Mechanisms of the escape phase of myeloma

Mechanisms of the Escape Phase of Myeloma

D. E. Joshua, J. Gibson, R. D. Brown S UMMA R Y. In multiple myeloma the duration of plateau is an important clinical aud biological determinaut of quality of life and survival. During plateau phase, the tumour is in an indolent state, as manifested by a low labelling index of plasma cells and other proliferative markers, e.g. the thymidine kinase level. The mechanism by which plasma cells escape from this indolent pbase to a more aggressive phase of this disease is unknown, but a number of possible mechanisms have been postulated. These include loss of immunoregulation, clonal evolution, cytokine dysfunction and oncogene activation or tumour suppressor gene dysfunction. As current chemotherapy protocols do not appear to be able to eradicate the malignant clone, uuderstanding the nature of the indolent phase of the malignant clone and the reasons for its escape from this phase are very important and may provide new options for disease control.

Nature of Plateau In patients with multiple myeloma treated with conventional therapy, entry into plateau and the duration of plateau, rather than the tumour response, is a major prognostic factor. Plateau phase is usually defined as 3 to 6 months of clinical stability, stable paraprotein levels and transfusion independence. Patients who attain plateau after chemotherapy or who are in plateau at diagnosis have a similar survival, in contrast to patients who fail to attain plateau irrespective of their initial ‘response’ to treatment who fare badly.’ An essential accompaniment of plateau phase is a low proliferative state of the malignant plasma cells. Thus plateau is always associated with a low labelling index (LI)2 or low levels of other proliferative markers such as serum thymidine kinase (STK).3 Markers of cell death, for example urinary spermidine, are also 10w.~ Plateau is therefore a state of tumour quiescence in which the malignant clone appears to be dormant. No known chemotherapeutic agent is D. E. Jo&m, Royal Prince Australia.

J. Gibson, R. D. Brown, Haematology Dept, Alfred Hospital, Camperdown, NSW 2050,

Bkwd Reviews (1994) 8, 13-20 Q 1994 Longman Group UK Ltd

able to prolong plateau with the possible exception of cr-interferon.5 The mechanism of plateau is unknown, but experimental data suggests that either immunoregulatory mechanisms and/or the intrinsic heterogeneity of the myeloma clone may be involved. It is most likely that a ‘plateau-like’ state occurs in patients who have received intensive chemotherapy and attained complete remission, as these patients almost invariably relapse, and anti-idiotype studies demonstrate the presence of residual paraprotein, even in otherwise apparent clinical complete remissions.6 Thus plateau phase, attained either by chemotherapy or present at diagnosis (smouldering myeloma), is a clinically and kinetically quiescent state of this disease. Escape from plateau (progressive disease) is associated with a poor outlook and understanding the mechanisms of this phenomenon is clearly important for planning strategies for the control of myeloma. This is particularly important given the current debate over the role of high dose therapy and/or autologous transplantation. If such therapy is incapable of eradicating the clonogenic cell in myeloma, then efforts to maintain plateau will become a major therapeutic endeavour.

14 MECHANISMS OF THE ESCAPE PHASE OF MYELOMA Escape from Plateau Phase Disease In general there are two principal scenarios seen when patients escape from plateau phase. The first is a slow indolent escape, often initially unassociated with clinical symptoms. The first indication of plateau escape in such patients is a slow but progressive rise in the paraprotein and/or Bence-Jones protein level, These patients, although clearly again in a progressive phase of their disease, may not require immediate chemotherapy. The second scenario is a much more fulminant escape, often associated with the excretion of Bence-Jones protein of a different electrophoretic mobility from that initially seen, or Bence-Jones protein unassociated with the original heavy chain. This latter phenomenon is the so-called ‘light chain escape’, and occurs in approximately one third of patients. In patients who are relapsing from plateau, the labelling index increases, especially in the early phase and is mirrored by a rising STK. Rarely, the LI of plasma cells does not appear to have risen dramatically despite the onset of fuhninant disease, and it has been postulated that the escape is due to proliferation of the non-plasma cell stem cell component of the disease. It is our experience, however, that a labelling index above that seen in plateau is an invariable feature of escape. Other clinical features of plateau escape include new or progressive bone lesions, increasing anaemia and bone marrow failure. Escape phase is also associated with resistance to chemotherapy and, in its fulminant form, is in many ways akin to blastic transformation of chronic granulocytic leukaemia or the development of aggressive large cell lymphoma after a prolonged phase of indolent follicular lymphoma or chronic lymphatic leukaemia. A number of mechanisms have been postulated to be responsible for escape from plateau, and are listed in Table 1. It is currently unclear to what extent any or all of these postulated mechanisms contribute to in escape from plateau. Clearly some derangements may just be epi-phenomena or a reflection of a more fundamental disturbance of the host/myeloma balance. Before a causative role in plateau escape can be attributed to any one phenomenon, certain prerequisite characteristics would need to be defined. Any mechanism which causes escape must be linked to the proliferative status of the malignant clonogenic cell and it must by definition be inactive during Table 1 Possible mechanisms responsible for loss of plateau 1. 2. 3. 4. 5. 6.

