Leukemia (2005) 19, 336–343 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu
DEBATE ROUND TABLE Reliable quantification of hematopoietic chimerism after allogeneic transplantation for acute leukemia using amplification by real-time PCR of null alleles and insertion/ deletion polymorphisms A Jime´nez-Velasco1, M Barrios1, J Roma´n-Go´mez2, G Navarro1, I Bun˜o3, JA Castillejo2, AI Rodrı´guez1, G Garcı´a-Gemar1, A Torres2 and AI Heiniger1 1 Hematology Department, Carlos Haya Hospital, Ma´laga, Spain; 2Hematology Department, Reina Sofı´a Hospital, Co´rdoba, Spain;and 3Bone Marrow Transplantation Unit, Gregorio Maran˜o´n Hospital, Madrid, Spain
Increasing mixed chimerism (MC) after allogeneic stem cell transplantation (SCT) has been associated with a high risk of relapse in acute leukemia. We evaluated a new method for chimerism detection, based on the quantitative real-time PCR (qrt-PCR) amplification of null alleles or insertion/deletion polymorphisms (indels). All qrt-PCR assays with null alleles and indels attained a sensitivity of at least 104, as well as good intra- and interassay concordance, and a high accuracy in experiments with cell mixtures. Informativeness was found in 80.3% of the 61 donor/recipient pairs tested. Nonrelapsed patients showed a progressive decrease in peripheral blood chimerism to values below 0.01% (complete chimerism (CC)). Bone marrow chimerism failed to reach CC more than 4 years after SCT. Increasing MC was observed prior to relapse in 88.2% of patients. Compared with conventional PCR amplification of variable number of tandem repeats, qrt-PCR predicted a significantly higher number of relapses (88.2 vs 44.4%) with a median anticipation period of 58 days. In conclusion, chimerism determination by qrt-PCR amplification of null alleles and indels constitutes a useful tool for the follow-up of patients with acute leukemia after SCT, showing better results than those obtained with conventional PCR. Leukemia (2005) 19, 336–343. doi:10.1038/sj.leu.2403622 Published online 20 January 2005 Keywords: chimerism; real-time PCR; acute leukaemia; allogeneic SCT; insertion/deletion polymorphisms
Introduction Relapse is the most frequent cause of failure after allogeneic stem cell transplantation (SCT) in patients with acute leukemia.1,2 Mixed chimerism (MC) increases in patients with acute leukemia who are at high risk for relapse, enabling its anticipation by about 2 months.3–7 Consequently, the determination of hematopoietic chimerism to monitor grafting and prevent relapse3,7–9 has become a useful method in the followup of SCT patients. DNA-based chimerism determination, mainly PCR amplification of polymorphic genes, such as nucleotide tandem repeats (VNTR or STR), has replaced previous less sensitive and accurate immunological or cytogenetic procedures.10,11 However, the sensitivity of conventional PCR methods is still no higher than 1%.9 As a consequence, a considerable number of patients still relapse shortly after demonstrating complete donor chimerism (CC),3,6,7 probably because the percentage of host cells does not reach the minimum level for detection. Correspondence: Dr A Jime´nez-Velasco, Hematology Department, Carlos Haya Hospital, Avda Carlos Haya, 29010 Malaga, Spain; E-mail:
[email protected] Received 10 July 2004; accepted 27 October 2004; Published online 20 January 2005
Procedures based on quantitative real-time PCR (qrt-PCR) offer important advantages with respect to previous amplification strategies that have been thoroughly described. In addition to higher sensitivity and accuracy, they show faster performance, less competition of products with different length and exclusion of nonspecific signals.12–14 Chromosome Y-related loci, readily detected by other chimerism methods, such as fluorescent in situ hybridization (FISH),9,15 have also proved suitable for qrt-PCR determination. These methods have been successfully used for the detection of small amounts of cells,16,17 although sex mismatch between donor and recipient constitutes its main limiting factor for routine application. Other recently designed methods are based on the quantification of single-nucleotide polymorphisms (SNP).13,14,18 Alizadeh et al14 evaluated 19 polymorphisms differing by at least two consecutive variable bases, and Maas et al18 also managed to distinguish donor and recipient using seven SNPs. These methods detect residual host cells up to levels of 0.1–0.01%, but they require identification of small differences between alleles, which reduces their discriminative ability and increases assay complexity. Recently, a great number of insertion–deletion diallelic polymorphisms (indels) have been characterized in the human genome.19,20 We propose a new approach for the determination of chimerism, based on the existence of long genomic sequences (indels), occasionally complete genes (null alleles), which are either present or absent in the individual genotype. The detection of a donor–recipient DNA mismatch in these sequences is especially useful for chimerism follow-up when the donor, but not the recipient, presents the null or the deleted genotype. We tested a set of 10 markers, four null alleles and six indels, and hypothesized that they predict acute leukemia relapse more accurately than conventional PCR methods. We therefore undertook a retrospective analysis in a series of consecutive patients allotransplanted for acute leukemia at our institution, using qrt-PCR Light-Cycler technology with sequence-specific primers and hybridization probes, and compared the results with those obtained by conventional PCR with VNTR markers.
