Articles. Funding National Institute of Health Research, Medical Research Council, and Cancer Research UK.

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1 Response comparison of multiple myeloma and monoclonal gammopathy of undetermined significance to the same anti-myeloma therapy: a retrospective cohort study John P Campbell*, Jennifer L J Heaney*, Sankalp Pandya, Zaheer Afzal, Martin Kaiser, Roger Owen, J Anthony Child, David A Cairns, Walter Gregory, Gareth J Morgan, Graham H Jackson, Chris M Bunce, Mark T Drayson Summary Background Multiple myeloma is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS), which is usually only treated by a form of anti-multiple myeloma therapy if it is causing substantial disease through deposition of secreted M proteins. However, studies comparing how MGUS and multiple myeloma plasma cell clones respond to these therapies are scarce. Biclonal gammopathy multiple myeloma is characterised by the coexistence of an active multiple myeloma clone and a benign MGUS clone, and thus provides a unique model to assess the responses of separate clones to the same anti-multiple myeloma therapy, in the same patient, at the same time. We aimed to identify how MGUS and multiple myeloma plasma cell clones responded to anti-multiple myeloma therapy in patients newly diagnosed with biclonal gammopathy multiple myeloma. Methods In this retrospective cohort study, we identified patients with biclonal gammopathy multiple myeloma by central laboratory analysis of 699 newly diagnosed patients with multiple myeloma enrolled in three UK clinical trials (Myeloma IX, Myeloma XI, and TEAMM) between July 7, 4, and June, 5. In addition to the inclusion criteria of these trials, our study necessitated at trial entry the presence of two distinct M proteins in immunofixation electrophoresis. The primary endpoint was difference in response achieved with anti-multiple myeloma therapy on MGUS (which we defined as ) and multiple myeloma (M) clones overall, within patients, and between therapy types with international therapy response criteria assessed with χ² analyses. We analysed by intention to treat. Findings 44 patients with biclonal gammopathy multiple myeloma with IgG or IgA MGUS clones were subsequently identified from the three trials and then longitudinally monitored. 4 (9%) of M clones had a response to therapy (either complete response, very good partial response, partial response, or minor response) compared with only 8 (64%) of clones (p= ). For the patients who received intensive therapy, there was no difference between the proportion of responding clones in M (9 [95%]) and (5 [75%], p= ). However, for the 7 patients who received non-intensive therapy, 6 (94%) of M clones had a response compared with ten [59%] of clones (p= ). When examining clones within the same patient, (68%) of 44 individual patients had different levels of responses within the M and clones. One patient exhibited progression to myeloma and subsequently died. Interpretation These results show that, in patients with biclonal gammopathy multiple myeloma, anti-multiple myeloma therapies exert a greater depth of response against multiple myeloma plasma cell clones than MGUS plasma cell clones. Although some MGUS clones exhibited a complete response, many did not respond, which suggests that the underlying features that render multiple myeloma plasma cells susceptible to therapy are present in only some MGUS plasma cell clones. To determine MGUS clone susceptibly to therapy, future studies might seek to identify, with biclonal gammopathy multiple myeloma as an investigative model, the genetic and epigenetic alterations that affect whether MGUS plasma cell clones are responsive to anti-multiple myeloma therapy. Funding National Institute of Health Research, Medical Research Council, and Cancer Research UK. Introduction Multiple myeloma, a cancer of immunoglobulin-secreting plasma cells, is the most common cause of blood cancer deaths worldwide and is consistently preceded by an asymptomatic precursor termed mono clonal gammopathy of undetermined significance (MGUS)., International guidelines do not recommend treatment of MGUS and instead a watch-and-wait approach or clinical study enrolment is advocated until multiple myeloma arises. Prevalence of MGUS increases with age and is 4% in adults older than 5 years in the general population, 4 7 is more common in men than in women, is twice as common in black people than in white people, 7,8 and progresses to multiple myeloma at a rate of 5 % each year. 9, Smouldering multiple myeloma an intermediate disease stage between MGUS and multiple myeloma has a % risk of multiple myeloma progression each year initially, but therapy is not recommended because studies of intervention with conventional chemotherapy have shown little benefit. Studies 4 of modern therapy methods in high-risk patients with smouldering multiple Lancet Haematol 7; 4: e Published Online November, 7 S5-6(7)9- See Comment page e565 *These authors contributed equally Institute of Immunology and Immunotherapy (J P Campbell PhD, J L J Heaney PhD, S Pandya MRes, Z Afzal MSc, Prof M T Drayson MD) and School of Biosciences (Prof C M Bunce PhD), University of Birmingham, Birmingham, UK; Department for Health, University of Bath, Bath, UK (J P Campbell); Institute of Cancer Research, London, UK (M Kaiser MD); St James s University Hospital, Leeds, UK (R Owen MD, Prof J A Child MD); Clinical Trials Research Unit, University of Leeds, Leeds, UK (D A Cairns PhD, Prof W Gregory PhD); The Myeloma Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA (Prof G J Morgan MD); and Northern Institute of Cancer Research, University of Newcastle, Newcastle, UK (Prof G H Jackson MD) Correspondence to: Prof Mark T Drayson, Institute of Immunology and Immunotherapy, Clinical Immunology Service, University of Birmingham, Birmingham B5 TT, UK m.t.drayson@bham.ac.uk Vol 4 December 7 e584

