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© 2000 American Society for Clinical Oncology Molecular Remission After Allogeneic or Autologous Transplantation of Hematopoietic Stem Cells for Multiple MyelomaFrom the Institute of Hematology and Medical Oncology "Seràgnoli," University of Bologna, Italy.
PURPOSE: To assess the clinical relevance of minimal residual disease (MRD) in patients with multiple myeloma (MM), 50 patients were monitored while they were in complete clinical remission (CCR) after autologous or allogeneic stem-cell transplantation. PATIENTS AND METHODS: Stringent molecular monitoring using clonal markers based on rearranged immunoglobulin heavy-chain genes was performed in 44 of 50 MM patients in CCR. Molecular clinical remission (MCR) was defined as more than one consecutive negative polymerase chain reaction (PCR) test result. RESULTS: Twelve (27%) of 44 molecularly monitored patients achieved MCR; four of the 12 became PCR-positive, and one of these four relapsed. In comparison with patients who did not achieve MCR, patients who achieved MCR had a significantly lower relapse rate (41% v 16%; P < .05) and longer relapse-free survival (35 v 110 months; P < .005). Fourteen of 26 patients in CCR who had received allografts were evaluated on a molecular basis: seven (50%) of the 14 achieved MCR and did not relapse; one of the seven remaining patients relapsed. Thirty of 47 patients in CCR who received autografts were evaluated on a molecular basis: five (16%) of the 30 achieved MCR; two of these five became PCR-negative, and one of these two relapsed. Ten of the 25 remaining patients later relapsed. For these nonrandomized groups, the higher MCR rate after allograft procedures was statistically significant (P < .01; Fisher’s exact test). CONCLUSION: MCR can be obtained in a relatively high proportion of MM patients who have achieved CCR after undergoing allograft procedures and in a smaller fraction of patients after undergoing autograft procedures. In approximately one fourth of MM patients who achieve CCR after transplantation, it may be possible to keep the disease burden constantly below the PCR threshold. Because MCR was associated with prolonged relapse-free survival, these patients could have a relatively favorable clinical outcome.
MULTIPLE MYELOMA (MM) is a B-cell malignancy that affects terminally differentiated B cells (plasma cells) and runs a progressive clinical course. The median survival time of MM patients treated with conventional chemotherapy is usually 3 to 4 years. Recently, several advances in the management of MM have been reported, including the use of high-dose chemo(radio)therapy followed by autologous1-5 or allogeneic transplantation of hematopoietic stem cells.6 Although autografting with bone marrow (BM) or peripheral-blood stem cells (PBSCs) has improved the clinical outcome and lengthened the survival of MM patients, in comparison to conventional chemotherapy, stringently defined complete remission (CR) is achieved in only 20% to 40% of patients, most of whom subsequently relapse.7-9 The reinfusion of residual myeloma cells that contaminate the autologous graft may contribute to disease recurrence, as has been previously demonstrated in other hematologic malignancies and solid tumors10: this notion has recently led to phase I and II clinical trials aimed at exploring the role of purging techniques.11,12 In comparison to autografting, allogeneic stem-cell transplantation offers two-fold advantages that include the use of a tumor-free source of hematopoietic progenitors and the existence of a graft-versus-myeloma effect.13-15 However, allotransplants are associated with a high risk of early mortality,16,18 and the relapse rate of patients who obtain CR, albeit lower than that observed after autografting,19 still remains at approximately 50%.16,17 In MM, the rearranged immunoglobulin heavy-chain (IgH) gene can be regarded as a sensitive tumor marker for the detection of residual myeloma cells below the limits of conventional methods of analysis.20 Moreover, using polymerase chain reaction (PCR)–based strategies, several groups have recently demonstrated that autologous PBSC collections are frequently contaminated by MM cells.11,21,22 However, little is currently known about the molecular status of MM patients after autologous or allogeneic stem-cell transplantation2,23-25; in particular, the significance of molecular remission (MCR) is at present unclear. In study presented here, we performed a PCR analysis26 of minimal residual disease (MRD) in a large series of patients in CR after allogeneic or autologous transplantation in order to determine the MCR rate and to assess its clinical relevance in terms of survival and relapse rate. In this regard, the rearranged variable region (VDJ) of the IgH gene was identified using two primers designed from the patient-specific IgH complementarity-determining regions (CDRs) II and III, as previously described.26 Our data indicate that patients who achieved MCR had a longer duration of disease control and a lower relapse rate, in comparison with those who had disease that was persistent at the molecular level. Furthermore, although the comparison between the two groups was not randomized, our results seem to suggest that MCR was obtained more frequently after allogeneic stem-cell transplantation.