Materials and methods
Clinical characteristics of patients A total of 61 consecutive patients with acute leukemia who received an allogeneic SCT at our institution between January 1997 and June 2003 were included in the study. In all cases, informed consent was obtained according to institutional guidelines. Collection of outcome data was ended as of
Real-time PCR and indels chimerism A Jime´nez-Velasco et al
337 December 2003, in order to guarantee a minimum follow-up of 6 months. The mean age of the 61 patients was 26.0 years (range: 4–54), 19 of whom were children (31.1%). The male/female ratio was 28/33, and myeloid leukemia was the most frequent diagnosis (54.1% of cases). Most patients (72.1%) were transplanted in complete remission and from an identical sibling (86.9%). The source of stem cells was peripheral blood (PB) in 43 of cases, bone marrow (BM) in 17 and in one case umbilical cord blood. Conditioning was based mainly on chemotherapy (47 cases), except for 14 patients with lymphoblastic leukemia in whom total body irradiation was used. Four patients (UPNs 2147, 2176, 2180 and 2181) had to be excluded from further chimerism analysis because of early death (within 1 month of SCT). The median follow-up in survivors was 41.8 months (range: 9.6–87.5).
DNA samples Genomic DNA was extracted from fresh whole PB or BM using standard procedures (Puregene DNA isolation kit, Gentra Systems, Minneapolis, MN, USA), and stored at –801C until PCR experiments were performed. Clinical criteria determined the rate of sample collection, although during the first post-SCT year, measurements were made about monthly in PB and every 2–3 months in BM; this period gradually lengthened thereafter. The median number of samples available in patients analyzed was four (range: 1–12) in BM and seven (range: 2–21) in PB. In order to homogenize the time schedule for all patients, samples were grouped in the same time intervals from the moment of SCT to the end of follow-up.
VNTR amplification Amplification by conventional PCR of five different minisatellite regions: D1S80, APO-B, DXS52, 33.6. and 33.1 was used for determination of VNTR chimerism. Primer sequences and conditions for each reaction have been described previously.7,21–23 To evaluate chimerism status after allo-SCT, we used a semiquantitative approach based on densitometric measurements, which has been fully described by our group.7,21 This assay allows the detection of a minor recipient cell Table 1
Real-time PCR amplification IN all, 10 sequences, corresponding to four null alleles (GSTM1, GSTT1, SRY and RhD) and six indels (DCP1, Xq28, R271, rs4399, FVII and THYR) were amplified using LightCycler technology. These markers were selected from different sources (Table 1), depending on their chromosome position, the length of the insertion sequence and the frequency of the different alleles.
Primers and probes: The sequences of the qrt-PCR primers and probes (TIB MolBiol, Berlin, Germany) used for amplification of the wild-type genes GSTM1, GSTT1, RhD and SRY have been published.18,24–26 The remaining primers and probes were designed according to the gene DNA sequence available in the GeneBank database by the LightCycler Probe Design program (Roche, Mannheim, Germany), in order to optimize the size, melting temperature and position of the nucleotides (see Table 1).27–30 Indel forward primers were always designed to hybridize the indel sequence, whereas the reverse primer and both probes were complementary of the nonvariable sequence of the gene; in null alleles, all primers and probes hybridize within the corresponding gene. The sequences of primers and probes used for each polymorphism have been included as ‘Supplementary Information’. LightCycler run conditions: Similar conditions were set up for all runs: 100 ng of genomic DNA was added to 20 ml reaction volume containing 0.4 mmol/l each primer, 0.2 mmol/l. anchor and sensor probe and 2 ml of LightCycler FastStar DNA Master Hybridization probes (Roche Molecular Biochemicals) that included buffer, Taq DNA polymerase and MgCl2. The final Mg2 þ concentration in the reaction mixture was adjusted to 3.5 mmol/l in all cases, except for SRY which was 4.5 mmol/l. b-Globin, used as a reference gene, was amplified in the same run and following the same procedure described for each marker. The primers and probes for b-globin amplification were provided in a commercial kit (LightCycler control kit DNA, Roche). The program conditions applied for qrt-PCR were previously tested
Characteristics of the null allelles and indel polymorphisms, employed in the LightCycler experiments GenBank Chromosome accession no.
