Minimal residual disease Molecular monitoring of the tumor ... - Nature

Bone Marrow Transplantation (2001) 28, 957–962  2001 Nature Publishing Group All rights reserved 0268–3369/01 $15.00 www.nature.com/bmt

Minimal residual disease Molecular monitoring of the tumor load predicts progressive disease in patients with multiple myeloma after high-dose therapy with autologous peripheral blood stem cell transplantation E Lipinski, FW Cremer, AD Ho, H Goldschmidt and M Moos Medizinische Klinik und Poliklinik V, Universita¨t Heidelberg, Heidelberg, Germany

Summary: The clinical relevance of the assessment of minimal residual disease (MRD) in patients with multiple myeloma (MM) to predict disease recurrence has not been proven. In the present study, we retrospectively analyzed the tumor load in peripheral blood (PB) and bone marrow (BM) samples of 13 patients with MM both in remission after high-dose therapy (HDT) with autologous PBSC transplantation (PBSCT) and at the time of progressive disease (PD). For six patients, subsequent samples obtained in remission could be included in the study. Tumor cells were assessed by means of quantitative PCR with allele-specific oligonucleotides (ASOqPCR) based on the method of limiting dilutions. PD was documented with ASO-qPCR in BM samples (median concentration of tumor cells in remission vs at PD: 0.18% vs 4.6%) representing a significant increase by a median factor of 8.7. In PB, the median tumor load was 799 cells/ml in remission and 23 400 cells/ml at PD. With a median factor of 45, the increase was much more pronounced. Comparing the results of the molecular monitoring in PB with those of the determination of the monoclonal protein, routinely applied as parameter for the course of the disease, revealed a superiority of the molecular monitoring because of the significantly higher increase in the tumor load. Analyzing the subsequent remission samples showed an increase of the malignant cells in four out of six PB samples and in all four BM samples available, indicating the potential of ASOqPCR for an early PD recognition. Bone Marrow Transplantation (2001) 28, 957–962. Keywords: myeloma; quantitative PCR; ASO-PCR; kinetics of tumor load; prediction of progressive disease

(HDT) with PBSC transplantation (PBSCT) leads to higher complete remission (CR) rates and prolonged overall survival in comparison to conventional chemotherapy for patients with MM,1 in most patients it fails to prevent recurrence. Further improvement might be achieved by novel strategies such as antiangiogenic and immunotherapeutic approaches after HDT. Assessment of minimal residual disease (MRD) could assist in the choice for the commencement of such treatment. So far this kind of monitoring has been hampered by the fact that in MM patients even in CR PCR negativity was only found in exceptional cases.2,3 This makes it necessary to quantificate the tumor load using elaborate methods in order to establish patterns in the kinetics of the tumor burden. The importance of such continued measurement of the tumor cells has been demonstrated recently by Cremer et al4 who provided evidence for a prognostic value of a determination of the tumor load in the course of sequential HDT. Quantitative PCR assays using allele-specific oligonucleotide primers (ASO-qPCR) are increasingly used for the sensitive and specific assessment of tumor cell numbers in PB and BM of patients with MM. In this retrospective study we analyzed the tumor load in PB and BM samples of 13 patients with MM after HDT and PBSCT using ASOqPCR. PB and BM samples of the patients were analyzed at the time of remission and at the time of progressive disease (PD). The hypothesis is that molecular monitoring is suitable for the recognition of PD. Samples from a subsequent point in time during remission from six patients were monitored to assess whether the kinetics of tumor load can be used for an early prediction of PD.

Materials and methods Patient characteristics

Multiple myeloma (MM) is a B cell malignancy characterized by a monoclonal expansion of plasma cells in bone marrow (BM) secreting a monoclonal protein measurable in peripheral blood (PB) or urine. Although high-dose therapy Correspondence: Dr M Moos, Department of Internal Medicine V, University of Heidelberg, Hospitalstr. 3, 69115 Heidelberg, Germany Received 31 May 2001; accepted 18 September 2001

Thirteen patients who achieved remission after HDT with subsequent PD and for whom an ASO primer was available were included in this study. Ten patients were treated with two sequential cycles of melphalan 200 mg/m2, three patients received one cycle of HDT (two patients (D, G) with melphalan only (200 mg/m2) and one patient (B) with melphalan (140 mg/m2) and total body irradiation (14.4 Gy)). The patients were enrolled either in a phase II study

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evaluating the toxicity and efficacy of sequential HDT and PBSCT in the therapy of MM5 or in a monocenter phase II study evaluating that of a single cycle of HDT and PBSCT.6 After HDT, three patients showed minimal response (MR), nine patients achieved partial remission (PR) and one patient achieved CR according to EBMT/IBMTR criteria.7 The median event-free survival was 15 months (range, 6–68 months after the last cycle). Patient characteristics are shown in Table 1.

