Molecular monitoring of tumour load kinetics predicts disease progression after non-myeloablative allogeneic stem cell transplantation in multiple myeloma

M. S. Raab1,*, F. W. Cremer2, I. N. Breitkreutz1, S. Gerull1, T. Luft1, A. Benner3, M. Goerner4, A. D. Ho1, H. Goldschmidt1 and M. Moos1

1 Department of Internal Medicine V and 2 Institute of Human Genetics, University of Heidelberg; 3 Central Unit Biostatistics, German Cancer Research Center, Heidelberg; 4 Department of Hematology and Oncology, Staedt. Kiniken Bielefeld GmbH, Germany

* Correspondence to: Dr Marc S. Raab, Department of Internal Medicine V, University of Heidelberg, INF 410, D-69120 Heidelberg, Germany. Tel: +49-6221-568072; Fax: +49-6221-565609; Email: marc.raab{at}med.uni-heidelberg.de


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background: Non-myeloablative allogeneic stem cell transplantation followed by immunomodulatory therapies is considered a potentially curative approach in the treatment of multiple myeloma and most effective in a minimal residual disease setting.

Patients and methods: The aim of this study was to find the most sensitive real-time PCR assay (TaqMan), based on the IGH rearrangement, to quantify the tumour load of 11 patients with multiple myeloma after non-myeloablative allogeneic transplantation. Patient-allele specific primers (ASO) and the TaqMan probe were derived from CDR2 and CDR3 hypervariable regions of IGH, while consensus primers were located within the FR3 and FR4/JH regions. Four different approaches of primer combinations were tested.

Results: ASO-forward and -reverse primers together with the clone-specific TaqMan probe were the most sensitive approach compared with the JH (P=0.071) or the FR3 consensus primer (P <0.001). The detection limit amounted to 1/104–1/105 cells. Consecutively, 120 samples from 11 patients prior and post allogeneic transplantation were analysed. Three patients reached complete clinical remission accompanied by molecular remission. Disease progression or relapse was seen in six patients. In five, molecular progressive disease was detected prior to the clinical diagnosis of progression or relapse.

Conclusion: Patient-specific real-time IGH-PCR provides the opportunity for earlier treatment intervention.

Key words: IgH rearrangement, minimal residual disease, multiple myeloma, non-myeloablative allogeneic transplantation, real-time PCR


    Introduction
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Multiple myeloma (MM) is a B cell malignancy characterised by a clonal expansion of plasma cells. Autologous stem cell transplantation (ABSCT) is considered the standard therapy for patients younger than 65 years of age [1Go, 2Go]. Nearly all patients will eventually relapse despite complete remission rates of up to 50% [3Go, 4Go]. Allogeneic hematopoietic stem cell transplantation (allo SCT) is postulated to be a potentially curative treatment that has been shown to be associated with a significantly lower risk of relapse and increased long-term disease-free survival [5Go]. These positive effects are probably due to the combination of high-dose chemotherapy and the capacity of donor lymphocytes to eradicate recipient myeloma cells, but are diminished by the high treatment-related mortality of myeloablative attempts in MM. Therefore, great effort is currently made to reduce these adverse effects while maintaining a potential graft-versus-myeloma (GvM) effect by the use of non-myeloablative conditioning regimens [6Go–9Go]. In this context, post-transplant therapeutic interventions are mainly based on immunomodulation, such as early discontinuation of immunosuppression or the infusion of donor lymphocytes (DLI) [10Go]. Since immunotherapy is most effective when tumour load is low, the kinetics of residual tumour cells might be predictive of early progression [11Go, 12Go]. In the autologous setting molecular monitoring of the tumour load is able to predict progressive disease [13Go, 14Go]. We therefore established a quantitative real-time PCR-assay (TaqMan technology) after having determined the most sensitive strategy of primer/probe design. Here, we demonstrate the ability for guidance and monitoring of post-transplant immunomodulation and the precocious detection of clinical relapse.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients and treatment characteristics
Patient characteristics are summarised in Table 1. Eleven patients with MM (stage III according to Salmon and Durie [15Go]) were retrospectively enrolled in this evaluation. All patients underwent non-myeloablative conditioning therapy prior to allogeneic transplantation. Written informed consent was obtained and ethical approval of the study was granted. The median age at diagnosis was 50 years (range 38–55), and 52 years (range 40–57) at the time of non-myeloablative allo SCT. The median number of cycles of chemotherapy prior to allo SCT was nine (range 4–13) involving at least one high-dose therapy followed by ASCT. Remission criteria defined by EBMT (European Group for Blood and Marrow Transplant) guidelines were applied [16Go].