Loss of anti-idiotype immunoregulation Loss of T cell and NK cell immunoregulation IL6 excess and/or IM-receptor dysfunction Nucleoside transporter dysfunction Sequential clonal evolution and chromosome changes Oncogene activation or tumour suppressor gene inactivation or dysfunction 7. Multi-drug resistance phenotype 8. Plasma cell homing receptor dysfunction

plateau. Recent molecular findings have indicated that the proliferative cell of origin of myeloma is a pre-plasma cell with homing characteristics to the bone marrow.‘~’ The fact that progressive disease is always associated with a rise in the labelling index of plasma cells, is a reflection of the close ontological proximity of the proliferative cell to the apparently mature plasma cell.

Mechanisms Responsible for Loss of Plateau Is Escape Phase Due to Loss of Anti-idiotypic Immunoregulation? In the murine plasmacytoma model, several groups have shown that anti-idiotypic immune responses and idiotype specific T-cells can block idiotypic secretion and tumour growth. In the MOPC315 model in particular, idiotypic reactive T-cells have been shown to recognise determinants on the heavy and light chain region of the immunoglobulin.’ Recently the presence of T-cells potentially instrumental in anti-idiotypic control of plateau have been reported in human myeloma, although at this stage their significance is uncertain.” In these studies, the isolation of either CD4 or CD8 T cells which bind fluorescein-labelled F(ab)’ fragments from autologous immunoglobulin but not isotype matched homologous immunoglobulin has been reported. The number of auto-reactive T-cells detected was generally low (S-15%), although in some patients they comprised up to 25% of total T-cells. It is also likely that myeloma patients not only have T-cells which bind unprocessed immunoglobulin, but also have T-cells that recognise macrophage processed idiotype. These cells will not be detected by the above technique, but have been demonstrated in a majority of patients using delayed type hypersensitivity reactions against macrophage processed idiotype. These investigators are now immunising patients with their own autologous idiotype in an attempt to stimulate host mediated control of the turnour.” If anti-idiotypic control is responsible for plateau maintenance it is possible that a change in idiotype induced by somatic mutation could permit escape from anti-idiotypic control mechanisms and hence plateau escape. Arguing against this, however, is recently published evidence that the idiotype does not alter as the patient enters progressive disease.7*8 In lymphoproliferative malignancies, the rearranged immunoglobulin heavy chain gene (IgH) which together with the light chain genes defines the idiotype, provides a valuable marker of the malignant clone. Changing or evolving idiotypes have been demonstrated as part of the natural progression of follicular non-Hodgkin’s lymphoma and acute lymphoblastic leukaemia. l1 We have studied the immunoglobulin gene in myeloma in patients at diagnosis and when they enter progressive disease in order to

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determine if disease progression is associated with change in the rearranged immunoglobulin gene and thus the idiotype of the paraprotein. Our results and those of other investigators clearly demonstrate that the idiotype in myeloma is stable.7,s Furthermore, since there is no active hypermutation in myeloma, clonal succession, in which patients relapse and show heavy IgH rearrangement not present at diagnosis, as seen in ALL, does not occur. This argues that genetic instability in the immunoglobulin gene and thus change in idiotype, with subsequent loss of antiidiotypic control, does not appear to be a mechanism of escape. Is Escape Due to Loss of T-cell and NK Cell Immunoregulation?