Patient Population Between 1984 and 1998, 229 patients with confirmed diagnoses of active MM received myeloablative treatments with the support of hematopoietic stem cells at the Institute of Hematology and Medical Oncology "Seràgnoli" at the University of Bologna. Of these 229 patients, 68 underwent allogeneic transplantation from HLA-identical sibling donors, and the remaining 161 were enrolled onto clinical trials of autologous PBSC transplantation. Allografting was performed using BM in 52 patients and PBSCs in the remaining 16 (Fig 1). Preparations for engraftment included total body irradiation plus chemotherapy in 48 patients and the association of busulfan and cyclophosphamide in the remaining 20, as reported elsewhere.25 Graft-versus-host disease prevention was performed using either cyclosporine (CsA) with or without methotrexate (31 patients) or T-cell depletion with or without CsA (37 patients).
Of the 161 patients who received autografts, 82 received a single transplant: 71 with unmanipulated PBSCs (subgroup A) and 11 with selected CD34+/Blin- PBSCs (subgroup B) (Fig 1). Double selection of CD34+/Blin- cells was performed by enrichment of CD34+ cells, as previously reported,11 followed by immunomagnetic depletion of CD10+, CD19+, CD20+, and CD56+ cells.12 The remaining 79 patients who received autografts were enrolled onto double transplant programs: of these 79, 62 were reinfused with unselected PBSCs (subgroup C) and 17 with enriched CD34+ cells (subgroup D) (Fig 1), as previously described.11 Priming of the PBSC collections was performed with a combination of cyclophosphamide 7 g/m2 and granulocyte colony-stimulating factor 5 mg/kg/d, until the time of stem-cell collection.12 Patients who received a single autotransplant (subgroups A and B) or the first of tandem transplants (subgroups C and D) were treated with high-dose melphalan (200 mg/m2). The conditioning regimen for the second transplant of unselected PBSCs (subgroup C) or enriched CD34+ cells (subgroup D) consisted of the combination of high-dose melphalan (120 mg/m2) and busulfan 12 mg/kg. Maintenance therapy with interferon alfa-2a (3 x 106 units three times a week) was administered in subgroup A and C patients starting 3 months after the first and second transplant, respectively, and was maintained until relapse.
Selection of Patients for Molecular Monitoring
Nucleic Acid Extraction and cDNA Synthesis BM samples or smears were obtained after informed consent was obtained during standard diagnostic procedures. DNA was obtained by cell lysis and salting-out procedures, as reported26; DNA from smears was obtained by lysing scraped cells and by phenol extraction and ethanol precipitation. DNA was spectrophotometrically quantified. RNA was isolated as reported.27 One microgram of total RNA was reverse-transcribed using 20 pmol of random primers. A 50-µL reaction was carried out in 10 mmol/L dithiothreitol, 1 mmol/L desossinucleotide triphosphate (Pharmacia LKB Biotechnology, Uppsala, Sweden), and 1x reverse transcriptase buffer (50 mmol/L TrisHCl, 6 mmol/L MgCl2, and 40 mmol/L KCl), with an additional 20 units of ribonuclease inhibitor (Boehringer, Mannheim, Germany) and 200 units of Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Gaithersburg, MD). The reaction was incubated at 37°C for 1 hour.
Identification of VDJ Gene Rearrangement
Detection of Residual Myeloma Cells
VDJ Gene Rearrangement To identify the VH family used in VDJ gene rearrangement, the VDJ regions of MM patients were amplified with a set of seven VH family-specific primers or six consensus primers derived from the IgH leader, together with a JH-consensus primer. Twelve patients who had received allografts were not studied because of the absence of suitable samples. The VDJ region was amplified and directly sequenced in 44 (88%) of 50 of the remaining patients; in six patients, we obtained a polyclonal band and were not able to identify the monoclonal CDRII and CDRIII regions. To obtain patient-specific markers, two patient-specific primers were designed for each sequence: a sense primer on the CDRII region and an antisense primer on the CDRIII. The sensitivity and specificity of our assay was tested as previously described26: we obtained a median sensitivity of one tumor cell in 10-5 normal marrow cells (range, 10-4 to 10-7 cells), and no false-positive results were obtained when the DNA of B-chronic lymphoblastic leukemia patients was used for negative controls.
