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© 1999 American Society for Clinical Oncology Molecular and Clinical Remissions in Multiple Myeloma: Role of Autologous and Allogeneic Transplantation of Hematopoietic CellsFrom the Dipartimento di Medicina ed Oncologia Sperimentale, Azienda Ospedaliera San Giovanni Battista-Divisione Universitaria di Ematologia, Torino; Divisione di Ematologia II, Ospedale San Martino, Genova; Unità Trapianto Midollo Osseo, Ospedale Cervello Palermo; and Divisione Universitaria di Ematologia, Palermo, Italy. Address reprint requests to Paolo Corradini, MD, BMT Unit, Istituto Scientifico H.S. Raffaele, Via Olgettina 60, 20132 Milan, Italy; Email paolo.corradini{at}hsr.it
PURPOSE: To describe molecular monitoring of minimal residual disease in patients with myeloma who have achieved complete remission (CR) after autologous or allogeneic transplantation of hematopoietic cells. MATERIALS AND METHODS: Clonal markers based upon the rearrangement of immunoglobulin heavy-chain genes were generated for each patient and used for polymerase chain reaction (PCR) detection of residual myeloma cells. Fifty-one patients entered the program and 36 achieved CR. After transplantation, molecular monitoring was performed on 29 patients (15 autologous and 14 allogeneic transplants) who had molecular markers. RESULTS: Our data show that molecular remissions are rarely achieved (7%) with high-dose chemotherapy followed by single or double autografting. In addition, virtually all peripheral blood progenitor cell and bone marrow samples contained residual myeloma cells, even when sample collection was scheduled after repeated courses of high-dose chemotherapy. All patients autografted with PCR-positive cells remain positive, and eight of 15 have relapsed. Two patients were autografted with PCR-negative cells: one is in clinical and molecular remission, and one relapsed 25 months after the transplant. In the allografting setting, a higher proportion of patients (50%) achieved molecular remission; there were two relapses, one in the PCR-positive group and one in the PCR-negative group. CONCLUSION: This is the first large study of molecular remissions in myeloma patients to use a PCR-based approach utilizing patient-specific tumor markers. The sizeable fraction of patients who achieved molecular remission after allografting with peripheral blood progenitor cells represents a promising finding in an incurable disease.
MULTIPLE MYELOMA (MM) is a B-cell malignancy characterized by the expansion of plasma cells producing a monoclonal immunoglobulin. It is an incurable disease with a median survival time of approximately 36 months. The standard treatment for MM is the melphalan and prednisone regimen. Extensive clinical trials with other drug combinations have been conducted, but no other regimen has been definitively proven to be superior to melphalan and prednisone.1 In the search for a more effective therapy, several groups have recently tried high-dose chemoradiotherapy followed by autologous or allogeneic transplantation of hematopoietic cells. Autologous transplantation using bone marrow or peripheral blood progenitor cells (PBPCs) has improved the quality of life and clinical outcome of myeloma patients, providing good symptom control, tumor mass reduction, and longer survival. Complete remission (CR) of the disease is achieved in 20% to 40% of patients who undergo single or double autotransplants.2-6 In 1996, a prospective randomized study showed that autologous bone marrow transplantation is superior to conventional chemotherapy in terms of both event-free and overall survival.7 Allogeneic bone marrow transplantation (BMT) has been confined mainly to young refractory/relapsed patients who have an HLA-identical sibling donor. Such a procedure is characterized by a higher transplant-related mortality rate, but it seems to offer a lower rate of relapse and progression.8-10 A rationale for the use of allogeneic transplantation is the recent demonstration of a graft-versus-myeloma effect in patients receiving donor lymphocyte infusions for relapse after allografting.11-13 Using a polymerase chain reaction (PCR)-based approach, several groups demonstrated that PBPCs, collected for autografting purposes, are frequently contaminated by residual myeloma cells.14-16 In a previous pilot study, we showed that even after several courses of high-dose chemotherapy, PBPCs contained residual tumor cells.14 However, very few data on molecular status after autologous or allogeneic transplantation have been reported so far. In particular, minimal residual disease (MRD) has not been evaluated using patient-specific tumor markers in PCR amplifications.17,18 In the present study, a PCR-based analysis of MRD was performed in patients treated with single or double autografting, allogeneic BMT, or blood cell transplantation (BCT). A PCR-based strategy, using patient-specific sequences derived from the rearranged variable region (VDJ) of immunoglobulin (Ig) heavy-chain genes (IgH), was used to detect the presence of residual plasma cells. Tumor-specific primers and probes were designed from the second and third complementarity-determining regions (CDRs) of patients' VDJ.19 An analysis of MRD was performed on PBPCs and bone marrow (BM) samples from patients undergoing autografting and on serial BM follow-up samples of patients who achieved CR after autografting or allografting.
