Abstract

In the present study, we used a polymerase chain reaction–based (PCR-based) strategy to retrospectively analyze the presence of residual myeloma cells in serial posttransplant bone marrow samples obtained from 13 patients in remission after allogeneic hemopoietic stem cell transplantation (allo SCT). For this purpose, patient-specific primers were generated from complementarity determining regions 2 and 3 of the rearranged IgH gene. The level of sensitivity of the PCR-based assay ranged from 1 in 105 to 1 in 106 normal marrow cells. Following transplantation, 9 of 12 patients who attained stringently defined complete remission (CR) remained persistently PCR for a median of 36 months, and 4 of the patients remained PCR up to the latest analysis, which was performed at 48, 72, 72, and 120 months, respectively, after allo SCT. None of the patients in the PCR subgroup experienced a disease relapse, and only 1 of 4 PCR+ patients experienced a relapse. It is concluded that allo SCT has the potential ability to induce sustained serological and molecular CR in selected patients with multiple myeloma.

During the last decade, pilot reports by our group1 and other groups2 demonstrating the efficacy of allogeneic bone marrow transplantation (BMT) for selected patients with multiple myeloma (MM) has progressively created interest in the use of this treatment strategy.3 In a previously reported case-matched analysis comparing autologous and allogeneic BMT,4 the latter did not result in a consistent improvement in the complete remission (CR) rate and produced fatal complications in approximately 40% of the patients. Moreover, the risk of relapse, even for those patients who attained CR, was unfortunately high,5,6 a finding which raises the possibility that the myeloma clone cannot be eradicated.

To address this issue, analysis of minimal residual disease (MRD) below the detection limit of methods conventionally employed to define CR may be of clinical relevance.7 In the present study, we used a polymerase chain reaction–based (PCR-based) strategy that employed patient-specific primers designed from tumor complementarity determining regions (CDRs) 2 and 3 to retrospectively analyze MRD in serial bone marrow samples. The samples were obtained from 13 MM patients who had been in remission after allogeneic hemopoietic stem cell transplantation (allo SCT). Of these patients, 5 survived 82 to more than 180 months (median, 84 months) after transplantation.

Study design

Patient and treatment characteristics

The patients included in the present study were part of a larger series of 68 MM patients who received allo SCT from HLA-identical sibling donors at the Institute of Hematology and Medical Oncology “Seràgnoli,” University of Bologna, Bologna, Italy. Of these patients, 26 (38% of the total population) attained remission following transplantation, as assessed according to the criteria listed below, and 13 patients could be retrospectively evaluated for the presence of MRD by the PCR-based assay. Their clinical and treatment characteristics are summarized in Table1.

Table 1.