Clinical Outcome In the autograft setting, a total of 36 (22.5%) of 161 patients achieved CCR, and 11 (30.5%) of them relapsed (Table 2, Fig 1). Seventy-one patients received a single, unmanipulated autograft (subgroup A) and, with a median follow-up period of 15 months (range, 12 to 36 months), eight (11.2%) achieved CCR; four of them later relapsed. Eleven patients received a single autotransplant with double-selected autograft (CD34+/Blin- cells) (subgroup B) and, with a median follow-up period of 6 months (range, 3 to 12 months), seven attained CCR (63.6%); two of them later relapsed. Of the 62 patients who underwent double unmanipulated autografting (subgroup C), with a median follow-up period of 18 months (range, 3 to 36 months), 15 (24.1%) achieved CCR; four of them later relapsed. Of the 17 patients who were double-transplanted with selected apheresis (CD34+ cells) (subgroup D), with a median follow-up period of 24 months (range, 18 to 36 months), six (35.3%) achieved CCR; one of them later relapsed.
Apheresis Contamination
Molecular Outcome In the allograft setting, 14 patients were studied (Figs 2 and 3, Table 3). Of these 14, seven (50%) achieved MCR. Five of these seven (patients no. 1, 2, 3, 4, and 6) had received BM (median molecular follow-up period, 72 months; range, 36 to 120 months). Two of these seven (patients no. 10 and 12) had received PBSCs; both patients had molecular follow-up periods of 24 months. Two of the seven patients who achieved MCR (patients no. 4 and 12) became PCR-positive at 60 and 24 months after transplantation but remained in CCR. Median time to first becoming PCR-negative was 12 months (range, 6 to 36 months; Table 3). Seven patients (50%) never achieved MCR: of these seven, one (patient no. 7) relapsed at 36 months after transplantation, whereas the other six (patients no. 5, 8, 9, 11, 13, and 14) are in CCR at 72, 36, 6, 24, 12, and 12 months after transplantation, respectively. No statistically significant difference was found in the rates of MCR between patients who had received BM and PBSC allografts (31% v 20%, respectively).
In the autograft setting, 30 patients were studied (Figs 2, 4, and 5; Table 4). Five (16.6%) of these 30 achieved MCR (median molecular follow-up period, 24 months; range, 12 to 36 months). Patients no. 19 and 29) had received a single autograft, and both patients had a molecular follow-up period of 12 months. Three of these five (patients no. 31, 35, and 48 had received a double autograft and had molecular follow-up periods of 24, 24, and 36 months, respectively. Patients no. 35 and 48 became PCR-positive at 18 and 36 months after transplantation; patient no. 35 later clinically relapsed. Median time to first becoming PCR-negative was 3 months (range, 3 to 24 months; Table 4). Twenty-five patients (83%) did not achieve MCR. Ten of these 25 (patients no. 16, 17, 18, 21, 24, 27, 32, 34, 39, and 49) later clinically relapsed, with a median follow-up period of 18 months (range, 6 to 36 months), whereas the remaining 15 remained in CCR, with a median follow-up period of 12 months (range, 3 to 36 months).
Detailed analysis of the MCR rates, although not performed on a randomized basis, showed a difference in favor of MM patients who received allografts (50% v 16.6% for patients who received autografts; P < .01, Fisher’s exact test). Moreover, it is remarkable that those patients who achieved MCR after undergoing either auto- or allografting had significantly longer median relapse-free survival times than did patients who did not achieve MCR (110 months [range, 12 to 120 months] v 35 months [range, 3 to 72 months], respectively; P < .005) (Fig 6). The fact that the relapse rate was significantly lower in MCR patients than in those patients who never obtained MCR is also remarkable (16.6% v 41%, respectively; P < .05).