Despite the lack of evidence for an alteration in the idiotype of myeloma, there is considerable evidence that immunoregulation is active in this disease and that there is a definite host/tumour interaction. For instance, elevated numbers of peripheral CD8 cells and NK cells have been documented in stable disease, suggesting that elevated CD8 or NK cells may be able to suppress the tumour and maintain plateau phase. Many of these studies show that the increase in T cells is in the activated T cell compartment. It has been recently shown that elevated numbers of peripheral blood CD38 cells are an adverse prognostic finding in myeloma. I2 We have demonstrated that these CD38 + cells are T cells rather than pre-plasma cells as was previously presumed. In addition and perhaps paradoxically, patients with elevated levels of such cells at diagnosis have some features that suggest a better preservation of normal immune function, as assessed by IgM levels within the normal range, re-emphasising the presence of immunoregulatory mechanisms. (unpublished observations) In patients with untreated symptomatic myeloma, both the percentage and absolute number of peripheral blood CD4 cells are usually decreased compared to controls. On the other hand CD8 cells are slightly increased in relative terms although normal in absolute numbers. There exist correlations between high CD8 levels and IgG myeloma, while clinical Stage III disease is usually associated with a lower CD4 count, which in untreated patients is also correlated with anaemia and high /I-microglobulin (B2M) levels.‘3,‘4 Cells bearing the natural killer cell antigens, CD16, CD56 and CD57 have also been studied. There is a significant increase in bone marrow CD1 6 cells and an expansion of the CD56 +CD3 - subset is found.14,15 These data suggest that there is expansion of an immature NK population in the bone marrow. In the peripheral blood, the relative numbers of CD56 + CD3 - cells are significantly increased. In addition, the suppressor cytotoxic subsets CD8 + CD57 + and the total number of CD57 cells are also elevated. No major differences in distribution

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of NK cells according to clinical stage has been found.14 While these data point to the existence of marked alterations in both T cell subsets and NK subsets in myeloma, a relationship to the mechanism of escape disease remains problematical. Such diverse subset variations may represent an epi-phenomenon, rather than having a causal relationship with progressive disease.

Is Escape Due to IL6 Excess and/or IL&receptor Dysfunction?

There is little doubt that IL6 is an important factor in the proliferation of the malignant plasmablast in terminal myeloma. l6 Plasma cell lines can be stimulated by exogenous IL6 and their growth blocked by IL6 antibodies. The evidence for this effect in vitro is now overwhelming. Elevated levels of IL6 are found in the serum of many, though not all patients with myeloma and may be responsible for the suppression of myelopoiesis, activation of osteoclasts and proliferation of plasmablasts. In early myeloma, however, the tumour appears to be mixed and composed of some cells which proliferate in response to IL6 and mature plasma cells which have little proliferative capacity. l7 There is increasing evidence that one can distinguish immature and mature plasma cells within the myeloma clone on the basis of surface antigenic markers. la These subsets of plasma cells have different responsiveness to IL6 and different levels of nucleoside transporters concomitant with their high proliferative ability (see below). In most cases the proportion of proliferating cells is small, and is the probable explanation as to why only a small proportion of plasma cells in patients with early myeloma proliferate in response to IL6 in vitro. It is likely that in plateau phase disease, the plasmablastic component is not large enough to be detectable, but as the disease progresses, plasmablastic cells increase in number, proliferate in response to IL6 and can be blocked by IL6 antibody.” No defects have been reported in the expression of the IL6 receptor. 2o It has, however, been found that the soluble IL6 receptor is able to increase by lo-fold the sensitivity of myeloma cell lines to IL6. Higher levels of soluble IL6 receptor are associated with a poor prognosis. 21 Therefore, while it is clear that plasmablastic disease is associated with sensitivity to IL6 and may be the mechanism by which the cells proliferate, it is not yet clear that IL6 and IL6 receptor dysregulation is the cause of progressive disease, but rather a mechanism by way other genetic abnormalities allow the disease to progress. For instance, while the role of IL6 as a plasmablastic growth factor is supported by the ability to obtain myeloma cell lines whose proliferation is dependent on exogenous IL6, these cell lines are usually obtained from patients with extramedullary proliferation.22 This emphasises that additional genomic events are