Patient and treatment characteristics

UPN  Age/sex  Status at allo SCT  β2M (mg/L)  Conditioning  GVHD prophylaxis  Source of SC  GVHD Time to CR, mo  Survival, mo Latest disease status  
Acute  Chronic  
15 35/F  Resp  1.7  TBI + Cy + MEL  CsA  BM  limited  12  188+  CR  
95  49/F  Resp  1.9 BU-Cy 4  T depletion  BM  0  none  3  98+  CR 
116  37/F  Resp  2.1  BU-Cy 4  CsA + MTX  BM 1  limited  3  84+  CR  
142  36/F  Resp 1.4  BU-Cy 4  CsA + MTX  BM  0  none —  84+  Stable  
168  46/F  Resp  1.5 BU-Cy 4  CsA + MTX  BM  0  none  6  82+  CR 
215  53/M  Refr  3.4  TBI + Cy + MEL  T depletion  BM  0  none  5  37  Relapse 
231  30/F  Resp  1.3  TBI + Cy + MEL  T depletion  BM  0  none  3  46+  CR  
239  47/F Resp  1.6  TBI + Cy + MEL  CsA + MTX  PB  limited  2  45+  CR  
254  47/M  Refr  3.1 TBI + Cy + MEL  CsA + MTX  PB  0  limited  36+  CR  
260  43/F  Resp  1.1  TBI + Cy + MEL CsA + MTX  PB  1  none  Already in CR  36+ CR  
275  47/M  Refr  2.9  TBI + Cy + MEL CsA + MTX  PB  0  extensive  6  30+  CR  
304 33/F  Resp  1.2  TBI + Cy + MEL  CsA + MTX  BM 0  none  Already in CR  18+  CR  
306 54/F  Resp  1.4  TBI + Cy + MEL  CsA + MTX  PB 2  extensive  2  12  CR 
UPN  Age/sex  Status at allo SCT  β2M (mg/L)  Conditioning  GVHD prophylaxis  Source of SC  GVHD Time to CR, mo  Survival, mo Latest disease status  
Acute  Chronic  
15 35/F  Resp  1.7  TBI + Cy + MEL  CsA  BM  limited  12  188+  CR  
95  49/F  Resp  1.9 BU-Cy 4  T depletion  BM  0  none  3  98+  CR 
116  37/F  Resp  2.1  BU-Cy 4  CsA + MTX  BM 1  limited  3  84+  CR  
142  36/F  Resp 1.4  BU-Cy 4  CsA + MTX  BM  0  none —  84+  Stable  
168  46/F  Resp  1.5 BU-Cy 4  CsA + MTX  BM  0  none  6  82+  CR 
215  53/M  Refr  3.4  TBI + Cy + MEL  T depletion  BM  0  none  5  37  Relapse 
231  30/F  Resp  1.3  TBI + Cy + MEL  T depletion  BM  0  none  3  46+  CR  
239  47/F Resp  1.6  TBI + Cy + MEL  CsA + MTX  PB  limited  2  45+  CR  
254  47/M  Refr  3.1 TBI + Cy + MEL  CsA + MTX  PB  0  limited  36+  CR  
260  43/F  Resp  1.1  TBI + Cy + MEL CsA + MTX  PB  1  none  Already in CR  36+ CR  
275  47/M  Refr  2.9  TBI + Cy + MEL CsA + MTX  PB  0  extensive  6  30+  CR  
304 33/F  Resp  1.2  TBI + Cy + MEL  CsA + MTX  BM 0  none  Already in CR  18+  CR  
306 54/F  Resp  1.4  TBI + Cy + MEL  CsA + MTX  PB 2  extensive  2  12  CR 

SC indicates stem cells; Resp, responsive; Refr, refractory; TBI, total body irradiation; Cy, cyclophosphamiole; MEL, high-dose melphalan; BU, busulfan; CsA, cyclosporine A; MTX, methotrexate; BM, bone marrow; PB, peripheral blood.

Remission criteria

Criteria for remission following allo SCT included a decrease in bone marrow plasma cell infiltration to less than 3% on bone marrow aspirate, and the disappearance of serum and/or urine monoclonal (M) protein by standard agarose gel electrophoresis. Patients were considered to be in CR if they did not have a detectable M component by immunofixation analysis.

PCR amplification and sequencing of the myeloma VDJ

Bone marrow samples for the detection and sequencing of clonal immunoglobulin heavy chain (IgH) gene–CDRs (IgH-CDRs) 2 and 3 were available from the time of diagnosis in 10 patients and before allo SCT in the remaining 3 patients. The degree of bone marrow plasma cell infiltration ranged between 10% and 30%. Rearrangement amplification of the VDJ region was performed with a panel of VH family-specific primers together with a JH consensus primer, as previously described.8,9 Tumor-specific primers were designed from CDRs 2 and 3 of the VDJ region.10 

Molecular monitoring of MRD

Follow-up studies for the detection of MRD were performed on bone marrow specimens taken every 6 months during the first year after allo SCT and every year thereafter. Clonal myeloma cells were detected by amplifying 1 μg DNA, using the internal patient-specific primers designed from CDR 2 (sense) and CDR 3 (antisense). After 2 rounds of amplification, 15 μL PCR product was analyzed by 3% agarose gel. A rearranged band of approximately 150–base pair (150-bp) was obtained from each patient. The sensitivity level of the PCR-based assay ranged from 1 in 105 to 1 in 106 normal marrow cells. Sensitivity experiments were performed by diluting patient tumor cells with normal bone marrow cells (data not shown).