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Table 1. Patient characteristics, pretreatment and key variables of allogeneic transplantation

 
Minimal residual disease analysis by real-time PCR
Nucleic acid extraction, immunoglobulin heavy chain (IGH) consensus PCR and sequencing of VDJ segments were essentially performed as described previously [13Go]. Briefly, mononuclear cells from bone marrow (BM) and peripheral blood (PB) samples were obtained by Ficoll-Hypaque density centrifugation (Biochrom, Berlin, Germany) and genomic DNA was isolated using DNAzol Reagent (Gibco BRL, Eggenstein, Germany) while total RNA was extracted with the RNeasy kit (Quiagen, Hilden, Germany). Integrity of DNA and RNA was checked by amplification of the BCL-2 and ß2-microglobulin genes (Stratagene, Heidelberg, Germany), respectively. After reverse transcription total cDNA was amplified using CDR1 consensus primers plus LJH as JH-consensus primer [17Go, 18Go]. Sequencing was done on an automated DNA sequencer (ABI Prism, PE Biosystems, Weiterstadt, Germany).

TaqMan real-time PCR was performed with patient-specific (allele specific oligo, ASO) and consensus primers and probes derived from CDR2, CDR3 and J regions as described below. The concentration of each primer and probe was optimised for each assay. Amplification and fluorescence detection were carried out on the ABI Prism 7700 SDS analytical thermal cycler (Applied Biosystems, Weiterstadt, Germany). Forty-five cycles of PCR were performed. The cycle threshold (CT) is known as the cycle in which fluorescent emission reaches 10-fold the basal emission, a value that is proportional to the copy number of the target gene. Patient-specific standard curves were prepared by serial 10-fold dilutions of the plasmid DNA containing the tumour-specific VDJ sequence starting from 105 to 10 copies in a background of 600 ng of polyclonal genomic DNA. An independent BCL-2 quantitative TaqMan assay was performed for each patient to normalise each sample for DNA content by dividing IGH copies with the mean number of BCL-2 copies. Each reaction was carried out in triplicate.

Strategies of ASO and consensus primer design
Different strategies for the use of primers and probes located in the highly variable as well as in the conserved regions have been reported [12Go, 19Go–22Go]. Determination of the specific and most sensitive approach has not been addressed yet. Involvement of the most variable CDR3 region, at least for the fluorogenic probe, was assumed to be necessary to reach sufficient sensitivity in patients after allogeneic transplantation. Therefore patient-specific ASO primers and the TaqMan probe were derived using the CDR2 (forward primer) and CDR3 (reverse primer/probe) hypervariable regions of the IGH rearrangement, while the consensus primers were located within the FR3 (forward primer FR3A) and FR4/JH (reverse primer LJH) region [17Go, 18Go]. The median CT-value of serial 10-fold dilutions of 16 different clone-specific plasmids in polyclonal genomic DNA was assessed for each of the following approaches (Figure 1): (A) ASO-forward/ASO-reverse primer, (B) ASO-forward/consensus reverse primer, (C) consensus forward/ASO-reverse primer and (D) consensus forward/consensus reverse primer.