Samples From the 13 patients, PB and BM samples were available in remission (median, 4 months; range, 2–11 months after the last cycle of HDT) and at the time of PD before further therapies were initiated. The BM sample in remission of patient C was collected 7 months after the PB sample. From six patients (A–F) six PB and four BM samples were available from a subsequent point in time during remission

Table 1

Patients’ characteristics

No.

Age (year)

Sex

Ig type

Stage

B2-MG (mg/l)

Response

PD (months)

A B C D E F G H I K L M N

57 31 61 60 47 48 52 44 43 64 43 40 52

m m f m m f m f m m m m m

IgA kappa IgG kappa IgA kappa IgG kappa IgG lambda IgG kappa IgG kappa IgG kappa IgG kappa IgA kappa IgG kappa IgG kappa BJ kappa

III A II A III A III A III A III A II A III A III A III A II A II A III B

na na 3,1 2,4 4,1 na na 1,5 na 3,5 3,4 na 2,9

PR MR PR MR PR PR PR MR PR PR PR PR CR

12 (9) 68 20 (14) 21 17 (12) 24 (20) 22 16 (11) 10 (6) 18 (13) 10 (7) 20 (15) 19 (15)

f ⫽ female; m ⫽ male. B2-MG ⫽ beta2-microglobulin, levels determined at diagnosis; na ⫽ not available; Response ⫽ best response after HDT; BJ ⫽ Bence-Jones protein; PR ⫽ partial remission; CR ⫽ complete remission; MR ⫽ minimal response; PD ⫽ time after 1st HDT, (time after 2nd HDT). Stage: according to the classification of Durie and Salmon.18

Table 2 No.

Tumor load in BM and PB of the 13 patients with MM

Monoclonal protein (g/l) t1

A pos.a B 25 C 0.3 D 27.3 E 6 F 9.1 G 14.9 H 19 I 19.3 K 2.1 L 7.1 M 26.3 N 5f Median 9.1g Range 0–27.3

Increase

t3 pos.a 37.5 6.1 33.9 18.7 12.1 39.5 42.7 22.8 9.9 13.3 31.1 80f 22.8g 0–80

BM tumor load (% of MNC)

t1 1 1.5 20 1.2 3.1 1.3 2.6 2.2 1.2 4.7 1.9 1.2 16f 1.9 1–20

t2

Increase

t3

0.03 0.22 0.3 0.13 3.4 6.57 0.71b — 6.2 0.57 1.6 0.5 0.041 — 0.076 0.22 2.06 9.8 0.04 — 10.6 0.083 — 9.3 0.8 — 1.4 0.061 — 0.61 1.4 — 2.9 1.2 — 4.6 1.3 — 6.7 0.18 1.8 4.6 0.03–1.4 0.22–3.4 0.076–10.6

PB tumor load (cells/ml)

t1 10 51 8.7 0.88 1.9 44.5 250 112 1.7 10 2.1 3.8 5.2 8.7 0.88–250

798 116.4 0c 1356 765.6 266 900 4550 799 6952 1125 516.8 2027.4 762 799 0–266 900

t2

Increase

t3

32 800 96 250 1438.1 24 120 108.1 12 880 1020 64 960 3068 77 000 5943 14 112 — 17 030 — 2096 — 3 447 360e — 11 900 — 23 400 — 62 920 — 18 060 2253 23 400 108.1–32 800 2096–3 447 360

121 207 ⬎119d 48 101 0.05 3.7 2.6 496 11 45 31 24 45 0.05–496

MNC ⫽ mononuclear cells; t1 ⫽ first time point of sample collection in complete remission, partial remission or minimal response; t2 ⫽ second time point of sample collection in complete remission, partial remission or minimal response; t3 ⫽ time point of sample collection in progressive disease; Increase ⫽ tumor load in t3/tumor load in t1. a Monoclonal protein only detectable by immunofixation; no increase. b t1 of BM sample ⫽ t2 of PB sample. c Below detection level. d Increase of 119 ⫽ tumor load in t3/tumor load in t2. e Concomitant acute myeloid leukemia occurred. f Analysis in urine (mg/l). g Nine cases of IgG type, three cases of IgA type monoclonal protein and one case of Bence-Jones protein in urine. Bone Marrow Transplantation