Patients and Response Criteria Fifty-one patients under the age of 55 (43 patients at diagnosis, two at relapse, four with refractory disease, and two with plasma cell leukemias) were enrolled in a high-dose chemoradiotherapy program: 15 patients entered a single-autografting program, 19 patients were enrolled in a double-autografting program, five patients underwent allogeneic BMT, and 12 patients underwent BCT. Patients had advanced stage disease; there were 29 IgG, 13 IgA, seven Bence Jones, and two nonsecretory myelomas. The high-dose sequential (HDS) chemotherapy regimen used in the single-autografting program has previously been described.5,14 The double-autografting program was organized as follows: patients received two courses of vincristine, doxorubicin, and dexamethasone (VAD), cyclophosphamide 5 g/m2, etoposide 2 g/m2, three cycles of dexamethasone 25 mg/m2 for 4 consecutive days, and cyclophosphamide 7 g/m2. Granulocyte colony-stimulating factor (G-CSF, 5 µg/kg) was infused after each high-dose drug was given. After etoposide and the last cyclophosphamide doses were given, two to three leukaphereses were performed to collect PBPCs. Bone marrow was collected from all patients for back-up purposes after cyclophosphamide 7 g/m2 was administered and in most of them it was not reinfused. Myeloablative regimens consisted of melphalan 200 mg/m2 in the first autografting and mitoxantrone 60 mg/m2 plus melphalan 180 mg/m2 in the second autografting. Patients undergoing allogeneic BMT received three to four VAD courses and were conditioned using total body irradiation (120 Gy) plus cyclophosphamide 120 mg/m2. Patients receiving allogeneic BCT were treated with three to four VAD courses or with cyclophosphamide 4 g/m2 followed by two courses of melphalan 80 mg/m2 with support of autologous PBPCs (patient nos. M229, M232, M237, and M261); the conditioning regimen consisted of busulfan 14 mg/kg plus melphalan 140 mg/m2. The mobilization regimen for normal donors consisted of G-CSF (10 µg/kg) or granulocyte-macrophage colony-stimulating factor (5 µg/kg) for 2 days followed by G-CSF (16 µg/kg) (four patients). Treatment for the prevention of graft-versus-host disease (GVHD) consisted of cyclosporine plus methotrexate. Patients were hospitalized in laminar airflow rooms and received prophylactic systemic antibiotics. Disease response was defined as follows: CR, 1% or fewer plasma cells of normal morphology on bone marrow smears and absence of serum and/or urine M-protein by immunofixation; partial remission, 50% decrease in serum M-protein and/or 75% decrease in Bence Jones protein levels; refractory disease, absence of an M-protein decrease during treatment.