Results and discussion

After allo SCT, stringently defined CR was documented in 12 patients (Table 1). At the censoring date of September 1999, the median follow-up time for all patients was 45 months, and the longest follow-up extended to 188 months after transplantation. In September 1999, 11 patients were alive; 10 patients were in continued CR, and 1 patient had a stable residual M component. Two patients died: one had chronic graft-versus-host disease (GVHD) while in CR, and the other experienced cholangiocarcinoma while in relapse.

Detailed analysis of transplantation outcome in the subgroup of 5 patients who survived more than 6 years revealed that 4 of the patients remained in CR up to the latest follow-up at 82, 84, 98, and 188 months, respectively, from transplantation (Table 1). No M component was detected at routine electrophoresis in the fifth patient, however, serial immunofixation analyses performed at time-points ranging from 6-84 months after transplantation showed the persistence of a small, albeit stable, M protein of the same isotype that was detected at diagnosis.

The results of MRD molecular monitoring are summarized in Figure1. All patients were PCR+before allo SCT. Of 12 patients who attained stringently defined CR, 9 were PCR at a median of 6 months after transplantation. Of these 9 patients, 4 received bone marrow SCT, and 5 patients received peripheral blood SCT. Notably, in 1 patient after transplantation, persistence of a clonal product was demonstrated in every sample analyzed up to 72 months, after which time PCR results converted from positive to negative. All PCR patients were serially monitored for MRD detection for a median of 36 months, and they always tested PCR. In particular, in 7 of the patients, there were no residual myeloma cells detected by PCR up to 36, 36, 36, 48, 72, 72, and 120 months, respectively, after allo SCT. In contrast, in the remaining 4 patients, a clonal product was demonstrated in every sample that was analyzed up to 12, 32, 36, and 72 months, respectively, after transplantation. Three of these 4 patients were in CR, and the other did not meet the stringent CR criteria. One patient had clinical signs of relapse 20 months after allo SCT.

Fig. 1.

Results of PCR analysis for MRD detection on serial bone marrow samples taken before and after allo SCT.

The figure represents positive (•) and negative (□) results of immunofixation analysis and positive (▴) and negative (▵) results of the PCR-based assay.

Fig. 1.

Results of PCR analysis for MRD detection on serial bone marrow samples taken before and after allo SCT.

The figure represents positive (•) and negative (□) results of immunofixation analysis and positive (▴) and negative (▵) results of the PCR-based assay.

Results from this study add important information to the poorly defined issue of MRD detection after allo SCT for MM.11,12 Using a sensitivity ranging from 1 in 105 to 1 in 106normal marrow cells, we demonstrated that MRD could no longer be detected in 69% of patients who were evaluated. Obviously, these patients could still continue to harbor residual myeloma cells below the detection limit of the PCR strategy. However, if this were the case, PCR positivity and/or M protein should have appeared with time. Theoretically, 2 other factors could lead to false-negative PCR results. First, residual myeloma cells could be distributed unevenly in a patient's bone marrow,13 in which case they might not be detected in a single marrow aspirate. However, the finding of persistent PCR negativity on serial posttransplantation bone marrow samples argues against this possibility. Second, the PCR strategy could fail to detect MRD because there were multiple clones at presentation14 or because a modified IgH gene rearrangement emerged during the course of the disease.15 This event, generally termed as clonal evolution, has been well documented in acute lymphoblastic leukemia but not in MM.16 