16 MECHANISMS OF THE ESCAPE PHASE OF MYELOMA necessary for the IL6 plasma cell growth nexus to be established and maintained. The source of IL6 in plasmablastic myeloma remains unclear. The original hypothesis of autocrine production by myeloma cells has not been confirmed and it seems more likely that autocrine producing IL6 lines occur by secondary mutation. IL6 is most likely produced by paracrine cells, i.e. not of the malignant clone. In addition, other cytokines may be involved in the proliferation of plasma cells by IL6. While the IL6 responsive plasma cell growth nexus is established as a mechanism of proliferation of endstage disease, the mechanism(s) by which patients with plateau phase myeloma develop this sensitivity to IL6 is unclear, but it may be due to interacting cytokines. For example, some authors have demonstrated that a-interferon can cause the development of autocrine IL6 loops. ” The underlying defect may, however, be the intrinsic heterogeneity of plasma cells and ongoing genomic instability, which allows further oncoprotein expression and autonomous growth (vide infra). Is Escape Due to Nucleoside Transporter Dysfunction?

Nucleosides are required for proliferation of mammalian cells. These are provided either by de novo synthesis of purine and pyrimidine nucleosides or by salvage of pre-formed nucleobases and nucleosides. Nucleoside transport across the plasma membrane is mediated by a diffusion process that can be inhibited and measured by the compound nitrobenzyhnercaptopurineriboside (NBMPR). This analogue binds tightly with a kd< 1 nmol, and with a 1: 1 stoichiometry to the equilibrative (es) nucleoside transporter polypeptide. NBMPR can be used to provide a convenient measure of transporter numbers.23 Studies of nucleoside transporter expression in acute leukaemia and lymphomas have suggested that increased nucleoside transporter expression is associated with an increased proliferation of malignant cells.24 Alterations in transporter levels in plasma cells and pre-plasma cells might therefore confer a growth advantage on such cells and be a mechanism of escape. We have recently measured nucleoside transporter expression in plasma cells and have correlated levels of transporter with cellular proliferation and clinically with escape from plateau phase.25 Nucleoside transporters were measured by the fluorescent ligand for the nucleoside transporter S-( SAENTA x 8) fluorescein. In general we found that transporters were not particularly high in myeloma plasma cells compared to plasma cells in normal controls. We did find, however, that nucleoside transporter levels were related to the proliferation of plasma cells, with a significant relationship between transporter levels and both the labelling index and STK. There was, however, no relationship to the expression of c-myc oncoprotein. Transporter levels were higher in primi-

tive plasma cells, identihed as CD38hi CD45 + , than mature plasma cells (CD38hi CD45 negative) and there was a significant trend for increased transporter levels in progressive disease compared to stable disease. Increased transporter levels, however, may be secondary to other intrinsic changes in the plasma cell. Up-regulation of transporters may be the mechanism rather than the cause of progressive disease. The overall low levels of nucleoside transporter levels in myeloma plasma cells has important implications for the use of drugs such as cytosine arabinoside in this disease and may explain the lack of activity of 2-chlorodeoxyadenosine and fludarabine, as all these drugs gain entry to the cell via the nucleoside transporter. Is Escape Due to Cytogenetic Sequential Evolution and Chromosomal Changes?