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Figure 1. Localisation of oligonucleotides within the IgH locus. Consensus primers were located within the FR3 and the JH region of the IGH locus. ASO primers were derived from the patient-specific CDR2 and CDR3 hypervariable regions. Each strategy incorporated a patient-specific CDR3 TaqMan probe. (A) ASO-forward/ASO-reverse primer, (B) ASO-forward/consensus reverse primer, (C) consensus forward/ASO-reverse primer and (D) consensus forward/consensus reverse primer.

 
Statistical analysis
A linear regression model was used to analyse the dependency of CT on copy number (log 10 transformed values) and strategy (A–D). For pairwise comparisons of the four strategies, P values were adjusted according to the method proposed by Tukey. An effect was judged as statistically significant at a P value not larger than 5%. All statistical calculations were performed using R, Version 1.8 [23Go].

Chimerism analysis
After nucleic acid extraction, as described above, small tandem repeats were amplified and analysed. PCR was performed using the Amp-FISTR Profiler PCR amplification kit (Applied Biosystems, Weiterstadt, Germany) as recommended by the manufacturer. Separation and detection of the amplified PCR products was done on an ABI 310 automated sequencer (Applied Biosystems). The analysis of the results was performed using the Genescan 2.1 software (Applied Biosystems). The threshold for quantification of the ratio of donor/recipient chimerism was 2.5%.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Evaluation of primer/probe design
To identify the most sensitive approach, comparison of the strategies A and B showed significantly lower CT-values than approaches C and D (P <0.001; Figure 2). With 10 copies/600 ng human genomic DNA of 16 patient-specific plasmids, the median CT-value reached was 40.1 for approach A (Figure 1), and below detection limit (>45) for B, C and D. The detection limit of approach A amounted to 1/104–1/105 cells within a linear range. Comparison of CT scores adjusted for the different copy numbers showed best values for A, followed by B (estimated CT difference {Delta}CT=1.7, P=0.071), C ({Delta}CT=5.3, P <0.001) and D ({Delta}CT=7.0, P <0.001). The use of ASO-forward and -reverse primers together with the allele-specific TaqMan probe results in the most sensitive strategy of primer design for the quantification of clonal tumour cells. Thus, this approach was applied for measurement of tumour load kinetics in patients after allogeneic transplantation following non-myeloablative conditioning.



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Figure 2. Cycle threshold (CT)-values of the four different approaches of primer localisation. CT-values obtained with the four different approaches are shown. 10, 102 and 104 copies of 16 clone-specific plasmids in a polyclonal background of 600 ng DNA were amplified. Median CT values corresponding to the plasmid dilutions are depicted by bars. Strategy A revealed the highest sensitivity with the lowest CT values of all dilutions. Second ranked was strategy B with a slight increase of CT. Comparison of strategy A with approaches C and D showed significantly higher CT values (P <0.001), corresponding to lower sensitivity.

 
Clinical and molecular follow-up of patients after allo SCT
A total of 51 BM and 69 PB samples from 11 patients were assessed for the presence of the clonal IGH rearrangement. Forty-one BM and 56 PB follow-up samples were obtained. The median time of molecular follow-up was 231 days (range, 69–440).

After allogeneic transplantation two patients (HM and SH) achieved clinical complete remission (CCR) on day +39 and +177, respectively. One additional patient (BG) remained in continued pre-transplant CCR. These three patients converted to molecular remission post-allo SCT on day +39 (HM, three BM and six PB samples revealing PCR negativity), +177 (SH, two BM and one PB samples) and +71 (BG, two BM and nine PB samples). Two of these patients (SH and BG) are currently in long-term CCR with last clinical data from day +440 and +1298, respectively. One patient (HM) relapsed after achieving a complete remission. Treatment with steroids and DLI resulted again in CCR and molecular remission.

Another three patients (BH, FK, TR) remained in partial response (PR) (last clinical data from day +364, +567 and +867, respectively) with a tumour load in BM between 0.005% and 0.5% (11 samples) while no tumour load was detectable in PB (nine samples).

