Cancer 48:853,1983 125. Pearce NE. Smith AH. Fischer DO: Malignant lymphoma and multiple myeloma linked with agricultural occupations in a New Zealand Registry-based study. Am J Epidemiol I21:225. 1985 126. Howard AD, Moore J Jr, Tomaszweski MM: Occurrencenf multiple myeloma three years after successful renal transplantation. Am J Kidney Dis 10147,1987 127. Vandermolen LA, Fehir KM. Rice L Multiple myeloma in a homosexual man with chronic lymphadenopathy. Arch Intern Md 145:745,1985 128. Thomas MA, Ibels LS,Wells JV, Isbister JP, Cooper DA. McMahon C: IgA kappa multiple myeloma and lymphadenopathy syndrome associated with AIDS virus infection. Aust NZ J Med 16:402,1986 129. Barlogie B, Alexanian R. Jagannath S.Zagars G. Spitzer6. Dunphy F. Yau J. LeMaistre F, Dicke K:High dose therapy with autologous marrow transplantation for multiple myeloma (Mhl) Blood, Abstract Proceeding 862,I988 130. Werne A, Joshua DE, Kronenbcrg H: Light chain irotyp associated suppression of surface immunoglobulin expression on peripheral blood lymphocytesin myeloma during plateau phase. Rr J Haematol 58:483,1984 131. Knowling MA. Haarwood AR, Bergsagel DE: Cornparim of extramedullary plasmacytomas with solitary and multiple plasma cell tumors of the bone. J Clin Oncol 1:255.1983 132. Alexanian R,Barlogie B, Dixon D: Prognosisof asymptom. atic multiple myeloma. Arch Intern Med 148:1963,1988 133. Alexanian R:Diagnosisand management of multiple myel* ma, in Wiernik PH (ed): Neoplastic Diseases of the Blood. Ncw York, Churchill Livingstone. 1984,p 529 134. Durie BG: Staging and kinetics of multiple myeloma.Semin Oncol 13300,t986 135. Salmon SE.Smith BA: Immunoglobulin synthesis and told body tumor cell number in IgG multiple myeloma. J Clin lnval 49:1I14,1970 136. Durie BGM,Salmon SE: A clinical staging system fa multiple myeloma. Correlation of measured myeloma cell mass with presenting clinical features, response to treatment and survival. Cancer 36:842. 1975 137. Merlini G, Waldenstrom JG. Jayakar SD: A new improved clinical staging system for multiple myeloma based on analynii of 123 treated patients. Blood 55:1011, 1980 138. Child JA. Norfolk DR, Cooper Eil: Serum beta-2 microplobulin in myelomatosis: Potential value in stratification and monitoring. Br J Haematol 63:406,1986 139. Garewal H,Durie BGM, Kyle R A , Finley P, Rower R. Serokman R: Serum beta-2 microglobulin in the initial stagin? nnd PLASMA CELL MYELOMA-INSIGHTS AND ADVANCES 879 rubrequent monitoring of monoclonal plasma cell disorders. J Clin Oricol 2:5 I , 1984 140. Alexanian R, Barlogie B, Fritsche H: Beta 2 microglobulin in multiple myeloma. Am J Hematol20:345, 1985 141. Bataille K, Grenier J, Sany J: Beta-2-microglobulin in myeloma: Optimal use for staging, prognosis and treatment-a prospective study of 160 patients. Blood 63:468, 1984 142. Alexanian R, Barlogie B: VCAD-VAD as initial chemotherapy for multiple myeloma. Proc ASH Ann Meet, vol72, 1988 (abstr m59) 143. Boccadoro M, Durie BG, Frutiger Y, Gavarotti P, Redoglia V. Massaka M. Dalberto M, Marmont F, Gallamini A.Tribalto M et ai: Lack of correlation between plasma cell thymidine labellingindex dnd serum beta-2-microglobulin in monoclonalgammopathies. Acta IhKIlYdtd 78:239, 1987 144. Greipp PR, Katzmann JA, O'Fallan WM, Kyle RA: Value of beta-2 n~icroglobulinlevel and plasma cell labeling indices as prognostic factors in patients with newly diagnosed myeloma. Blood 72~219.1988 145. Salmon SE, Wampler SE: Multiple myeloma: Quantitative staging and assessment of response with a programmable pocket calculator. Blood 49:379. 1977 146. Chronic Leukemia-Myeloma Task Force, National Cancer Institute: Proposed Guidelines for Protocol Studies. I1 Plasma Cell Mycloma. Cancer Chemother Rep 4:145, 1973 147. Alexanian R, Bonnet J, Gehan E, Haut A, Hewlett J, Lane M, Monto R, Wilson ti: Combination chemotherapy for multiple myeloma. Cancer 30:382. 