A recent study in a large series of MM patients has shown that high-dose chemotherapy plus auto- or allograft treatment is capable of inducing stringently defined CCR rates of 33% and 41%, respectively.2,29 Nevertheless, the percentage of MM patients who obtain MCR is much lower, and only a few such cases have been reported so far.2,24 Therefore, it is not clear yet whether MM can be eradicated on a molecular basis nor whether this is of clinical relevance. The study presented here addresses this issue in the context of the largest reported series of MM patients in CCR after undergoing allo- or autografting who are monitored for MRD by a highly sensitive and specific PCR-based assay. In our series, 27% of the patients in CCR after undergoing allo- or autografting obtained MCR. Moreover, the patients in this groups who achieved MCR had a significantly lower relapse rate (16% v 41%, respectively) and longer relapse-free survival time (110 v 35 months, respectively). Whether MCR does actually represent a goal of MM therapy remains to be demonstrated, but our data support the concept that, as with other hematologic malignancies,30-34 MCR in MM could also represent a first step in clinical-outcome improvement. Our data indicate that the improvement induced by transplantation procedures includes not only extended survival in7 but also a significant reduction of the relapse risk in the subset of patients who achieve MCR. Concerning the prognostic value of molecular evaluation of MRD, only one (2%) of the patients who achieved MCR in our series relapsed. This suggests that stringently defined MCR may have important prognostic significance. Our stringent definition of MCR as two consecutive PCR-negative results and the high sensitivity and specificity of our PCR assay reduced the effective MCR rate among our patients but also allowed us to demonstrate the prognostic potential of molecular monitoring. On the other hand, we found that approximately 50% of patients who either never achieved MCR or who had failed to maintain MCR remained in CCR for at least 1 year anyway. Studies on larger series of patients are needed in order to confirm the utility of such molecular monitoring, as shown by our data, and to investigate by means of a quantitative assay35 whether a threshold exists beneath which there is no significant risk of clinical relapse. With regard to the roles of the different transplantation procedures on MRD status, we found that the proportion of patients in CCR who also achieved MCR was 50% in the allograft group versus 17% in the autograft group. This statistically significant difference is consistent with the clinical observation that the relapse rate after allogeneic transplantation is lower than that after autologous transplantation. This could be a result of either the use of a tumor-free source of hematopoietic cells or the existence of a graft-versus-myeloma effect,14,15 both of which would ultimately result in a longer duration of disease control. Moreover, the preparative regimens of the two transplantation procedures could also have played a role in the results of the two groups. When considered in light of the reduced transplant-related mortality rate in MM patients who received allografts, these findings provide a rational basis for offering allografts to patients with an HLA-identical donor at an early stage in the disease. Nevertheless, because the two groups evaluated in this study were not randomized, and because some of the CCR patients could not be monitored on a molecular basis, all these evaluations should be regarded as indications that require confirmation in larger, randomized studies. For all four different autograft procedures we performed, MCR was rarely achieved. In the patients who received single and double unselected autografts, only one (16%) in six and two (16%) in 12 patients, respectively, obtained MCR. In the patients who received single and double selected autografts, only one (14%) in seven and one (20%) in five patients, respectively, obtained MCR. When we evaluated the tumor contamination in the nonselected apheresis samples after the first and the second purification steps, we found that all the unselected apheresis samples were PCR-positive, whereas only in a small fraction of the manipulated or double-manipulated apheresis samples were we able to obtain a PCR-negative, virtually tumor-free graft. A supposed advantage of purging procedures is the possibility of being able to reinfuse tumor-free grafts, but only two of the patients who were reinfused with PCR-negative apheresis samples achieved MCR. On the other hand, in the group of patients who underwent transplantation with double selected apheresis samples, CCR was obtained in a relatively high number of patients (seven of 11 patients). It should be noted that we have only used the purging procedures in relatively few patients; this could explain the lack of increase in the MCR rate. Nevertheless, although confirming the concept that the reduction of tumor burden obtained by different autotransplant procedures (double transplantation, reinfusion with CD34+-selected cells or CD34+/Blin- double selected cells) may help to increase the CCR rate, as has been also reported by others,36 our data suggest that this may still not be enough to obtain the disappearance of disease on a molecular level in a consistent number of patients.
Supported by the Italian Association of Cancer Research, by Italian National Council of Research grant no. 98.00526.CT04, by target projects from the Ministero Dell’Università e Della Ricerca Scientifica e Tecnologica (40%, S.T.; 60%, M.C.), by "30 Ore per la Vita" Associazione Italiana Contro le Leucemie grants, and by the University of Bologna funds for selected research topics. We thank Robin M.T. Cooke for assisting us in revising the manuscript and Chiara Conslovi, Maria Stella Zagarella, and Serena Mangianti for their technical assistance.
G.M. and C.T. contributed equally to this study. Giovanni Martinelli, MD, Molecular Biology Unit, Institute of Hematology and Medical Oncology "Seràgnoli," University of Bologna, Via Massarenti, 9-40138 Bologna, Italy; email gmartino@ kaiser.alma.unibo.it.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
), PCR-negative; (•), PCR-positive; (
), immunofixation-negative; (
), immunofixation-positive.