Persistent PCR bone marrow samples from MM patients in long-term CR is reminiscent of data previously reported in other malignancies.17-21 In these instances, analysis of MRD by PCR was found to closely correlate with posttransplantation clinical outcome and to provide important information for patient management. Whether this also holds true in MM remains, as yet, unanswered. Larger molecular monitoring studies and, importantly, quantitative measures of residual tumor cells are required to further define the real value of PCR-based strategies. Results of these studies must also be compared with conventional methods of analysis to clarify the prognostic relevance of MRD detection in MM. Recently, the existence of a graft-versus-myeloma effect was formally demonstrated by several groups,22 who reported favorable responses to donor lymphocyte infusion in patients who experienced a relapse after transplantation. This finding suggests that in MM, as in other diseases, immunological mechanisms mediated by donor leukocytes may operate in vivo and, ultimately, concur to control23 and eventually eradicate the tumor clone. Obviously, the sample size of the presently described series is too small to allow any indirect measure of the graft-versus-myeloma effect by correlating posttransplantation outcome with acute and chronic GVHD. Studies involving larger numbers of allografted patients evaluated for the presence of the clonal IgH gene rearrangement may help clarify this clinically relevant issue in the near future.

In conclusion, the data herein reported demonstrate that allo SCT induces sustained serological and molecular remission in selected patients with MM. Although a longer follow-up is required to determine if these patients are truly cured, it is unusual for relapses to occur more than 5 years after allo BMT.7,8,24 This finding deserves consideration and should be discussed with each patient under 50 years of age for whom a matched related and perhaps unrelated donor is available. In the meantime, efforts to reduce transplant-related mortality should continue and should focus on more careful patient selection, earlier timing of transplantation, improved supportive care, and investigational use of peripheral blood hemopoietic stem cells in an attempt to lower the risk of fatal infectious complications.25 

Supported in part by Università di Bologna, Progetti di Ricerca ex-60% (M.C.) and MURST (cofin. 99) (S.T.).

Submitted October 20, 1999; accepted February 15, 2000.

Reprints:Michele Cavo, Institute of Hematology and Medical Oncology “Seràgnoli,” via Massarenti 9, 40138 Bologna, Italy; e-mail: mcavo@med.unibo.it.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