Quantification of clonotypic tumor cells The proportion of tumor cells in the PB and BM samples was determined using a quantitative ASO-PCR assay based on the method of limiting dilutions.8,9 Briefly, DNA from 330 000 cells per sample was serially diluted in 0.5 log steps and amplified using the appropriate ASO primers. At each dilution level, at least five identical PCR reactions were processed simultaneously. The PCR was performed from the dilution level in which all reactions were positive up to the dilution level in which all reactions were negative. A reaction without DNA was always used as negative control. The proportion of clonotypic cells in a sample was calculated based upon Poisson distribution statistics from positive and negative PCR reactions at each dilution level using the MAXLIKE computer program.9 The absolute numbers of circulating clonotypic cells per ml were calculated by multiplying the percentage determined by quantitative PCR by the difference of the number of leukocytes and the number of neutrophils per ml PB. All ASO primers included in this study generated a PCR signal only with the target DNA but not with buffy-coat DNA. The sensitivity of the ASO primers was tested for four patients (A, I, L, N), and the detection of a single copy of the target DNA in a background of DNA from 330 000 polyclonal cells was demonstrated.4 Clinical parameters of PD PD was defined by an increase of the M-component of the serum of at least 25% up from the time of remission after HDT in 10 patients and in one patient (N) by reappearance of the Bence-Jones protein in urine by immunofixation. One patient (A) was diagnosed with an extramedullary plasmacytoma. Patient F revealed an increase of the plasma cells in the cytology of the BM aspirate (⬍5% plasma cells in remission after HDT; 40% at PD). The amounts of the monoclonal protein at the first time of the PB and BM sample collection in remission and at the time of PD are shown in Table 2 and Figure 1a, and the kinetics of the monoclonal protein of patients A–F in Figure 2.

Monoclonal protein (g/l)

Mononuclear cells from BM and PB samples were obtained by Ficoll–Hypaque density centrifugation (Biochrom, Berlin, Germany). Genomic DNA was isolated using DNAzol Reagent (Gibco BRL, Eggenstein, Germany). The concentration of DNA was determined by optical density measurement. The concentration and the integrity of DNA was verified by gel electrophoresis of defined amounts of DNA. Integrity and quality of the DNA was checked by amplification of 2 ␮g of DNA using primers complementary to sequences of the bcl2-gene.

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P = 0.0022

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Clonotypic cells in BM (% of MNC)

Nucleic acid extraction

Median factor of increase: 1.9

Clonotypic cells in BM (% of MNC)

(median, 9 months; range, 6–61 months after the last cycle of HDT; and median, 4 months; range, 2–12 months before PD).

(2) Remission

Progressive disease

Median factor of increase: 8.7 P = 0.0024

100 10 1

(3)

0.1 0.01 0.001

Remission

Progressive disease

Median factor of increase: 45 P = 0.019

1e6 1e5 10000 1000 100 10

(4) Remission

Progressive disease

Figure 1 Intraindividual comparison of the amount of the monoclonal protein in PB (a) and of the proportion of clonotypic cells in BM (b) and of the number of clonotypic cells per ml PB (c) at the time of remission and at the time of PD of the 13 patients with multiple myeloma after high dose therapy. MNC ⫽ mononuclear cells. (1) Analysis in urine (mg/l); (2) monoclonal protein only detectable by immunofixation; no increase; (3) the BM sample in remission collected 7 months after the PB sample in remission; (4) below detection level.

Statistical analysis The intraindividual differences between the tumor load in BM and PB and the monoclonal protein at the time of remission and at the time of PD were determined with the Wilcoxon matched pairs test. The same method was used to analyze the increase of the tumor load in BM and PB and of the monoclonal protein from the time of remission after HDT to the time of PD. Bone Marrow Transplantation

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PD

Figure 2 Kinetics of the tumor load in BM and PB and of the monoclonal protein in PB of the six patients (A–F) with additional samples from a subsequent point in time during remission. The monoclonal protein (MP) is indicated with a dotted line, the proportion of the clonotypic cells in BM with a solid line and the number of the clonotypic cells in PB with a dashed line. In patient A and F the diagnosis of PD was not based on the monoclonal protein determination. MNC ⫽ mononuclear cells. (1) Monoclonal protein only detectable by immunofixation, no increase; (2) tumor load in the first PB sample below detection level.

Results Tumor load in remission and at the time of PD The monoclonal protein in PB at the time of remission was in a median of 9.1 g/l (range, 0–27.3 g/l) and at PD 22.8 g/l (range, 0–80 g/l) (Table 2). The monoclonal protein was in nine cases of the IgG type, in three cases of the IgA type and in one case of the Bence-Jones type (measured in Bone Marrow Transplantation

urine). The increase of the monoclonal protein was significant (P ⫽ 0.0022) with a median factor of 1.9 (range, 1– 20) as determined by the Wilcoxon matched pairs test (Figure 1a). At the time of remission, the proportion of clonotypic cells as assessed by ASO-qPCR in BM was 0.18% (range, 0.03–1.4%) and at PD 4.6% (range, 0.076–10.6%) (Table 2). In 12 of the 13 patients, there was an increase of tumor load in BM from the time of remission to PD. The increase