Nucleic Acid Extraction and cDNA Synthesis
Amplification and Sequencing of the Tumor VDJ
Detection of Residual Myeloma Cells
Oligonucleotide Synthesis
Patient Outcome A total of 51 young myeloma patients were enrolled in a program of autologous or allogeneic transplantation: 34 underwent single or double autografting and 17 underwent allografting. In the single-autografting program, two of 15 patients did not receive full treatment for pulmonary fungal infection (patient no. M249) and disease progression (patient no. M154). Seven of 13 patients who underwent single autografting were in CR at treatment completion. In the double-autografting program, four of 19 patients received only single autografting because of acute mental disorder (patient no. M162) and low-yield PBPC collections (patient nos. M163, M165, and M204). Twelve patients achieved CR, two after single autografting (patient nos. M196 and M272) and 10 after double autografting. In the allografting program, five patients received BMT and 12 received BCT; all patients were in CR after transplantation (patient no. M232, 6 months after BCT). Because BMT patients were studied retrospectively, and we then selected patients in CR who had samples for the molecular analysis, the data on treatment-related mortality and overall response rate cannot be given (see clinical characteristics in Table 1). All myeloma patients who received BCT in the Palermo and Torino hospitals were enrolled in the study. There were two treatment-related deaths, a transplant-associated thrombotic microangiopathy at day 53 (patient no. M237) and a case of interstitial pneumonia at day 117 (patient no. M229). There were two cases of grade 3 acute GVHD in the BCT group (Table 1).
Identification of Myeloma VDJ
Detection of Residual Myeloma Cells Overall, 19 of 34 patients were in CR after autografting, and molecular monitoring of MRD was performed in the 15 who had a molecular marker. Four patients were in the single-autografting program and 11 were in the double-autografting program. In the latter program, two patients (M196 and M272) achieved molecular remission; they were reinfused with PCR-negative cells and remained PCR-negative for a period of 20 and 21 months (Fig 1). However, patient no. M196 experienced a clinical and molecular relapse at month 25 after autografting. Hence, only one patient from the autografting series is in continuous clinical and molecular remission (median follow-up, 26.5 months; range, 6 to 63 months). So far, eight of the 13 patients who had PCR-positive bone marrow samples have already relapsed.
Seventeen patients from the allografting program were in CR, and molecular monitoring was possible in the 14 who had a molecular marker (five BMT and nine BCT patients). Only one of five patients who underwent allogeneic BMT became PCR-negative, several years after transplantation (median follow-up, 26 months; range, 9 to 59 months). Six of nine patients who underwent allogeneic BCT became PCR-negative (median follow-up, 18 months; range, 1.5 to 42 months), and all became negative shortly after transplantation (Fig 2). Patient no. M232 had only one PCR-negative result out of six marrow samples; he is considered to be in the cohort of patients who have not achieved molecular remission. Among the patients who became PCR-positive after BMT, there was one clinical and molecular relapse (patient no. M265). In the PCR-negative group, there was one clinical and molecular relapse: patient no. M311 had an extramedullary relapse (9 months after transplantation) followed by a marrow relapse. Fisher's exact test showed a statistical significance (P < .014) between the number of molecular remissions after allogeneic transplantation (seven of 14) and autologous transplantation (one of 15).