2 Research in context Evidence before this study Multiple myeloma consistently arises from a premalignant plasma cell clone called monoclonal gammopathy of undetermined significance (MGUS) that is present in 4% of the population older than 5 years. International guidelines do not recommend screening for or treatment of MGUS unless it is directly causing substantial morbidity eg, arising from M protein deposition diseases, such as monoclonal gammopathy of renal significance; polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome; or light-chain amyloidosis. In the treatment of MGUS, anti-multiple myeloma therapy is used, but only a few studies have compared responses of MGUS and multiple myeloma clones with these therapies. Consequently, whether the spectrum of response of MGUS plasma cell clones will be similar or different to that of the spectrum of response of multiple myeloma plasma cell clones in relatively rare conditions like light-chain amyloidosis is not known. The need for randomised studies of the efficacy of different therapies against diseases such as light-chain amyloidosis are hindered by small patient numbers and so there is strong reliance on the results of randomised trials in multiple myeloma on the assumption that the results will largely translate to MGUS plasma cell clones. However, few comparisons exist of the efficacy of anti-multiple myeloma therapies against MGUS versus multiple myeloma, which reflects the difficulty of comparison between two sets of diseases and associated morbidities that are very different, that in themselves are very heterogeneous, and within which patient tolerance of therapy also exhibits great variability. Before this study, we did literature searches using MEDLINE for studies in English published before Aug, 7, and found minimal investigation into the effects of anti-multiple myeloma therapy on a rare type of multiple myeloma termed biclonal gammopathy multiple myeloma. Biclonal gammopathy multiple myeloma represents the simultaneous presence of a multiple myeloma plasma cell clone and an MGUS plasma cell clone. As such, biclonal gammopathy multiple myeloma serves as a unique model to assess both MGUS and multiple myeloma plasma cell clone responses to the same therapy, at the same time, in the same microenvironment with both clones sharing almost all of the patient s genes. Added value of this study Both MGUS and multiple myeloma are characterised by monoclonal plasma cells in the bone marrow and monoclonal antibody in blood, and accordingly, changes in the concentration of blood monoclonal antibody provide a unique biomarker of a patient s disease activity and are central to monitoring response to therapy and for relapse from remission. By contrast with most blood cancers, this makes frequent longitudinal measurement of disease activity cheaply and very effectively available by frequent blood sampling. Our blood monoclonal antibody concentration results show similar MGUS and multiple myeloma plasma clone responses in only a third of patients with biclonal gammopathy multiple myeloma, highlighting to our knowledge, for the first time the high prevalence of this disparity, and, by nature of the biclonal gammopathy multiple myeloma model, that the explanation is most likely to be in genetic or epigenetic differences between the MGUS and multiple myeloma plasma cell clones of the same patient. Implications of all the available evidence Many MGUS plasma cell clones were unresponsive to available anti-multiple myeloma therapies, including intensive therapy, immunomodulatory drugs, and proteasome inhibitors, despite the same therapies in the same patients being much more effective against multiple myeloma plasma cell clones. This result highlights the need for caution when translating anti-multiple myeloma therapy to the rare patients with MGUS who require treatment and the need to find alternative therapies for these patients. The biclonal gammopathy multiple myeloma model, although clinically uncommon, has important and unique facets that will facilitate better understanding of MGUS and multiple myeloma and the search for new therapies. myeloma have shown variable effects and are under continued investigation. 5 Studies 6 that investigate an even earlier intervention in patients with MGUS have so far been restricted to nutritional compounds. However, MGUS needs to be treated clinically when it is causing substantial morbidity by immunoglobulin deposition as monoclonal gammopathy of renal significance (MGRS); polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome; or light-chain amyloidosis. 7 9 In these situations, anti-multiple myeloma therapies are used selectively to treat each condition, even though the efficacy of these treatments against MGUS compared with multiple myeloma plasma cell clones is not well characterised. This knowledge gap reflects the difficulty of comparison between two sets of diseases and associated morbidities that are very different, that in themselves are very heterogeneous, and within which patient tolerance of therapy also shows great variability. For further insight, we aimed to evaluate responses of both multiple myeloma and MGUS plasma cell clones to exactly the same anti-multiple myeloma therapy, in the same patient, at the same time, with multiple myeloma with biclonal gammopathy as an investigative model. We have recently confirmed that up to % of newly diagnosed patients with multiple myeloma have two M proteins in serum immunofixation electro phoresis, termed biclonal gammopathy multiple myeloma. Both MGUS and multiple myeloma are characterised by M proteins monoclonal whole antibody and free light-chains in e585 Vol 4 December 7

3 blood, and accordingly, changes in the concentration of blood monoclonal antibodies provide a unique biomarker of a patient s disease activity and are central to monitoring response to therapy and identification of relapse from remission. As MGUS evolves into multiple myeloma, intraclonal heterogen eity increases, and at diagnosis multiple myeloma often has ten or more parallel subclones with different combinations of somatic mutations. These compete and can each manifest as being dominant at different stages of disease. 4 In some patients, this subclonal evolution can be observed by changes in the relative amounts of whole monoclonal antibodies and free-light-chain monoclonal antibodies that are secreted, but importantly the heavy-chain and light-chain types and their electrophoretic mobilities remain the same and identical between subclones. 5 As such, it has been proposed that, in most cases of biclonal gammopathy multiple myeloma, the largest monoclonal antibody is a product of the active multiple myeloma clone and its subclones, and the usually ten to times smaller monoclonal antibody represents a separate MGUS clone that is a relic of previous biclonal gammopathy of undetermined significance (BGUS). 6 BGUS is a much more common condition than biclonal gammopathy multiple myeloma, 6 and has a prevalence among all MGUS of 5% in black patients, 7% in white patients, and % in Hispanic patients. 