Five patients (BK, HH, MK, SW, VP) experienced progressive disease (PD), diagnosed by an increase of the monoclonal protein post allo SCT on day +290, +118, +31, +57 and +189, respectively. One relapsed on day +116 (HM, as mentioned above). Figure 3 depicts the clinical and molecular follow-up of the six patients with PD or relapse. By molecular follow-up, an increase of tumour load was detected in five of these cases, on day +100 in PB confirmed by a second PB sample on day +171 for patient BK, on day +95 in PB confirmed by BM on day +109 (HM), on day +27 in BM and PB (HH), on day +30 in BM confirmed by a second BM sample on day +56 (SW) and on day +30 confirmed by BM and PB on day +56 (VP). Thus, the first increase of the malignant plasma cell clone preceded the clinical diagnosis of PD by a median of 95 days (range, 21–190).



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Figure 3. Kinetics of tumour load in patients with relapse/PD. Kinetics of the tumour load are depicted by rhombs (bone marrow samples, BM) and squares (peripheral blood, PB) while the monoclonal protein is represented by triangles. The bottom line indicates the detection limit (<0.001%). Clinical (CPD) and molecular progressive disease (MPD) are depicted by arrows.

 
Since a precocious molecular detection of disease progression should lead to early therapeutic intervention, a reliable definition is necessary. Therefore, we defined molecular PD as an increase in tumour load in at least two different samples taken either of the same compartment (BM or PB) at a consecutive or of the other compartment at a corresponding point of time.

According to this definition, molecular PD (BK, HH, SW, VP) or relapse (HM) could be detected 119, 91, 1, 133 and 7 days, respectively, before PD was diagnosed clinically by kinetics of the monoclonal protein (Figure 3). The remaining patient (MK) entered early clinical PD at day +31 after transplantation due to increase of urine light chain excretion. At that time, the patient showed a persistently high amount of BM tumour load of 9.6%. However, molecular PD could not be clearly diagnosed according to the above mentioned criteria.

After diagnosis of clinical PD, five patients received steroids (BK, HH, HM, SW, VP), in two cases followed by DLI (SW and HM). After DLI, a significant reduction of tumour load was observed, with patient HM entering CCR and molecular remission again, accompanied by extensive intestinal Graft-versus-Host disease (GvHD) (WHO grade III) with fatal outcome. For the remaining patient (MK) immunosuppression was reduced as sole measure, resulting in an ongoing reduction of tumour load accompanied only by mild GvHD of the skin (WHO grade I) and an overshooting polyclonal production of immunoglobulin (Figure 3).

In summary, we were able to detect molecular PD preceding clinical PD or relapse in five out of six patients. Furthermore, all patients of this study in whom an increase of tumour load was recognised by molecular follow-up after allo SCT eventually went into clinical PD.

Recipient/donor chimerism and correlation to the tumour load
Each of the 41 BM and 56 PB follow-up samples obtained after allo-SCT was analysed for the percentage of recipient/donor chimerism. Patients in CCR achieved full donor chimerism (FDC) in the bone marrow on days +252 (BG) and +177 (SH), while the patient relapsing from CCR (HM) never reached FDC (best <2.5%, day +67). Those in PR showed constant low level recipient chimerism of <2.5% with two of them (FK, TR) reaching FDC on days +221 and +307, despite a detectable tumour load in the BM. However, no clear correlation of recipient/donor chimerism and tumour load kinetics was observed in patients with PD or relapse (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Allogeneic stem cell transplantation holds promise as a possible curative approach in the treatment of MM. Since treatment-related mortality impairs the clinical outcome, and a GvM effect seems to be substantial for the postulated curative efficacy, reduced intensified conditioning is currently considered the treatment of choice within the field of allogeneic transplantation [7Go, 8Go, 24Go, 25Go]. These therapeutic regimens are mainly based on peri- and post-transplant immunomodulation. However, a successful GvM effect curing a clinically progressing MM after allo SCT is mostly associated with a life-threatening GvHD, whereas milder immunologic reactions are rarely efficient [26Go]. This is paralleled by observations with chronic myeloid leukemia which best responds to Graft-versus-Leukemia reactions in the stage of minimal disease. Here, early immunomodulatory intervention can retain curative activity while minimising the procedure-associated side effects [27Go]. Therefore patients with MM may also benefit from monitoring molecular remission and especially kinetics of tumour load after non-myeloablative allo SCT.