Initial cytogenetic analysis of plasma cell disorders was hampered by the low mitotic index of the cells under study. Despite this, a number of studies have been performed (see ref. 25, for review). Overall the incidence of a cytogenetically abnormal clone in myeloma is 40 to 50%. This is much higher in patients who have plasma cell leukaemia compared with patients who have monoclonal gammopathy of undetermined signmcance (MGUS) or smouldering myeloma. There is a clear trend towards a poorer prognosis in patients who have more karyotypic abnormalities, however the karyotype of the abnormal clones in myeloma is extremely complex and no systematic pattern of evolution reflecting disease progression has been determined. In general most authors believe that monoclonal gammopathies begin with one chromosomal abnormality and the karyotype becomes more complex through chromosomal evolution during tumour progression. The most common abnormalities are in chromosomes 1, 14q + and 6q-, such abnormalities occurring in approximately 40%, 20% and 7% of patients respectively. 26*27A small number of 5q- abnormalities, Philadelphia chromosome and t( 8; 14) abnormalities have also been described although they are fairly rare. The significance of the chromosome 1 abnormality is not clear. The 14q+ abnormalities are consistent with the B cell nature of these disorders, as the break-point on 14 may involve the immunoglobulin heavy chain locus. However, in myeloma unlike other B cell lymphomas, no consistent abnormality appears to be direct related to progressive or escape disease. Chromosomal abnormalities appear to randomly accumulate as disease progresses and to what extent new subclones are actually induced by mutator mechanisms, chromosoma1 duplication errors, ongoing therapy or natural evolution of the disease, remains to be elucidated. A number of cytogenetic abnormalities have been reported to be related to clinical features and

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phenotype. For example the 6q- abnormality is associated with lytic bone lesions and osteoclast activating factor. 28The relationship between chromosome 1, the most commonly abnormal chromosome which carries the n-ras oncogene, and the relationship between chromosome 14 abnormalities and the immunoglobulin heavy gene is of interest, but of uncertain significance. The retroblastoma (Rb) gene which has occasionally been found to be abnormal is associated with chromosome 13 abnormalities,2g but the high level of c-myc oncoprotein overexpression and the c-myc changes in myeloma (see below), and the lack of chromosomal changes at chromosome 8 are yet to be explained. Again, while these chromosomal changes and rearrangements, which may increase the probability of subsequent errors during mitosis, are associated with progressive disease, whether they are the cause or a result of the neoplasia is still unclear. Such changes may, however, prove useful in determining the prognosis in myeloma as they do in acute myeloid leukaemia. Is Escape Due to Oncogene Activation or Tumour Suppressor Gene Inactivation or Dysfunction?

Unlike other B cell malignancies, no consistent abnormalities in oncogenes have been found in human myeloma. However, a range of different abnormalities of oncogenic expression have been described in a number of individual patients. These include mutations or deletions involving the oncogenes c-myc, 30-32and ras32 or the tumour suppressor genes Rb and ~53. 34 Early studies with c-myc suggested it was associated with increased proliferative activity in B cell malignancies with high mitotic indices, however, it is now clear that c-myc is also elevated in some patients with myeloma and chronic lymphatic leukaemia, both malignancies with a low mitotic index. Gene rearrangements of the c-myc locus are rare in myeloma, but rearrangement of the MLVl-4 locus which maps to 20 kb 3’ of c-myc have been reported in 16% of patients35 and increased c-myc messenger RNA expression has been found in 64% of patients with progressive disease.32 There are a number of drawbacks, however, in associating c-myc expression with progressive disease. Most reports are restricted to analysis of the oncogene at DNA or RNA level and are based on the application of blotting techniques. The inability to assign gene expression to a distinct cell type has led to patients with more advanced disease and high marrow plasmacytosis to be preferred in such analyses. Thus, a causative relationship between tumour mass and biological function of the gene may have been prematurely assumed. The loss and/or rearrangement of the Rb gene has been found to be common in patients with myeloma, but ~53 mutations have been reported to be uncommon.36 Several studies have demonstrated the high