7 A study 7 of 59 biclonal gammopathies including patients with biclonal gam mopathy multiple myeloma and BGUS diagnosed in one centre from 98 to 9, found that in patients receiving either multiple myeloma or Waldenstrom s macroglobulinaemia therapy, the MGUS clones in biclonal gammopathy multiple myeloma responded to therapy overall. However, to what degree the response of multiple myeloma and MGUS clones correlate within the same patient and vary between patients requires further detailed study in a larger group of patients treated more recently with conventional therapies. Here, following central laboratory analysis and screening of monoclonal antibodies in serum from 699 newly diagnosed patients with multiple myeloma entered into three multicentre UK clinical trials, we have investigated the responses of multiple myeloma and MGUS plasma cell clones to conventional anti-multiple myeloma therapies in 44 patients diagnosed with bi clonal gammopathy multiple myeloma. We aimed to provide a comprehensive longitudinal assessment of a biclonal gammopathy multiple myeloma cohort through the course of disease from diagnosis, response to therapy, and relapse from remission. Methods Study design and participants Patients included in our study were all enrolled in a multicentre, phase trial: either the UK Medical Research Council Myeloma IX trial (ISRCTN68454), the Cancer Research UK Myeloma XI trial (ISRCTN494785), or the UK National Institute of Health Research Tackling EArly Morbidity and Mortality in myeloma (TEAMM) trial (ISRCTN57976). From these trials, we identified 58 patients with a biclonal gammopathy multiple myeloma diagnosis, as described elsewhere, and all of whom had assessable longitudinal data. We excluded patients whose secondary biclonal gammopathy multiple myeloma monoclonal antibody exhibited an IgM isotype (4 [4%] of 58 patients) on the basis that these IgM monoclonal antibodies are most likely to be secreted from lymphoplasmacytic clones that might progress to lymphoma rather than the IgG-secreting or IgA-secreting plasma cell MGUS clones that might progress to multiple myeloma. As such, 44 patients were eligible for inclusion in this study. The Myeloma IX trial evaluated the effects of bisphosphonate and thalidomide therapy on progressionfree survival and overall survival. All enrolled patients had newly diagnosed symptomatic multiple myeloma and were aged 8 years or older. The study protocol and findings have been described in detail previously. 8 Patients were assigned to bisphosphonate (oral clodronic acid 6 mg/day or intravenous zoledronic acid 4 mg every 8 days with induction chemotherapy, and every 8 days thereafter) and induction treatments via an intensive or non-intensive treatment pathway. The intensive pathway consisted of between four and six -day cycles of either cyclophosphamide, vincristine, doxorubicin, and dexamethasone (CVAD; 5 mg oral cyclophosphamide per week, 4 mg vincristine daily combined with 9 mg/m² doxorubicin daily as a 4-day continuous infusion, and 4 mg dexamethasone daily on days 4 and 5) or oral cyclophosphamide, thalidomide, and dexamethasone (CTD; 5 mg cyclophosphamide per week, mg thalidomide daily and increasing to mg daily as tolerated, and 4 mg dexamethasone daily on days 4 and 5). After completion of induction therapy, patients underwent peripheral blood stem-cell mobilisation and harvest, intravenous high-dose melphalan treatment ( mg/m²), and autologous stem-cell transplantation. The non-intensive pathway consisted of between six and nine 8-day cycles of either oral melphalan and prednisone (7 mg/m² melphalan and 4 mg prednisone, both on days 4), or attenuated oral CTD (CTDa; 5 mg cyclophosphamide per week, 5 mg thalidomide daily initially and increasing to mg/day as tolerated, and mg dexamethasone daily on days 4 and 5 8) until best response. After initial therapy, all eligible patients were randomly assigned a second time to no maintenance or low-dose thalidomide maintenance therapy given until disease progression (5 mg daily for 8 days, increasing thereafter to mg daily if well tolerated). eligible patients with biclonal gammopathy multiple myeloma from Myeloma IX were identified; seven patients were in the intensive pathway (n=4 CTD; n= CVAD) and six were in the non-intensive pathway (n= melphalan and prednisone; n=5 CTDa). Vol 4 December 7 e586

4 The Myeloma XI trial completed recruitment in 7. All enrolled patients had newly diagnosed symptomatic multiple myeloma and were aged 8 years or older. Myeloma XI had two treatment pathways, intensive and non-intensive, which both had induction, consolidation, and maintenance therapy components. In the intensive pathway, Myeloma XI compared oral cyclophosphamide, lenalidomide (Revlimid; Celgene Corporation, Summit, NJ, USA), and dexamethasone (CRD; 5 mg cyclophosphamide on days and 8, lenalidomide 5 mg daily for days, and dexamethasone 4 mg daily on days 4 and 5) with oral CTD (cyclophosphamide 5 mg weekly, thalidomide initially mg daily for weeks increasing to mg daily, and dexamethasone 4 mg daily on days 4 and 5) or carfilzomib (Kyprolis; Amgen, Thousand Oaks, CA, USA) and CRD (CCRD; cyclo phosphamide 5 mg on days,, 8, 9, 5, and 6, carfilzomib mg/m² administered on days and of cycle and dose capped at a body surface area of m², lenalidomide 5 mg daily for days, and dexamethasone 4 mg on days 4, 8, 9, 5, and 6), repeated every 8 days for up to six cycles, followed by high-dose melphalan and autologous stem-cell transplant, as per local practice. In the non-intensive pathway, attenuated oral CRD (cyclo phosphamide 5 mg on days and 8, lenalidomide 5 mg daily for days, and dexamethasone mg daily on days 4 and 5 8) was compared with CTDa (cyclophosphamide 5 mg daily, thalidomide initially 5 mg daily for 8 days and increasing every 8 days by 5 mg increments to mg daily, and dexamethasone mg daily on days 4 and 5 8), repeated every 8 days for six cycles or longer. In patients who showed a suboptimal response to induction therapy, the use of bortezomib, cyclophosphamide, and dexamethasone was investigated. Patients were ran domly assigned further to no maintenance, oral lenalidomide, or oral lenalido mide and vorinostat main tenance therapy (lenalidomide mg daily and vorinostat mg daily on days 7 and 5 ) on a repeating cycle every 8 days until disease progression. 