Gerard et al. [28Go] first showed the feasibility of real-time IGH PCR assays to quantitate residual myeloma cells in clinical samples after high-dose therapy and ABSCT. Thereafter, several studies determined the kinetics of tumour cells after ABSCT as well as allogeneic transplantation. In these studies different approaches of primer and probe design were used, such as ASO-forward primer and probe/JH consensus reverse primer [18Go], ASO-forward primer/JH consensus reverse primer and probe [21Go], ASO primers/FR3 consensus probe [21Go], ASO primers and probe [12Go]. Each of these groups reported a sensitivity of 10–4–10–5. However, a comparison of the different strategies has not been reported yet.

In the present study, we found the most sensitive approach of primer and probe design using forward and reverse patient-specific primers derived from the CDR2 and CDR3 regions, the latter representing the most variable part of the IGH sequence in which we located our patient-specific fluorescent probe as well. Compared to consensus forward primers (FR3A) within the FR3 region replacing the ASO-forward primer (P <0.001), JH region consensus primers (LJH) replacing the ASO-reverse primer showed just a slightly lower sensitivity (P=0.071). When analysing the individual clonal sequences with respect to the primer sequence, all but one showed at least one base-pair mismatch in the FR3 region. One clone-specific plasmid containing an FR3 region matching the FR3A consensus sequence showed comparable CT results for strategy C and strategy B.

Molecular remission assessed by non-quantitative ASO-PCR has been reported to be achieved after myeloablative allogeneic transplantation [11Go, 29Go–31Go] and to predict superior relapse-free survival [11Go]. Recently, the group of Voena et al. [12Go] reported the feasibility of real-time PCR to evaluate the GvM effect after allogeneic transplantation following conventional myeloablative conditioning in three patients. The assessment of molecular remission and kinetics of residual myeloma cells in the non-myeloablative setting has not been described yet.

In order to characterise the kinetics of myeloma cells after non-myeloablative allogeneic transplantation, we used strategy A for evaluating 120 patient samples. We report the molecular follow-up of 11 patients with focus on progressive disease or relapse of six cases after allo SCT. In five of these cases a molecular progression of the disease could be detected prior to clinical onset of PD/relapse. However, in two cases this succeeded only a few days before PD was diagnosed clinically, due to a lack of appropriately timed samples.

One patient went into clinical PD shortly after allo SCT accompanied by a high level of tumour load. Reduction of immunosuppression led to an ongoing decrease of clonal genomes, indicating an enabled GvM effect.

In summary, our findings demonstrate that (i) real-time PCR can provide a useful tool to detect molecular PD prior to clinical PD allowing early treatment intervention such as DLI, and (ii) patients with stable molecular tumour load without signs of PD could benefit from optimising immunotherapeutic intervention to maintain curative activity while minimising the associated side effects.

Prospective studies should evaluate if the monitoring of molecular disease can guide early post-transplant therapeutic intervention, reduce life-threatening side-effects and prevent clinical relapse or PD.


    Acknowledgements
 
We want to thank Julia Gobbert, Renate Bangert, Edith Ehrbrecht, Hildegard Bethäuser and Carmen Kröner for excellent technical assistance. This work was supported by the Deutsche Jose-Carreras-Foundation and the Heidelberg University Young Investigators Award.

Received for publication June 19, 2004. Revision received November 24, 2004. Accepted for publication November 29, 2004.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
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