GSTM1 GSTT1 SRY RhD Xq28 rs4399 DCP1 FVII R271 THYR
population at the 1–2.5% sensitivity level, depending on the informative locus and the band pattern of each patient.7,21
NT_019273 AB057594 X53772 AF187846 AF003626 AL008720 X62855 J02933 AC009286 AY053519
1p13 22q11.2 Yp11 1p36 Xq28 22q13.3 17q23 13q34 22q11.1 8q24
Polymorphism size (bp)
5925 12090 2151 57932 20 18 287 10 9100 1464
Deleted homozygote (frequency)
47% 17% 50% 10% 30% 16% 32% 63% 25% 22%
Refs.
24 25 17 26 MID1540a NCBIb 27 28 29 30
Observed frequencies Deleted homozygote (donor/host)
Mismatches (%)
Informativeness (%)
43%/48% 18%/19% 45%/45% 18%/13% 43%/33% 54%/62%c 33%/41% 69%/75% 13%/26% 16%/20%
37 21 50 17 30 38 18 30 26 20
15 10 25 12 20 15 5 12 7 8
a
ID code at Human Insertion/Deletion Polymorphisms Database, Marshfield Clinic, Winsconsin. ID code rs4399 at the NCBI-SNP Database. Discrepancy with respect to previously reported percentage may be explained by the non-Caucasian origin of the reference population.
b c
Leukemia
Real-time PCR and indels chimerism A Jime´nez-Velasco et al
338 and the optimized Light-Cycler run parameters for each polymorphism are added as ‘Supplementary Information’.
Sample quantification: In order to reduce the variation between different assays and samples, we used a procedure based on the relative quantification of target genes vs their control in relation to the reference gene.31 Calculations were automatically performed by LightCycler software (RelQuant, version 1.0, Roche). The normalized ratio was obtained from the following equation and expressed as a percentage of the control: Normalized ratio ¼ ðEtarget ÞDCp target ðcontrolsampleÞ ðEref ÞDCp ref ðcontrolsampleÞ Efficiencies (E) of each gene were calculated from the slopes of the crossing point (Cp) vs DNA concentration plots, according to the formula E ¼ 10(1/slope). DCp corresponds to the difference between control Cp and sample Cp, either for the target or for the reference genes. The selected control corresponded to the pre-SCT host sample of each patient; this was considered as 100% recipient chimerism and was repeatedly used for all determinations of the same patient.
linear correlations, Pearson coefficients were obtained and linear regression parameters were estimated. Differences in quantitative variables were analyzed by nonparametric tests: Mann–Whitney for independent data or Wilcoxon rank test for paired data. Categorical data were compared by the w2 test. In all cases, P-values below 0.05 were considered significant.
Results
Clinical outcome In our series, 24 patients (39. 3%) relapsed, most (58.3%) within the first 6 months post-SCT. The mean overall survival in this group was 17.0 months and, at the end of the recorded period, only three (14.3%) of the subjects who relapsed remained alive. The majority of deaths in the relapsed group were due to disease progression. In all, 12 (36.4%) of the nonrelapsed patients died, six within the first 100 days after SCT, mainly because of infectious complications. Overall survival in the nonrelapsed patients was significantly longer (55.6 months) than in the relapsed patients (P ¼ 0.026, log-rank test).