4 eligible patients with biclonal gammopathy multiple myeloma ( patients in the intensive pathway and in the nonintensive pathway) were found to be eligible for the study herein. TEAMM was a randomised, double-blind, placebocontrolled trial assessing the benefits of antibiotic prophylaxis (levofloxacin) and its effects on infections associated with health care. All enrolled patients had newly diagnosed symptomatic multiple myeloma and were aged years or older. All anti-multiple myeloma therapies were eligible for use in TEAMM. Seven eligible patients with biclonal gammopathy multiple myeloma from TEAMM were identified and received CTD (n=4), CTDa (n=), and melphalan, prednisolone, and thalidomide (n=). The administration of these therapies was delivered as per local practice. Procedures All serological laboratory testing was done at the Clinical Immunology Service, University of Birmingham (Birmingham, UK). Monoclonal antibodies in serum were identified by immunofixation electrophoresis (Sebia, Lisses, France) and quantified by protein electrophoresis and densitometry (Interlab, Rome, Italy). If accurate quantitation of monoclonal antibodies was not feasible eg, when a pair of monoclonal antibody bands shared the same position on serum protein electrophoresis (ie, IgGκ and IgGλ) or when the size of the monoclonal antibodies was too small to be detected by densitometry (limit of detection is around g/l) monoclonal antibody concentration was estimated from immunofixation electrophoresis, taking into account the size of the monoclonal bands as a proportion of total immunoglobulin of the heavy-chain isotype (ie, taking into account background polyclonal immunoglobulin). This exercise was done by three experienced immunofixation electrophoresis users, independently and without knowledge of the sample timepoint, before agreement was reached per sample. Serum IgG, IgM, IgA, creatinine, β microglobulin (all Roche, Basel, Switzerland), and free light-chains (Binding Site, Birmingham, UK) were measured on a Roche Hitachi Modular analyser. In patients with a light-chain monoclonal antibody identified by immunofixation electrophoresis without a heavy-chain component (light-chain-only myeloma), monoclonal antibody size was measured and monitored by concentrations of involved free light-chains and expressed in g/l. Outcomes Our primary objective was to determine whether antimultiple myeloma therapy exerted differences in the depth of response between MGUS and multiple myeloma clones in biclonal gammopathy multiple myeloma, and our secondary objective was to identify whether the frequency of relapse among MGUS and multiple myeloma clones differed after anti-multiple myeloma induction therapies. We used internationally accepted response criteria 9, to define and categorise depth and duration of response according to changes in M protein concentrations between biclonal gammopathy multiple myeloma diagnosis and the date of maximum response to anti-multiple myeloma therapy and disease progression. Statistical analysis We hypothesised that multiple myeloma and MGUS clones would respond differently to anti-multiple myeloma therapy. Patient responses to therapy were categorised with international response criteria on the basis of the percentage decrease in monoclonal antibody size (%=complete response, 9%=very good partial response, 5 9%=partial response, 5 5%=minor response, <5% change=stable disease, and >5% increase=progressive disease). 9, Response codes were further aggregated into good (complete response or very e587 Vol 4 December 7

5 good partial response) versus moderate (partial response or minor response) versus poor (stable disease or progressive disease) responses; or responders (complete response, very good partial response, partial response, or minor response) versus non-responders (stable disease or progressive disease). We analysed frequency differences between groups (eg, M [the largest monoclonal antibody (in g/l), which is categorised as the multiple myeloma monoclonal antibody] or [the smaller monoclonal antibody, which is categorised as the MGUS monoclonal antibody] monoclonal antibodies and treatment pathways) with the following statistical tests: for factors within the same patients (ie, response or no response M or ) by McNemar s test; for factors within the same patients (ie, good, moderate, or poor response M or ), and for 6 factors within the same patients (ie, complete response, very good partial response, partial response, minor response, stable disease, or progressive disease M or ) by Stewart Maxwell s test; and for factors between independent groups (ie, intensive or non-intensive therapy response or no response) by Pearson s χ² test. We assessed correlations between change in M and concentrations from diagnosis to maximum response with Spearman s correlational coefficient. We present data as median (IQR), unless otherwise stated. Analyses were by intention to treat. We analysed data with IBM SPSS (version 4) and R (version..) software. Role of the funding source The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. Results Patients were enrolled between July 7, 4, and June, patients with biclonal gammopathy multiple myeloma were initially identified from 699 newly diagnosed patients with multiple myeloma. Of these, we found that 4 patients with biclonal gammopathy multiple myeloma (4%) had a secondary biclonal IgM M protein. On the basis that we could not discern whether these IgM M proteins were secreted by a lymphoplasmacytic clone (which might progress to Waldenstrom s macroglobulinaemia or lymphoma) or an MGUS plasma cell clone (which might progress to the rare entity IgM multiple myeloma), we excluded these patients from our analyses. As such, 44 (76%) patients were eligible for inclusion in this study. 8 (64%) patients were men and 6 (6%) were women, with a median age of 69 years (IQR 64 75), serum β-microglobulin concentration of 5 µg/ml ( 5 5 5), and serum creatinine concentration of 9 µmol/l (85 ). Regarding the baseline isotype and size characteristics of M and monoclonal antibodies, M monoclonal antibodies were around ten to 5 times larger in size than their counterparts (table ). Four (9%) patients presented with a free light-chain monoclonal antibody that did not have an associated intact immunoglobulin monoclonal antibody of the same light-chain isotype (ie, light-chainonly myeloma); each of these free light-chain monoclonal antibodies were larger than 5 g/l (range 6 9 g/l). In each of these patients, the other biclonal monoclonal antibody which was a different light-chain isotype to the free light-chain monoclonal antibody was small in size (range 4 5 g/l). Comparatively, in ten other patients with biclonal gammopathy multiple myeloma with free light-chain monoclonal antibody