1972 148. McLaughlin P. Alexanian R: Myeloma protein kinetics following chemotherapy. Blood 60:851, 1982 149. Hobbs JR: Monitoring myelomatosis. Arch Intern Med 135:125, 1975 150. Salmon SE, Haut A, Bonnet J, Amare M, Weick J, Durie BGM. Dixon D: Alternating combination chemotherapy improves burviva1 in multiple myeloma. A Southwest Oncology Group Study. J Clin Oncol 1:453, 1983 151. Durie BG, Dixon DO, Carter S, Stephens R. Rivkin S. Bonnet J. Salmon SE, Dabich L, Files JC, Costanzi JJ: improved rurvival durdlion with combination chemotherapy induction for niultiph myeloma: A Southwest Oncology Group study. J Clin Oncol4:1227, 1986 152. Barlogie B. Smith L, Alexanian R: Effective treatment of advanced multiple myeloma refractory to alkylating agents. N Engl J Mcd 3101353,1984 153. Barlogie E. Hall R. Zander A. Dicke K. Alexanian R: High dosc melphaian with autologous bone marrow transplantation for multiple myeloma. Blood 67: 1298. 1986 154. Barlogie B, Alexanian R. Smallwood L. Cheson B, Dixon D, D i k e K, Cabanillas F Prognostic factors with high dose melphalan fur rrfractory multiple myeloma. Blood 72:2015, 1988 155. Vadhan-Raj S. Keating M, LeMaistre A, Hittelman WN, McCredie K, Trujillo JM, Broxmeyer HE, Henny C, Gutterman J: EAects of recombinant human granulocyte-macrophage colonyrtiniulating factor in patients with rnyelodysplastic syndromes. N Engl J Med 317:1545. 1987 156. McCredie KB. Freireich EJ: Cells capable of colony formation in the peripheral blood of man. Science 171:293,1971 157. Korbling M, Dorken B, Ho AD, Peuuto A, Hunstein W, Fliedner TM: Autologous transplantation of blood-derived hemopoietic stem cells after myeloablative therapy in a patient with Burkitt's lymphoma. Concise Report. Blood 67529, 1986 158. Osserman EF, DiRe LB, DiRe J. Sherman WH, Hersman JA. Storb R: Identical twin marrow transplantation in multiple myeloma. Acta Haematol68:215, 1982 159. Fefer A, Cheever MA, Greenberg PO: Identical twin (syngeneic) marrow transplantation for hematologic cancers. JNCI 76: 1269, I986 160. Tura S,Cavo M; Baccarani M. Ricci P, Gobbi M: Bone marrow transplantation in multiple myeloma. Scand J Haematol 36:176, 1986 161. Gahrton G, Tura S , Flesch M. Gratwohl A. Gravett P, Lucarelli G, Michallet M, Reiffers J, Ringden 0, van Lint MT, Vernant JP, Zwaan F E Bone marrow transplantation in multiple myeloma. Report from the European Cooperative Group for Bone Marrow Transplantation. Blood 69:126, 1987 162. Gahrton G: Allogenic bone marrow transplantation studies in multiple myeloma. Acta Haematol (in press), 1989 163. Kernan NA, Byers V, Scannon PJ, Mischak RP, Brochstein J, Flomenberg N. Dupont B, OReilly RJ: Treatment of steroidresistant acute graft-vs-host disease by in vivo administration of an anti-T-cell ricin A chain immunotoxin. JAMA 259:3 154, 1988 164. Quesada JR, Alexanian R. Hawkins M, Barlogie B, Borden E, Itri L. Gutterman JU: Treatment of multiple myeloma with recombinant alpha-interferon. Blood 67:275, 1986 165. Mandell; F, Tribalto M, Avvisati G. Cantonetti M, Petrucci MT, Boccadoro M. Pileri A. Marmont F. Resengotti L, Lauta V, Dammacco F Recombiaant interferon alfa-2b (Intron A) as postinduction therapy for responding multiple myeloma patients. M84 protocol. Cancer Treat Rev I S(suppl A):43, 1988 166. Oken MM. Kyle RA. Greipp PR. Kay NE. Tsiatis A, OConnell MJ: Alternating cycles of VBMCP with interferon (rIFN-alpha 2) in the treatment of multiple myeloma. Proc Am Soc Clin Oncol 7:868A. 1988 167. McElwain TJ, Powles RL: High-dose intravenous melphalan for plasma-cell leukaemia and myeloma. Lancet 1922, 1983 ' 168. Selby PF. McElwain TJ. Nandi AC. Perren TJ, Powles RL, Tillyer CR. Osborne RJ, Slevin ML, Malpas JS: Multiple myeloma treated with high dose intravenous melphalan. Br J Haernatol66:SS. 1987 169. Selby P, Zulian G, Forgeson G, Nandi A, Milan S , Meldrum M. Osborne R. Malpas JS, McElwain TJ: Developments in treatment of myeloma. Acta Haematol (in press), 1988 170. Durie BGM, Russell DH. Salmon SE: Reappraisal of plateau phase in myeloma. Lancet 2:65. 1980 171. Bergsagel DE: Use a gentle approach for refractory myeloma patients, editorial. J Clin Oncol6:757, 1988 172. Young DC, Griffin JD: Autocrine secretion of GM-CSF in acute myeloblastic leukemia. Concise Report. Blood 68:1178, 1986