In this article, we describe PCR-based monitoring of MRD in patients in CR after autologous or allogeneic transplantation of hematopoietic cells. Our data show conclusively that molecular remissions are rarely achieved (7%) with high-dose chemotherapy followed by autografting. In addition, we demonstrated that virtually all PBPC and bone marrow used for transplantation contained residual myeloma cells even when cell collection was scheduled after repeated courses of high-dose chemotherapy. All patients who were reinfused with their PCR-positive cells remained positive; eight of them have already had a relapse. So far, one of two patients who were reinfused with PCR-negative cells has relapsed. After allografting, a higher proportion of patients (50%) achieved molecular remission; there were two relapses, one in the PCR-positive group and one in the PCR-negative group. The data on tumor contamination of PBPC samples are in accordance with our previous findings and with reports from the literature showing the persistence of myeloma cells in the grafts used for autotransplantation.14-16 In our series, PBPCs were not less contaminated than bone marrow samples, because the only two patients who achieved a PCR-negative status had negative PBPC and bone marrow cells.22 Furthermore, patients' harvests remained PCR-positive despite the fact that two VAD cycles and two to three courses of high-dose chemotherapy were delivered before leukaphereses. In our double-autografting program, two rounds of leukapheresis were planned: no patient who was PCR-positive in the first round became PCR-negative in the second round. The only two patients collecting PCR-negative cells had all PBPC harvests that were negative. The rationale for scheduling PBPC collections at the end of the program was based upon a previous work showing that plasma cell content (identified by flow cytometry) is lowered by repeated courses of high-dose therapy.23 Analysis of follow-up samples of patients who have achieved CR showed that 13 of 15 patients were not in molecular remission. The two patients in molecular remission after autografting were already PCR-negative after the first transplantation (Fig 1); however, disease recurred in one of them 25 months after autografting. Using a less intense chemotherapy program, Bjorkstrand et al17 reported molecular remissions in four of five patients after double autografting. The discrepancy with our results can be explained by the difference in PCR methods. The IgH fingerprinting assay has a best sensitivity of 10-4, which is one to two logs lower than ours. In addition, the specificity of IgH fingerprinting relies not on the use of clone-specific oligonucleotide primers but only on VH consensus primers.17 Our results indicate that only a minority of patients in CR may achieve molecular remission, and this is in accordance with the clinical outcome of myeloma patients. In fact, most myeloma patients relapse after autografting. In this article, we do not question the beneficial effect of autografting in terms of symptom control and prolonged survival. In other hematologic malignancies, however, the achievement of molecular remission has represented a first step to improved outcome.24-28 It is worth noting that one of two patients reinfused with PCR-negative cells relapsed; in this patient, PCR analysis performed 5 months before relapse was negative. This case of relapse after PCR negativity was achieved in vivo suggests that disease recurrence was caused by the persistence of myeloma cells in the patient and not by reinfusion with the graft. Alternatively, both patient and graft harbored myeloma cells below the threshold of the PCR assay. Our findings indicate that alternative approaches, other than high-dose chemotherapy alone, are required to increase the fraction of patients with PCR-negative hematopoietic cells and possibly to eradicate the disease. A longer follow-up of a large panel of patients who have achieved molecular remission is necessary to conclusively assess the role of PCR in predicting relapse in myeloma. In other mature B-cell malignancies, PCR negativity has been correlated with a better outcome.24,25,28 In the setting of allogeneic transplantation, we showed that molecular remissions could be achieved in a sizeable fraction of cases: seven (50%) of 14 assessable patients became PCR-negative. One patient achieved PCR negativity several years after BMT; a late negativization of patients who have undergone BMT has been described. Using the IgH fingerprinting method, Bird et al18 described three patients in clinical and molecular remission after allogeneic BMT. They showed that a PCR-negative status could be achieved in a period ranging from 1 to 4.5 years after transplantation. Bird et al, however, were not using patient-specific primers, so their data cannot be fully compared with ours in terms of sensitivity and specificity. It is interesting to note that in allogeneic BCT, the conversion to PCR negativity seemed to occurr earlier. Furthermore, we showed that molecular remissions were achieved in transplant patients with a chemosensitive disease (for partial remission or CR status before transplantation, see Table 1). Such a finding, along with a low treatment-related mortality rate, may indicate that BCT should be offered early during the