concentrations of more than 5 g/l, all had a light-chain-matched intact immunoglobulin M protein that was larger than 8 g/l. As such, in the four patients mentioned before with biclonal gammopathy multiple myeloma with a lightchain-only M protein, the free light-chain monoclonal antibody was selected as the M monoclonal antibody and the intact immunoglobulin monoclonal antibody as the monoclonal antibody. In (7%) of 44 patients with biclonal gammopathy multiple myeloma, the light-chain isotype of the monoclonal antibody was different to Frequency Concentration (g/l) Multiple myeloma (M) IgG Total 7 6 ( 5 ) IgGκ 7 6 ( 5 7) IgGλ 4 ( ) IgA Total 9 6 ( 4 5) IgAκ 6 6 ( 8 5 4) IgAλ 6 ( ) IgD Total 6 IgDκ IgDλ 6 FLC Total 4 ( 6 6) FLCκ 4 ( 9 6) FLCλ 7 Monoclonal gammopathy of undetermined significance () IgG Total 6 ( 6 4 9) IgGκ 7 5 ( 5 4 5) IgGλ 6 8 ( 5 ) IgA Total 7 ( 4) IgAκ 5 ( 8 7) IgAλ 6 5 ( 8) Data are n or median (IQR). M=multiple myeloma monoclonal antibody. =monoclonal gammopathy of undetermined significance monoclonal antibody. FLC=free light-chain. Table : Characteristics and frequencies of M and monoclonal antibodies at trial entry Vol 4 December 7 e588

6 See Online for appendix M patients patients All patients (n=44) Response* 4 (9%) 8 (64%) Good 7 (6%) (45%) Complete response 4 (%) 8 (4%) Very good partial response (%) (5%) Moderate 4 (%) 8 (8%) Partial response (5%) 7 (6%) Minor response (7%) (%) Poor (7%) 6 (6%) Stable disease (5%) 6 (6%) Progressive disease (%) No response (7%) 6 (6%) Intensive therapy (n=) Response 9 (95%) 5 (75%) Good 5 (75%) (55%) Complete response 8 (4%) (5%) Very good partial response 7 (5%) (5%) Moderate 4 (%) 4 (%) Partial response (5%) 4 (%) Minor response (5%) Poor (5%) 5 (5%) Stable disease (5%) 5 (5%) Progressive disease No response (5%) 5 (5%) Non-intensive therapy (n=7) Response** 6 (94%) (59%) Good (59%) 8 (47%) Complete response 5 (9%) 7 (4%) Very good partial response 5 (9%) (6%) Moderate 6 (5%) (%) Partial response 5 (9%) (%) Minor response (6%) Poor (6%) 7 (4%) Stable disease 7 (4%) Progressive disease (6%) No response (6%) 7 (4%) Data are n (%). M=multiple myeloma monoclonal antibody. =monoclonal gammopathy of undetermined significance monoclonal antibody. *p= ; χ²=8 between M and responses to all therapies in all patients (n=44) when responses were assessed using international response codes. p= when we assessed using response or no response composite response codes. p= ; χ²= 4 when we analysed with the good, moderate, and poor composite response codes. p= 74; χ²=7 4 between M and responses to all therapies in all patients (n=44) when responses were assessed using international response codes. p= when we assessed using response or no response composite response codes. p= 7; χ²=4 57 when we analysed with the good, moderate, and poor composite response codes.**p= 9; χ²= 6 between M and responses to all therapies in all patients (n=44) when responses were assessed using international response codes. p= when we assessed using composite response or no response codes. p= 49; χ²=6 4 when we assessed using good, moderate, or poor composite response codes. Table : Serological responses of M and monoclonal antibodies at the time of maximum response to anti-multiple myeloma therapy that of the M monoclonal antibody, and so the M and monoclonal antibodies that were associated with free light-chain secretion could be identified. (97%) of these patients had evaluable free light-chain data. The MGUS clone was associated with free light-chain monoclonal antibody secretion in only five (6%) of these patients; this outcome is consistent with MGUS usually having a whole monoclonal antibody without detectable free light-chain monoclonal antibody. By contrast and as expected, 89% of the patients without biclonal gammopathy multiple myeloma with evaluable free light-chain data in the Myeloma IX (n=8) and Myeloma XI (n=54) studies secreted free light-chain monoclonal antibody (data not presented herein), and we found that 8 (9%) of patients with biclonal gammopathy multiple myeloma had a free light-chain monoclonal antibody secreted by their M clone. Serum free lightchain concentrations at biclonal gammopathy multiple myeloma diagnosis (4 patients [95%] with evaluable data) are in the appendix (p ). Response to anti-multiple myeloma therapy was assessed in 44 patients. Four (9%) of the 44 patients did not receive a biological anti-multiple myeloma drug, while the other 4 (9%) patients all received either thalidomide or lenalidomide at diagnosis, with two patients subsequently receiving bortezomib after exhibiting a less than very good partial response to immunomodulatory drugs therapy. Of these 44 patients, 4 (98%) achieved a maximum and stable reduction in concentrations of M monoclonal antibody while one patient s multiple myeloma progressed during induction therapy. The median duration from diagnosis to M maximum response was 58 days (IQR 87 6). At diagnosis, M concentrations were greater than concentrations (table ); however, fewer clones responded to antimultiple myeloma therapy (p= ; table ). The M response was a very good partial response or better in 7 (6%) of 44 patients compared with (45%) patients for responses (p= ; table ). Insignificant monoclonal antibody responses (<5% reduction or an increase in monoclonal antibody concentrations) were seen in only three (7%) patients for M response but in 6 (6%) patients for response (p= ; table ). We assessed the effects of intensive versus non-intensive induction therapies (from the Myeloma IX and Myeloma XI trials only) on M and concentrations (table ). We observed that responses to non-intensive therapy were inferior to M responses to non-intensive therapy (p= 9; table ) while the number of M and monoclonal antibodies exhibiting a very good partial response or better to non-intensive therapy were similar, the number of stable disease or progressive disease responses were greater among clones (p= ). With regards to intensive therapies, M and responses did not differ (p= ), with similar frequencies of complete responses achieved by M and clones; however, we noted a higher frequency of non-responsive clones e589 Vol 4 December 7