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frequency of n-ras mutations and have also suggested that n-ras might play an important role in progression of the disease or in certain clinical features of the disease such as the occurrence of plasmacytomas.33 In a recent study,37 we used dual label flow cytometry to determine the incidence and intensity of expression of a variety of oncoproteins and tumour suppressor proteins in individual plasma cells of patients with myeloma. Oncoprotein expression was verified by comparison with mRNA expression detected by in situ hybridisation. We established an oncoprotein phenotype of patients with myeloma and were able to correlate the expression of these proteins with disease status and to determine the relationship between oncoprotein expression and markers of proliferation (labelling index and the STK). Using a panel of 8 monoclonal antibodies to oncoprotein and tumour suppressor genes, we found that 82% of patients studied had abnormal expression of at least one of these tumour markers. Increased oncoprotein expression was found in patients with active disease, but was absent in prolonged plateau phase. By using flow cytometry we were able to avoid the bias often seen in PCR and blotting studies. For example, in a recent publication of p53 gene mutations, all patients with < 10% plasma cells were excluded to avoid misrepresentation due to the presence of a normal cell population.38 While no specific oncoprotein was related to the onset of active disease, we found that a chaotic random association of oncoprotein or tumour suppressor gene expression is related to progressive disease. Thus it seems that in myeloma, there is a degree of genomic instability found in the oncogenes but not in the immunoglobulin gene. This could lead to dysregulation of oncoprotein expression, and infers that there is a genomic ‘destabiliser’ in myeloma which allows individual cells to enter proliferative states with the associated up-regulation of other proliferative markers. This phenomenon of additive step-wise, yet random accumulation of abnormalities ultimately results in disease which is both highly proliferative and refractory to drug therapy. The parallels between the situation in myeloma and colorectal cancer are obvious. In the latter situation ras abnormalities and abnormalities of chromosome 5, 17 and 18 all increase in a random fashion as colonic lesions progress from adenoma class I to carcinoma.3g This is consistent with a genetically unstable cell accumulating genetic changes with increasing malignancy. In myeloma, only plasma cells in plateau are negative for all oncoproteins, yet in advanced diseases a number of abnormalities occur. This acquired defect may result from activation of a mutator gene or other factors or abnormalities which increase the possibility of errors during mitosis. There is no evidence of an inherited defect of genetic instability in myeloma such as seen in Fanconi’s syndrome nor is there suggestion of an inherited susceptibility to myeloma per se such as is seen in

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MECHANISMS

OF THE ESCAPE PHASE OF MYELOMA

colon cancer.40 In other B cell malignancies such as lymphoma, specific changes can sometimes be related to progression but often only with clear change in morphology, e.g. follicular lymphoma to Burkitt-like lymphoma associated with c-myc translocation.41 In myeloma all the changes appear random and may represent irrelevant epi-phenomena. However, the constant associations of increased oncoprotein expression with progressive disease and their high incidence argue against this. Is Escape Due to Acquisition of the Multi-drug Resistant Phenotype? Resistance to previously effective chemotherapy is one of the clinical features strongly associated with escape from plateau phase. Although clinical resistance to anti-cancer agents can be evident at presentation, it is more commonly seen during treatment or, in particular, on relapse. In vitro cell culture systems have been extensively used to study this phenomena in numerous tumours and has led to the identification of several putative mechanisms by which malignant cells acquire drug resistance. Such mechanisms include alteration in topoisomerases, increased levels of glutathione S-transferase and metallothionen systems, and the multi-drug resistance (MDR) phenotype.42-45 Cells bearing the MDR phenotype exhibit crossresistance to a wide variety of ‘natural’ cytotoxic agents, including the vinca alkaloids, epidophyllotoxins, anthracyclines, actinomycin D, taxol and colchitine. The phenotype is associated with increased expression of the 170-kd membrane glycoprotein (P-170 or p-glycoprotein). The gene encoding p-glycoprotein belongs to a multi-gene family designated mdr. MDRl encloses p-glycoprotein and, as demonstrated in transfection experiments, is sufficient to produce the MDR phenotype. In contrast MDR2 has not been shown to convey cytotoxic drug resistance. The natural functions of mdr genes remain to be definitely elucidated, although the widespread expression on a large variety of normal tissues including secretary and absorptive epithelium implies normal physiological functions. For p-glycoprotein such functions may include an efflux pump, a ‘membrane vacuum clearer’ or a ‘flippase’, transferring molecules from one side of the plasma membrane to the other.42-45 The phenomenon of p-glycoprotein mediated multi-drug resistance in myeloma and has been studied by a number of groups. As a generalisation, the levels of p-glycoprotein in marrow or tissue biopsies of newly presenting patients are reported to be low. A small number, however, have high levels at presentation and this may, in part, be the explanation for patients who are primarily refractory to chemotherapy. Considerable data has accumulated on the development of drug resistance in chemotherapy treated patients. 46 For instance, in one study,