Polymorphism frequency and informativeness Other molecular analysis The p210BCR-ABL, p190BCR-ABL, TEL-AML1 and AML1-ETO fusion transcripts were analyzed by means of the reverse transcription nested polymerase chain reaction, according to the primers and protocols established by the European BIOMED 1 concerted action.32
Sensitivity, cell mixture and variability experiments Dilution experiments of host DNA in donor DNA were designed to obtain proportions of 1 to 105, always keeping a final DNA concentration for the assay of 100 ng. The mean Cp results of three experiments were plotted against DNA dilutions, and standard lines were constructed. Linear correlation coefficients were then calculated, and sensitivities and efficiencies estimated for each gene from their corresponding regression lines. In order to assess the accuracy of qrt-PCR measurements, known dilutions of host and donor cells were prepared in the range evaluated (1–10–4). DNA was extracted and host chimerism of two null alleles (SRY, RhD) and two indels (DCP1 and R271) was quantified in the mixtures with the LightCycler as described above. The chimerism results were plotted against the known concentrations. Intra-assay variability was analyzed introducing a sample in three different capillaries of the Light-Cycler within the same run; the means and the standard deviations (s.d.) of the Cps and the coefficients of variation (CV) after amplification were then obtained. The same process was repeated at different host DNA concentrations. For analysis of interassay variablity, a sample was studied in three independent runs, using different mastermix solutions. The experiment was also repeated in samples containing different concentrations of host DNA.
Statistical analysis Apart from standard dispersion measures, CVs were calculated by dividing the s.d. by the corresponding mean, in order to compare variability at different concentrations. To investigate Leukemia
The distribution of all the polymorphisms in the 61 donor– recipient pairs was studied. The percentages of recipients containing the inserted sequence or the null allele, pair mismatch and informativeness for each marker are shown in Table 1. Informativeness, described previously as the presence of a polymorphism in the recipient which was absent in the donor, reached 80.3% overall (49 of 61 patients). More than one marker could be found in 38.8% of informative pairs.
Sensitivity, accuracy and variability experiments A qrt-PCR run is depicted in Figure 1a. All polymorphisms showed a high linear correlation between concentration and Cp with negative slopes, as can be seen in Figure 1b. The results of the regression coefficients and slopes, and the highest sensitivity for each marker, as well as the estimation of efficiencies for each qrt-PCR, are shown in Table 2. Regarding sensitivity, although most polymorphism assays detected recipient DNA at the lowest host concentration used (105), linearity was frequently lost in these extreme conditions. We therefore established a common threshold value for all markers at the 104 concentration (0.01% host DNA in the total amount). Samples quantified under this level were considered as though host chimerism was not detectable (donor CC) and automatically assigned to the 104 value. Known dilutions of host and donor cells were quantified by qrt-PCR with four different markers, two null alleles and two indels; the results are shown in Figure 1c. Significant linear correlations were observed between the prepared dilutions and the measured chimerism, with slopes very close to 1. This high agreement between actual and qrt-PCR determined values was found in the whole range of concentrations used, and confirmed the accuracy of the procedure. Intra- and interassay variabilities were also examined for each polymorphism, as described previously. Ranges for the means, s.d.s and CVs corresponding to different concentrations are also included in Table 2. High reproducibility and accuracy of the different null allele and indel assays were observed for all markers with CVs always below 3%.
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339
a Fluorescence (640/530)
0.15
0.12
0.09 10-1
1
10-2
10-3
10-4
10-5
0.06
0.03
H2O
20
22
24
26
28
30
32
34
36
38
40
42
44
46
Cycle number
b
c
40
% Host chimerism
Crossing point
100
35
30
y = -3.10x + 23.9 r = 1.000
25
10
1
SRY DCP1 R271 RhD
0.1
0.01
20 10 -5
10 -4
10 -3
10 -2
10 -1
0.01
1
0.1
1
10
100
% Host cells concentration
GSTM1 host concentration
Figure 1 Evaluation of qrt-PCR sensitivity and accuracy. (a) Light-Cycler fluorescence-cycle plots obtained at different concentrations (1–105) of host DNA with the GSTM1 polymorphism. (b) Concentration-crossing point (Cp) plot and linear regression parameters corresponding to GSTM1 polymorphism (values represent the mean 7 standard error of the mean of three replicates from three different runs). The lowest concentration depicted (105, empty circle) was detected in all samples but was not used for linear estimations. Similar graphs were obtained for each polymorphism analyzed. (c) Chimerism of null alleles (SRY, RhD) and indels (DCP1, R271) was quantified in a range of known dilutions of host and donor cells (1–104). qrt-PCR chimerism values were plotted against actual cell dilution, and regression lines have been depicted. Correlation coefficient r ¼ 0.994 for SRY, 0.996 for RhD, 0.998 for DCP1, and 0.994 for R271. Slopes close to 1 (0.93 for SRY, 0.94 for RhD, 0.99 for DCP1 and 0.92 for R271). Black circle, SRY; black diamond, RhD; empty circle, DCP1; empty diamond, R271.