7 (five [5%] of ) than M clones (one [5%] of ). In separate analyses, M responses (p= 9) and responses (p= ) between intensive and non-intensive therapies did not differ (data not shown). We next assessed whether M and clones responded differently within patients (figure ). Maximum response for was always achieved within the time taken to achieve maximum response for M. The percentage reduction of M and monoclonal antibodies from trial entry to time of maximum response to therapy was not correlated and neither were the absolute reductions of M and (in g/l). Further analyses (table ) revealed that ten (%) patients achieved a complete response of both M and, and, notably, complete responses were not limited to light-chain-matched monoclonal antibodies (figure ). Complete responses were achieved in seven (6%) patients in whom the M and monoclonal antibodies had different light-chain isotypes, showing that anti-multiple myeloma therapy can often achieve complete responses in two unequivocally independent plasma cell clones in the same patient (figure ). In three of these patients, free light-chain associated with the clone was elevated at trial entry (to 77 mg/l, 5 mg/l, and 86 mg/l; appendix p ) without evidence of renal damage (serum creatinine concentrations all < µmol/l), indicating more advanced neoplastic activity of these particular clones and perhaps particular susceptibility to anti-multiple myeloma therapies (n= for CVAD and n= for CTD, CRD, or CCRD). Despite these several dual complete responses among biclonal gammopathy multiple myeloma clones, M and responses were the same in only 4 (%) patients when using international response criteria (table ); in (5%) patients the response was greater than the M response, and in 9 (4%) patients the M response was greater than the response (table ). In separate analyses, M and responses in the same patient did not differ between the different types of induction therapy (figure ); although insufficient statistical power, due to the broad range of different treatment regimens implemented, restricted these analyses. Finally, responsivity to therapy was not related to the starting size at presentation, since complete responses of were achievable from a starting concentration of more than g/l at disease presentation (appendix p ). We monitored patients with follow-up results available during disease remission to assess M and inter-clonal competition and relapse over time. For these patients, the median follow-up duration was 8 days (IQR ; figure ). We segregated data into patients who achieved a complete response of at maximum response to therapy and those patients that did not (figure ). In the eight patients who achieved a complete response for with no M relapse, two patients had a return of (figure ): IgGκ 5g/L at trial entry and g/l at return (5 days after Percentage change of from trial entry to maximum response to therapy Matched light-chain isotype (n=) Different light-chain isotype (n=7) Responsive M Unresponsive (n=4 [%]) Responsive M Responsive (n=7 [6%]) Percentage change of M from trial entry to maximum response to therapy maximum response) and IgGλ 8 g/l at trial entry and g/l at return (8 days after maximum response). In the ten patients who did not achieve a complete response of at maximum response but had M relapse, one patient had a return of (IgGκ 5g/L at trial entry and g/l at return) and another patient had progression Unresponsive M Unresponsive (n= [5%]) Unresponsive M Responsive (n= [%]) M and light-chain isotype status Matched light-chain isotype Different light-chain isotype Figure : M and monoclonal antibodies from disease presentation to the time of maximum response to anti-multiple myeloma therapy Responses are coded on the basis of whether the M and clone exhibited matched (red circles) or different (clear circles) light-chain isotypes. Of the 44 patients, data for one patient were not plotted as the patient exhibited progression of M and stable in response to anti-myeloma therapy. M=multiple myeloma monoclonal antibody. =monoclonal gammopathy of undetermined significance monoclonal antibody. Complete response for Very good partial response for Partial response for Minor response for Stable disease for Complete response (%) (%) (7%) for M Very good partial 4 (9%) (%) 4 (9%) 4 (9%) response for M Partial response for M (7%) (5%) 6 (4%) Minor response for M (%) (%) (%) Stable disease for M (%) (%) Progressive disease for M (%) M=multiple myeloma monoclonal antibody. =monoclonal gammopathy of undetermined significance monoclonal antibody. Progressive disease for Table : Responses achieved by M and at the time of maximum response to anti-multiple myeloma therapies Vol 4 December 7 e59

8 5 CVAD MP MPT CTD CTDa CTDa or CRDa CTD, CRD, or CCRD Percentage change from trial entry to maximum response to therapy Response achieved (n) Complete response Very good partial response Partial response Minor response Response Stable disease Progressive disease No response M M M M M M M Figure : M and monoclonal antibodies from diagnosis to the time of maximum response to different anti-multiple myeloma therapies Data are from 44 patients with biclonal gammopathy multiple myeloma; one patient was tabulated but only partly plotted because the patient exhibited disease progression of M and stable disease of in response to melphalan and prednisone therapy. Boxplots represent median and IQR, and whiskers represent lowest and highest non-outlier values; outliers are represented by red dots (> 5 IQR) and clear dots (> IQR). M=multiple myeloma monoclonal antibody. =monoclonal gammopathy of undetermined significance monoclonal antibody. CVAD=cyclophosphamide, vincristine, doxorubicin, and dexamethasone. MP=melphalan and prednisone. MPT=melphalan, prednisolone, and thalidomide. CTD=cyclophosphamide, thalidomide, and dexamethasone. CTDa=attenuated CTD. CRD=cyclophosphamide, lenalidomide, and dexamethasone. CRDa=attenuated CRD. CCRD=carfilzomib and CRD. Complete response of achieved at maximum response 8/44 (4%) Complete response of not achieved at maximum response 6/44 (59%) 5/8 (8%) with follow-up data available after maximum response 6/6 (6%) with follow-up data available after maximum response Relapse of M No M relapse Relapse of M No M relapse No change to 7/5 (47%) 94 days (IQR 448) to relapse of M Increase of /5 No change to 6/5 (4%) Monitored for 49 days (IQR 75) Increase of /5 (%) 5 and 8 days to increase No change to 8/6 (5%) 7 days (IQR 478) to relapse of M Increase of /6 (%) 5 and 5 days to increase No change to 6/6 (8%) Monitored for 5 days (IQR 5) Increase of /6 Figure : Flow diagram Relapse among the 44 patients with biclonal gammopathy multiple myeloma after maximum response to anti-multiple myeloma therapy. M=multiple myeloma monoclonal antibody. =monoclonal gammopathy of undetermined significance monoclonal antibody. of to multiple myeloma ( IgGκ 5 g/l at trial entry and 6 g/l at progression; figure ); responded to subsequent therapy in this patient, before a relapse of M occurred and the patient died. Taken together, these results show that most monoclonal antibodies remained stable after a median follow-up of around year (figure ). Discussion The results of this study show that anti-multiple myeloma therapy is more effective against multiple myeloma clones than it is against MGUS clones in patients diagnosed with biclonal gammopathy multiple myeloma. Although we found that some MGUS plasma cell clones exhibited complete responses to anti-multiple myeloma e59 Vol 4 December 7