References

References
1
Tura
S
Cavo
M
Baccarani
M
Ricci
P
Gobbi
M
Bone marrow transplantation in multiple myeloma.
Scand J Haematol.
36
1986
176
2
Gahrton
G
Tura
S
Ljungman
P
et al. 
for the European Group for Bone Marrow Transplantation. Allogeneic bone marrow transplantation in multiple myeloma.
N Engl J Med.
325
1991
1267
3
Cavo
M
Benni
M
Gozzetti
A
Tura
S
The role of haemopoietic stem cell supported myeloablative therapy for the management of multiple myeloma.
Bailliere's Clin Haematol.
8
1995
795
4
Bjorkstrand
B
Ljungman
P
Svensson
H
et al. 
Allogeneic bone marrow transplantation versus autologous stem cell transplantation in multiple myeloma: a retrospective case-matched study from the European Group for Blood and Marrow Transplantation.
Blood.
88
1996
4711
5
Gahrton
G
Ljungman
P
Bladè
J
et al. 
Prognostic factors in allogeneic bone marrow transplantation for multiple myeloma.
J Clin Oncol.
13
1995
1312
6
Bensinger
WI
Buckner
CD
Anasetti
C
et al. 
Allogeneic marrow transplantation for multiple myeloma: an analysis of risk factors on outcome.
Blood.
88
1996
2787
7
Bladè
J
Samson
D
Reece
D
et al. 
Criteria for evaluating disease response and progression in patients with multiple myeloma treated by high-dose therapy and haemopoietic stem cell transplantation: Myeloma Subcommittee of the EBMT. European Group for Blood and Marrow Transplant.
Br J Haematol.
102
1998
1115
8
Martinelli
G
Terragna
C
Lemoli
RM
et al. 
Clinical and molecular follow-up by amplification of the CDR-III IgH region in multiple myeloma patients after autologous transplantation of haematopoietic CD 34+ stem cells.
Haematologica.
84
1999
397
9
Lemoli
RM
Martinelli
G
Olivieri
A
et al. 
Selection and transplantation of autologous CD 34+ B-lineage negative cells in advanced-phase multiple myeloma patients: a pilot study.
Br J Haematol.
107
1999
419
10
Voena
C
Ladetto
M
Astolfi
M
et al. 
A novel nested-PCR strategy for the detection of rearranged immunoglobulin heavy-chain genes in B cell tumors.
Leukemia.
11
1997
1793
11
Bird
JM
Russell
NH
Samson
D
Minimal residual disease after bone marrow transplantation for multiple myeloma: evidence for cure in long-term survivors.
Bone Marrow Transplant.
12
1993
651
12
Corradini
P
Voena
C
Tarella
C
et al. 
Molecular and clinical remissions in multiple myeloma: role of autologous and allogeneic transplantation of hematopoietic cells.
J Clin Oncol.
17
1999
208
13
Bartl
R
Frisch
B
Burkhardt
R
et al. 
Bone marrow histology in myeloma: its importance in diagnosis, prognosis, classification and staging.
Br J Haematol.
51
1982
361
14
Beishuizen
A
Hahlan
K
Hagemeijer
A
et al. 
Multiple rearranged immunoglobulin genes in childhood acute lymphoblastic leukemia of precursor B-cell origin.
Leukemia.
5
1991
657
15
Wasserman
R
Yamada
M
Ito
Y
et al. 
VH gene rearrangement events can modify the immunoglobulin heavy chain during progression of B-lineage acute lymphoblastic leukemia.
Blood.
79
1992
223
16
Ralph
QR
Brisco
MJ
Joshua
DE
Brown
R
Gibson
J
Morley
AA
Advancement of multiple myeloma from diagnosis through plateau phase to progression does not involve a new B-cell clone: evidence from the Ig heavy chain gene.
Blood.
82
1993
202
17
Roth
MS
Antin
JH
Ash
R
et al. 
Prognostic significance of Philadelphia chromosome-positive cells detected by the polymerase chain reaction after allogeneic bone marrow transplant for chronic myelogenous leukemia.
Blood.
79
1992
276
18
Diverio
D
Rossi
V
Avvisati
G
et al. 
Early detection of relapse by prospective reverse transcriptase-polymerase chain reaction analysis of the PML/RAR alpha fusion gene in patients with acute promyelocytic leukemia enrolled in the GIMEMA-AIEOP multicenter “AIDA” trial.
Blood.
92
1998
784
19
Nizet
Y
Van Daele
S
Lewalle
P
et al. 
Long-term follow-up of residual disease in acute lymphoblastic leukemia patients in complete remission using clonogeneic IgH probes and the polymerase chain reaction.
Blood.
82
1993
1618
20
Provan
D
Barlett-Pandite
L
Zwicky
C
et al. 
Eradication of polymerase chain reaction-detectable chronic lymphocytic leukemia cells is associated with improved outcome after bone marrow transplantation.
Blood.
88
1996
2228
21
Gribben
JG
Neuberg
D
Freedman
AS
et al. 
Detection by polymerase chain reaction of residual cells with the bcl-2 translocation is associated with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma.
Blood.
81
1993
3449
22
Mehta
J
Singhal
S
Graft-versus-myeloma.
Bone Marrow Transplant.
22
1998
835
23
Rondelli
D
Bandini
G
Cavo
M
et al. 
Discrepancy between serological complete remission and concomitant new bone lytic lesions after infusion of escalating low doses of donor lymphocytes in multiple myeloma: a case report.
Bone Marrow Transplant.
24
1999
685
24
Cavo
M
Bandini
G
Benni
M
et al. 
High-dose busulfan and cyclophosphamide are an effective conditioning regimen for allogeneic bone marrow transplantation in chemosensitive multiple myeloma.
Bone Marrow Transplant.
22
1998
27
25
Cavo
M
Bandini
G
Lemoli
RM
et al. 
Allogeneic transplantation with bone marrow or peripheral blood stem cells for multiple myeloma: a multivariate analysis of risk factors on outcome [abstract].
Bone Marrow Transplant.
21(suppl 1)
1998
S213