expression of p-glycoprotein in myeloma patients who had no prior chemotherapy was 6% but rose to 45% in treated patients.47 The level of p-glycoprotein expression strongly correlated with the use of chemotherapeutic agents known to induce this phenomenon in cell lines, particularly vincristine and adriamycin. In patients treated with these agents the incidence of multi-drug resistance reflected the total dose of those particular drugs. This effect is cumulative and patients who received high levels of both vincristine and adriamycin exhibited almost 100% p-glycoprotein expression on their tumour cells. In contrast, melphalan alone was a poor inducer of p-glycoprotein expression (11%). This study also demonstrated that p-glycoprotein expression did not correlate with either clinical parameters (age, sex or stage) or with immunophenotypic factors and, in particular, the proliferative rate.47 Some, but not all, investigators, however, believe that the clonogenic cell of myeloma constitutively expresses p-glycoprotein, and that this cell is present as part of a circulating B cell component. Such cells have been reported to be present in the majority of patients, both treated and untreated, even when the plasma cells of the marrow do not express p-glycoprotein. 48This phenomenon is of considerable importance especially in view of the widespread application of peripheral cell stem autotransplantation; if this cell is the cell responsible for ultimate relapse and escape from plateau, stem cell transplantation cannot be a curative procedure. It seems most likely that the phenomenon of multidrug resistance, at least as reflected in p-glycoprotein expression, is an induced genetic event which is a reflection of the classes of therapeutic agents used to treat the patient, and is not a phenomenon intrinsic to the tumour. The lack of a documented ongoing relationship between expression of p-glycoprotein and proliferation argues against the expansion of these cells as being responsible for loss of plateau. The correlation of p-glycoprotein expression with escape from plateau phase and the subsequent therapeutic implications are not inconsiderable, however, given the increasing use of combination chemotherapeutic protocols that rely heavily on ‘natural’ cytotoxic agents. Strategies to manipulate or modify the MDR phenotype are thus likely to become increasingly important in the management of patients who progress from plateau phase.49-51 Is Escape Due to Plasma Cell Homing Receptor Dysfunction? There is accumulating evidence that the bone marrow microenvironment may be abnormal in B cell malignancies. A pronounced feature of myeloma is its localisation in the bone marrow until the terminal stages of the disease. The homing properties of plasma cells are believed to be due to the expression of a number of adhesion molecules including those

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from the immunoglobin superfamily, beta-l and beta-2 integrins and selectins. There is clearly a difference between the expression of these molecules by malignant myeloma plasma cells and that of benign plasma cells. For instance N-CAM (CD56), a neural cell adhesion molecule appears to be exclusively expressed on malignant plasma cells whereas normal plasma cells and the plasma cells of patients with monoclonal gammopathy of uncertain significance (MGUS) are negative.53954 The disruption of the homing of plasma cells to the marrow in plasmablastic myeloma is well documented. In advanced disease plasma cells may circulate in the blood and extramedullary plasmacytomas are frequently formed. Changes in CD56 expression appear to correlate with the phenomenon and may be a factor responsible for progressive disease. Recent studies show that CD56 is absent in circulating plasma cells and that cells of endstage myeloma and plasma cells leukaemias and bone marrow plasma cells in refractory patients also have a tendency to be CD56-negative.55 It seems as if this adhesion molecule is down-regulated as the disease becomes progressive and its loss is responsible for plasma cell spread outside the bone marrow. These findings appear to be similar to the possible role adhesion molecules play in the dissemination of other B cell malignancies. For instance, there is a correlation between localised lymphoma and CD44 negative lymphoma cells and widespread disease and high CD44 expression. 56 Similarly, in non- Hodgkin’s lymphoma, the adhesion of cells to high endothelial venules is related to dissemination.57 Other molecules including fibronectin and VLAS may play a role in determining the biological properties of myeloma. Plasma cells can be separated into high proliferative, primitive and low proliferative, mature cells on the basis of their expression of the integrin VLAS.” However, while such changes in homing receptor expression may provide a mechanism for increased dissemination of myeloma, there is no evidence that they play an active part in the increased proliferation which is a necessary concomitant of escape phase and changes in homing receptors in advanced, compared to stable, myeloma have not been clearly defined.54 Conclusions In summary, escape from plateau in myeloma is associated with a bewildering array of biological abnormalities, including molecular, genetic and cytogenetic changes, kinetic abnormalities, nucleoside transporter abnormalities, changes in homing receptors, multi-drug resistance and abnormal cytokine and cytokine receptor dysfunction. These abnormalities are seen in cells which are far advanced in their clonal evolution. We believe that the basic abnormality is one of genomic destabilisation, which is not totally random, as immunoglobulin genes are appar-