9 therapies, many did not respond. We did not identify features of disease that predicted MGUS responsivity to therapy; however, the response patterns observed might indicate that some MGUS plasma cell clones have genetic and epigenetic alterations more akin to multiple myeloma that simultaneously renders them more likely to progress to multiple myeloma but also means they are more likely to respond to existing anti-multiple myeloma therapies. We did this novel study because of the increasing need to intervene, at an earlier stage, in high-risk asymptomatic monoclonal gammopathies, and to treat MGUS that causes significant disease usually through M protein deposition in tissues. For POEMS, light-chain amyloidosis, and MGRS, variations of available anti-multiple myeloma therapies are prescribed, 7 9 yet little is known about the relative efficacy of these therapies on MGUS clones versus multiple myeloma plasma cell clones. To provide more insight, we did this innovative evaluation of biclonal gammopathy multiple myeloma through assessment of anti-multiple myeloma therapy, remission, and relapse. To our knowledge, this is the largest study of its kind done to date. Although previous studies 4 have shown that in a few previous cases of biclonal gammopathy multiple myeloma, the monoclonal antibody pairs originate from clonally related plasma cells, most arise from two independent plasma cell clones producing unrelated monoclonal antibodies that exhibit either different lightchain isotypes, or the same light-chain isotype with no clonal relatedness. 4 Thus, in most cases, evaluation of biclonal gammopathy multiple myeloma allows investigation of clonally unrelated MGUS and multiple myeloma responses to therapy, and in doing so enables comparisons within individual patients in whom the two plasma cell clones share the same microenvironment, the same exposure to anti-multiple myeloma therapy, and almost all of their genes. A profound feature of multiple myeloma is the broad scale of subclonal heterogeneity and the evolution of the subclone hierarchy over time and in response to therapy. 4 In some patients, alterations to the subclonal architecture can be observed by changes in the relative amounts of whole monoclonal antibody and free light-chain monoclonal antibody that are secreted, but, importantly, the heavy-chain and light-chain types and their electrophoretic mobilities remain the same and identical between subclones. 5 Accordingly, in the present study, M and monoclonal antibody concentrations represent the total clonal substructure of multiple myeloma and MGUS and do not inform on evolution of intraclonal heterogeneity. Using international consensus response criteria, we found that anti-multiple myeloma therapy commonly achieves complete responses in both MGUS and multiple myeloma clones in biclonal gammopathy multiple myeloma. Moreover, a higher proportion of complete responses was achieved in MGUS monoclonal antibodies than in multiple myeloma monoclonal antibodies, and this MGUS response was not dependent on monoclonal antibody concentration at diagnosis or its heavy-chain or light-chain isotype. In a small subset of three patients exhibiting elevated free light-chain secretion (around mg/l) associated with the clone, complete responses were observed in all of them after anti-multiple myeloma induction therapy, suggesting that monoclonal plasma cells associated with light chain production akin to observations in light-chain amyloidosis 5 7 might be more susceptible to anti-multiple myeloma therapy. However, it could be that in MGUS with light-chain amyloidosis, the amyloidogenic nature of the free lightchain renders those plasma cells more susceptible to antimultiple myeloma therapies, particularly proteasome inhibitors 7 and none of these patients with biclonal gammopathy multiple myeloma had light-chain amyloidosis. Importantly, the overall number of MGUS clones responding to anti-multiple myeloma therapy was inferior to multiple myeloma clones, and insignificant monoclonal antibody responses (<5% reduction in monoclonal antibody concentrations) 9, were observed in more patients for MGUS response than patients who elicited an insignificant multiple myeloma monoclonal antibody response. This higher proportion of MGUS non-responders compared with multiple myeloma nonresponders contrasts with the complete responses of around 4% in MGUS and around % in multiple myeloma clones and indicates a dichotomous response to anti-multiple myeloma therapy in MGUS clones in biclonal gammopathy multiple myeloma. This duality of MGUS responsivity contradicts patterns observed for multiple myeloma responses, which were for the most part responsive to therapy. Taken together, these findings indicate that, unlike multiple myeloma clones that are entirely malignant, the benign nature of MGUS clones in biclonal gammopathy multiple myeloma is divided into those clones associated with resistance to anti-multiple myeloma therapies and those clones that are susceptible to anti-multiple myeloma therapies. Future research could investigate whether the clones that are susceptible to therapies are also those at greatest risk of progression to multiple myeloma. Biclonal gammopathy multiple myeloma provides a good model to investigate this hypothesis and to assess MGUS resistance to antimultiple myeloma therapies, since the nature of the model focuses attention on genomic and epigenetic differences between the MGUS and multiple myeloma clones in the same patient. In addition to the comparisons of therapy responses between groups of multiple myeloma and MGUS clones, our assessment of biclonal gammopathy multiple myeloma enabled the investigation of intrapatient multiple myeloma and MGUS responses. We observed that the multiple myeloma clone exhibited a higher response than the MGUS clone in 44% of patients, and vice versa in 5% of patients. Multiple myeloma and MGUS responses were the same in around % of patients. Most patients Vol 4 December 7 e59