disease course of patients who have an HLA-matched sibling donor. The low treatment-related mortality rate might have several explanations: (i) patients were not heavily pretreated; (ii) the toxicity of the busulfan/melphalan regimen seems quite low; (iii) the use of G-CSF–mobilized peripheral blood cells allows a rapid hematopoietic reconstitution; and (iv) the incidence of acute GVHD higher than grade 2 was low. Our data are the first report on PCR negativity after allografting, using patient-specific primers and probes for the detection of MRD. Molecular remissions, in 50% of a subset of myeloma patients, represent a novel finding and are worthy of further investigations. A possible explanation for the molecular remissions is the immunotherapeutic effect exerted by the donor T cells present in the graft. The existence of graft-versus-myeloma activity was demonstrated recently in patients receiving donor lymphocyte infusions for relapse after allogeneic BMT.12,13 The higher number of molecular remissions and the early conversion to PCR negativity that was achieved with BCT might be due to the large inoculum of donor CD34+ cells and T cells. It has been reported that patients who have undergone BCT for bcr/abl-positive leukemias have a greater number of molecular remissions and a lower relapse rate compared with patients who have undergone BMT. A higher rate of complete chimerism has been proposed as a possible explanation.29 In our series, however, molecular remissions do not seem to be correlated with better engraftment because there were no differences in the chimerism status between patients who underwent BMT and those who underwent BCT (data not shown). There was also no correlation between acute GVHD and molecular remission or relapse. Chronic GVHD was not significantly correlated with molecular remissions, probably because of the limited number of PCR-negative patients; however, extensive GVHD was quite common after BCT (58%) and might play an important role (Table 1). This lack of correlation is not a completely unexpected finding for several reasons: (i) more PCR-negative patients with acute GVHD higher than grade 2 and extensive chronic GVHD are required; (ii) after BCT, an increased incidence of acute GVHD was not reported; G-CSF, used for mobilization purposes, seems to be responsible for the lack of increase in acute GVHD cases while preserving the graft-versus-tumor effect30,31; and (iii) a higher incidence of chronic GHVD has been reported with the use of mobilized peripheral blood cells. The clinical response to donor lymphocyte infusions has also been correlated with chronic GVHD development in myeloma patients treated for relapse after allogeneic BMT.13,32 It has to be stressed that the achievement of clinical and molecular remission in myeloma patients represents a promising finding in an incurable disease. In the panel of therapeutic options for myeloma treatment, allogeneic transplantation and, in particular, BCT seem to represent a field worthy of investigation. The chemoresistance of myeloma cells might be overcome by the antitumor effect of the allograft. Future options should probably evaluate nonmyeloablative regimens with programmed delayed infusions of T lymphocytes.33 Such an approach might reduce treatment-related mortality and raise the age of patients who can benefit from allografting. In conclusion, our study shows that, first, molecular remission is very rare with single or double autografting, and that even in PCR-negative patients, there is a possibility of relapse. The goal of curing myeloma with high-dose chemotherapy and autografting remains questionable. Second, allografting offers a larger number of molecular remissions, and BCT seems particularly promising. Third, larger studies of molecular monitoring are required to fully explore the role of allografting in myeloma treatment.
Supported by Associazione Italiana Ricerca sul Cancro (AIRC, Milano Italy) and Consiglio Nazionale delle Ricerche (Progetto Finalizzato ACRO #9500416.PF39). M.A. is the recipient of a fellowship from AIRC We thank P. Bondesan for cryopreservation.
Present affiliation of Dr. Corradini: Bone Marrow Transplantation Unit, Istituto Scientifico H.S. Raffaele, Milan, Italy.
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Giralt S, Estey E, Albitar M, et al: Engraftment of allogeneic hematopoietic progenitor cells with purine analog-containing chemotherapy: Harnessing graft-versus-leukemia without myeloablative therapy. Blood 89:4531-4536, 1997 Submitted February 20, 1998; accepted September 1, 1998.
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Copyright © 1999 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
or C
) to obtain Ig cDNA. Five micrograms of total RNA were reverse-transcribed with 20 pmol of reverse transcription primer. A 50-µl reaction was carried out in 10 mM dithiothreitol, 1 mM dNTPs (Pharmacia LKB Biotechnology, Uppsala, Sweden), and 1x reverse transcriptase buffer (50 mM Tris-HCl, 6 mM MgCl2, and 40 mM KCl), final concentration, with an additional 20 units of ribonuclease inhibitor (RNasin; Promega, Madison, WI) and 200 units of Moloney murine leukemia virus reverse transcriptase (Superscript; Gibco BRL, Gaithersburg, MD). The reaction mixture was incubated for 1 hour at 37°C. 
], PCR-negative; [•], PCR-positive.