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ently unaffected. Once malignant plasma cells reach this clonally evolved state they are extremely difficult to eradicate with current chemotherapy. In contrast, during plateau phase the tumour has the characteristics of a benign or minimal deviation tumour, yet still possess a degree of genomic instability. In this state the malignant cells, like normal plasma cells, have a low labeling index, normal levels of oncoprotein expression, normal nucleoside transporter and normal IL6 receptors. It is, however, unlikely that such inactive cells will be able to be eradicated by current chemotherapy protocols. The problem for the physician is thus to keep patients in plateau phase and prevent genomic instability and clonal succession from occurring. The mechanism of the escape phase remains unknown but is probably an inherent abnormality of the malignant clone and may be common to all tumours which transform from indolent low grade disease to tumours of high grade malignancy.

Acknowledgements We wish to thank MS Barbara Hart for secretarial assistance. Supported in part by grants from National Health and Medical Research Council of Australia, State Cancer Council of New South Wales and the Leo & Jenny Leukaemia Foundations.

References 1. Joshua D E, Snowdon L, Gibson J et al. Multiple myeloma: Plateau phase revisited. Hematol Rev Commun 1988; 5: 59-66. 2. Boccadoro M, Gavarotti P, Fossati G. Low plasma cell 3 [H ]-thymidine incorporation in monoclonal gammopathy of undetermined significance (MGUS), smouldering myeloma and remission phase myeloma: A reliable indicator of patients not requiring therapy. Br J Haematoll984; 58: 689-696. 3. Brown R D, Joshua D E, Ioannides R, Kronenberg H. Serum thymidine kinase as a marker of disease activity in patients with multiple myeloma. Aust N Z J Med 1989; 19: 226-232. 4. Durie B G M, Salmon S E, Russell D H. Polyamines as markers of response and disease activity in cancer chemotherapy. Cancer Res 1977; 37: 214319. 5. Mandelli F, Awisati G, Amadori S. Maintenance treatment with recombinant interferon alfa-2b in patients with multiple myeloma responding to conventional induction chemotherapy. New England J Med 1990; 322: 1430. 6. Stevenson G T, Thompson J. Idiotypes and anti-idiotypes in myeloma. Hematol Oncol 1988; 6: 103-106. I. Bakkus M H C, Heirman C, Van Riet I, Van Camp B, Thielemans K. Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation. Blood 1992; 80: 2326-2335. 8. Ralph Q M, Brisco M J, Joshua D E, Brown R, Gibson J, Morley A A. Advancement of multiple myeloma from diagnosis through plateau phase to progression does not involve a new B-cell clone: Evidence from the Ig heavy chain gene. Blood 1993; 82: 202-206. 9. Hoover RG, Kornbluth J. Immunoregulation of murine and human myeloma. Hematol Oncol Clin North Am 1992; 6: 407-424. 10. Osterborg A, Yi Q, Bergenbrant S et al. In: Proceedings of the International Workshop on Multiple Myeloma, Rochester, Minnesota, 56-57. 11. Bird J, Galili N, Link M, Stites D, Sklar J. Continuing rearrangement but absence of somatic hypermutation in immunoglobulin gene of human B cell precursor leukaemia. J Exp Med 1988; 168: 229-245. 12. Omede P, Boccadoro M, Gallone G. Multiple myeloma:

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