10 had a response of both multiple myeloma and MGUS; however, many patients had a multiple myeloma response but no response in their MGUS clone. We found that the baseline size of the MGUS monoclonal antibody had no effect on the responsivity to therapy, nor did whether the multiple myeloma and MGUS monoclonal antibodies share the same light-chain isotypes. Because of the broad range of anti-multiple myeloma therapies given to patients across the three clinical trials included in our study, we were unable to yield sufficient statistical power to compare the effects of different therapies between multiple myeloma and MGUS monoclonal antibodies in biclonal gammopathy multiple myeloma. Comparisons between intensive and non-intensive therapies preliminarily indicate that inten sive therapies induced deeper responses against both multiple myeloma and MGUS monoclonal antibodies in biclonal gammopathy multiple myeloma. Future larger studies are needed to confirm this observation. Importantly, MGUS response of less than 5% was seen in around 5% of patients receiving a wide spectrum of therapies, including intensive therapy. These findings suggest that the genetic alterations that render a plasma cell clone neoplastic (ie, transformation from benign MGUS to multiple myeloma) are the dominant factor in determining a response to anti-multiple myeloma therapy over and above the individual s genetic make-up, the plasma clone s microenvironment, and the type of anti-multiple myeloma therapy used. Whether treated intensively or non-intensively, we found that MGUS monoclonal antibody concentrations were stable in almost 9% of patients with biclonal gammopathy multiple myeloma, with samples available for monitoring throughout multiple myeloma remission. In one patient, the MGUS monoclonal antibody progressed to multiple myeloma; this progression is broadly in line with known MGUS progression over a -year period in people of this age (median 69 years [IQR 64 75]). 6 By contrast, around 5% of patients had relapse of their multiple myeloma plasma cell clone. These findings indicate that MGUS response to anti-multiple myeloma therapy is of greater duration than multiple myeloma responses, but longer follow-up is needed to confirm duration of remission. A limitation of this study is that we were unable to characterise and evaluate change at the tumour cell level because bone marrow cells were not available at the time of this retrospective study. Consequently, we have not been able to assess two important areas that should be central to future studies of biclonal gammopathy multiple myeloma. These are detailed genetic and epigenetic signatures of MGUS and multiple myeloma clones. Furthermore, investigation into depth of responses to therapy was limited to monoclonal antibody concentrations and we were not able to assess for existence of low-level minimal residual disease. Future studies of biclonal gammopathy multiple myeloma should include flow cytometric identification of plasma cell phenotypes, including distinguishing MGUS and multiple myeloma clones through expression of heavy-chain and light-chain immunoglobulin isotypes. Subsequent single cell genomic and epigenomic analysis should identify the differences between the two neoplastic clones and normal cells, allowing focus on the differences between the MGUS and multiple myeloma clones that underlie their different malignancy and response to anti-multiple myeloma therapy in the same microenvironment. In summary, our findings show that anti-multiple myeloma therapy is more effective against multiple myeloma than it is against MGUS clones in biclonal gammopathy multiple myeloma. Moreover, anti-multiple myeloma therapy induced highly variable responses to MGUS monoclonal antibodies in biclonal gammopathy multiple myeloma, indicating that some MGUS clones are highly responsive to therapy and many are unresponsive; these responses to therapy might indicate that some of these plasma cell clones have genotypes more similar to multiple myeloma and thus are at higher risk of progression. Future biclonal gammopathy multiple myeloma studies on the neoplastic cells, rather than just their secreted M proteins, could provide an understanding of the cause of the high prevalence of MGUS resistance to available anti-multiple myeloma therapies and reveal alternative therapeutic strategies. Contributors JPC, CMB, and MTD wrote the manuscript. MTD, JPC, and JLJH designed the investigation into biclonal gammopathy multiple myeloma. JPC, JLJH, SP, and ZA did the experiments. JPC, JLJH, DAC, and MTD analysed the data. JPC, JLJH, SP, and MTD interpreted the data. JAC, GJM, WG, MTD, RO, GHJ, and MK designed the Myeloma IX, Myeloma XI, and TEAMM trials. All authors approved the manuscript. Declaration of interests MTD reports personal fees and other from Abingdon Health, outside the submitted work. JPC reports other from Abingdon Health, outside the submitted work. RO reports personal fees from Celgene, and personal fees and other from Janssen and Takeda, outside the submitted work. GJM reports personal fees from Amgen, Celgene, Janssen, and Takeda, outside the submitted work. All other authors declare no competing interests. Acknowledgments We are grateful to the National Cancer Research Institute Haematooncology subgroup and to all principal investigators for their dedication and commitment to recruiting patients to the Myeloma IX, Myeloma XI, and TEAMM trials. We thank the Clinical Trials Research Unit at The University of Leeds (Leeds, UK; Myeloma IX and XI) and the Clinical Trials Unit at the University of Warwick (Warwick, UK; TEAMM). We are grateful to the staff of the Clinical Immunology Service in Birmingham and Tim Plant, Karen Walker, Alison Adkins, and Nicola Newnham. Finally, we are grateful to all patients and their clinical teams at centres throughout the UK whose participation made this study possible. The National Institute of Health Research, Medical Research Council, and Cancer Research UK funded the study. References Landgren O, Kyle RA, Pfeiffer RM, et al. Monoclonal gammopathy of undetermined significance (MGUS) consistently precedes multiple myeloma: a prospective study. Blood 9; : Weiss BM, Abadie J, Verma P, Howard RS, Kuehl WM. A monoclonal gammopathy precedes multiple myeloma in most patients. Blood 9; : 548. Rajkumar SV, Dimopoulos MA, Palumbo A, et al. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncol 4; 5: e e59 Vol 4 December 7