was significant with a median factor of 8.7 (range, 0.88– 250; P ⫽ 0.0024; Figure 1b). In one patient (D) the tumor load in BM was lower at PD than at the time of remission. In PB samples, the number of clonotypic cells as assessed by ASO-qPCR was 799 cells/ml (range, 0– 266 900 cells/ml) at the time of remission and 23 400 cells/ml (range, 2096–3 447 360 cells/ml) at PD (Table 2). In 12 of the 13 patients the tumor load in PB was higher at the time of PD. The increase of the tumor load resulted in a median factor of 45 (range, 0.05–496) and was also significant (P ⫽ 0.019; Figure 1c). In one patient (F) the tumor load decreased. The increase of the tumor load in PB was significantly higher than that of the monoclonal protein (Wilcoxon matched pairs test: P ⫽ 0.003). The higher increase of the tumor load in PB than in BM in 10 of the 13 patients also confirmed the benefit of the PB measurement (P ⫽ 0.15). Figure 3 shows the results of the ASO-qPCR of the BM sample from patient B collected at the time of PD. Kinetics of the tumor load after HDT with PBSCT In all four patients (A, B, D, F) with an additional BM sample in remission the tumor load was already rising. In one patient (D) the tumor load at the subsequent time of remission was even higher than that at the time of PD. In four (A, B, C, E) of the six patients (A–F) with a Pos. Ctrl. Neg. Ctrl. 100 bp ladder

subsequent PB sample in remission there was already an increase in the number of malignant cells visible. In two patients (D, F) there was a decrease of the tumor load at the subsequent time of remission. The kinetics of the tumor load in PB and BM samples and the kinetics of the monoclonal protein of the six patients are shown in Figure 2.

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Discussion In MM, quantitative PCR with ASO primers is an appropriate method for assessing low numbers of tumor cells and thus for monitoring of minimal residual disease. The clinical significance for such monitoring in predicting PD has not been demonstrated. A predictive assessment could be relevant for further treatment decisions. In this study, clonotypic cells were detected in all patient samples except one. PD was documented with ASO-qPCR by a significant increase of the tumor load between the observation points not only in BM but also in PB samples. Interestingly, the increase in PB was much more pronounced than in BM, indicating that PB might be preferable to BM for monitoring MRD in MM. Additionally, the increase of the tumor load in PB in contrast to BM was significantly higher than that of the monoclonal protein. We observed an increase in the tumor load before the diagnosis of PD in PB in four of six patients and in BM

20 bp ladder 100 cells

33 cells

10 cells

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100 bp ladder

Specific PCR product (82 bp)

Specific PCR product (82 bp)

100 bp ladder Neg. Ctrl. Pos. Ctrl.

1 cell

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Figure 3 Analysis of the tumor load of the BM sample collected at progressive disease from patient B by ASO-qPCR. The specific PCR product is 82 bp in length. The result of the ␹2-approximation was a percentage of clonotypic cells of 6.6% of mononuclear cells. Bone Marrow Transplantation


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in all four patients. Thus, our results indicate that the assessment of the kinetics of the tumor load in PB and BM by ASO-qPCR might add predictive information for an early commencement of further therapy options. With these results we confirm previous ones of Kiel et al,10 who observed higher amounts of circulating CD19-positive and CD19-negative clonotypic cells at the time of PD in comparison to the time of remission. To date, studies focusing on a continuous determination of the tumor load up to the development of PD are limited. The study of Billadeau et al11 described that the PB tumor burden remains rather constant in six MM patients prior to and post transplantation with only one sample analyzed at the time of PD. In line with Billadeau’s results, Rasmussen et al12 reported a reduction of clonal cells in PB after induction therapy with no further changes in the course of the HDT in seven MM patients. Only in two patients was more than one sample after HDT measured, and no samples at PD were included.13,14 PB samples are not only more homogenous than BM samples, but are also more easily available. This facilitates more frequent sampling and thus tighter monitoring of MRD. Together with the higher factor of increase observed for the PB tumor load in PD, this indicates that PB is preferable over BM for monitoring MRD. Thus, in future – after evaluation of appropriate studies – the kinetics of tumor burden in PB could influence clinical decisions on an early onset of an antiangiogenic treatment with low doses of thalidomide or even on immunotherapeutic approaches like idiotype vaccination protocols. New developments in the quantification of tumor cells by PCR like real time assays 15,16 may allow evaluation of larger numbers of patients in a shorter time. This is a prerequisite for including monitoring of MRD in the stratification of patients in therapy protocols, as is already done in the management of acute lymphoblastic leukemia.17

Acknowledgements We are indebted to Axel Benner for his important information regarding the statistical analysis and to Ileana Wenzel for her critical reading of the manuscript. The authors thank Hildegard Betha¨ user, Edith Ehrbrecht, Carmen Kro¨ ner and Renate Schulz for excellent technical assistance.

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