Table 2 Regression parameters for the concentration vs crossing point (Cp) plot, and results of sensitivity and variability intra- and interassay of the analyzed polymorphisms Sensitivitya (maximum)
Regression line
GSTM1 GSTT1 SRY Xq28 rs4399 RhD DCP1 FVII R271 THYR
Slope
Origin
R
3.10 2.91 2.42 3.07 2.93 3.30 3.05 3.30 3.01 3.27
23.9 24.8 25.8 23.2 23.1 24.7 24.4 24.5 21.1 24.3
1.000 0.995 0.999 1.000 0.997 0.999 0.996 0.998 0.999 0.999
105 104 105 105 104 104 104 104 104 104
Intraassay
Interassay
Mean range
s.d.b range
CV rangec
Mean range
s.d. range
CV range
23.9–36.2 24.4–35.0 25.9–39.6 23.1–36.3 25.5–35.9 24.6–36.5 24.1–34.2 24.6–36.1 21.0–32.3 24.9–32.9
0.03–0.42 0.13–0.42 0.21–0.78 0.12–0.57 0.14–0.51 0.07–0.41 0.07–0.36 0.03–0.78 0.06–0.58 0.02–0.18
0.11–1.30 0.51–1.49 0.61–2.61 0.54–1.77 0.48–1.44 0.26–1.30 0.27–1.18 0.11–2.22 0.28–1.78 0.05–0.54
23.1–36.7 24.3–33.0 26.5–42.1 25.2–35.2 25.0–35.4 26.3–37.4 24.8–34.7 25.1–37.0 25.2–34.3 24.5–32.8
0.09–1.01 0.14–0.28 0.17–0.85 0.22–0.88 0.02–0.44 0.08–0.46 0.41–0.69 0.36–0.73 0.04–0.69 0.10–0.40
0.37–3.12 0.49–1.13 0.58–2.23 0.76–2.52 0.07–1.28 0.21–1.53 1.46–2.38 1.29–2.09 0.14–2.02 0.35–1.21
a
Maximum sensitivity, the highest DNA recipient concentration that could be detected in all replicates. SD, standard deviation, expressed as the maximum and minimum value in the range of concentrations used (1–105). c CV, coefficient of variation (s.d./mean), expressed as the maximum and minimum percentage in the range of concentrations used (1–105). b
Chimerism determination by qrt-PCR in patients Nonrelapsed patients: The analysis of chimerism by qrtPCR in nonrelapsed patients was performed in 24 of 33 cases (72.7%), who showed informativeness in at least one poly-
morphism (Figure 2a). In BM, recipient DNA remained at levels above 1% during the first 6 months after SCT, then descended progressively but without disappearing after more than 4 years follow-up. Thus, no CC could be detected in BM samples (Figure 2a). In five long-term survivors in this Leukemia
Real-time PCR and indels chimerism A Jime´nez-Velasco et al
340
a
100 BM
% Host chimerism
PB
10
1
0.1
0.01 0
Patients
b
12
24
36
Months post-SCT 10 14 12 13 10 11 11 16 14 12 10 11
BM PB
48 6 6
100 BM
% Host chimerism
PB
10 p = 0.002
1
0.1
0.01 5
4
3
2
1
REL
Relapsed patients: Of the 24 patients who relapsed, 21 (87.5%) showed an informative marker. However, chimerism study was ruled out in three cases (UPNs 2096, 2148 and 2162) in whom no remission was detected after SCT. Another patient (UPN 2196) could not be followed in either BM or PB because only one sample had been collected before the occurrence of early relapse. Of the remaining 20 patients, 17 had at least one informative marker and were included for analysis. The chimerism pattern (Figure 2b) was clearly different from that observed in nonrelapsed patients, especially in PB, where an ascending, instead of a descending, trend was observed. Despite starting this increasing trend earlier, no differences were found until the sample nearest relapse, whose mean host percentage in PB was 2.670.8, a level significantly higher than that observed in the previous sample (0.770.3; P ¼ 0.002, Wilcoxon test). In the relapsed group, increasing chimerism in PB samples was found significantly more frequently than in the nonrelapsed patients (45.2 vs 18.5%) (P ¼ 0.004, w2 test). Moreover, at all intervals between SCT and this last measurement, chimerism percentages were similar in relapsed and nonrelapsed patients, indicating that the behavior of both groups did not differ until this moment before relapse. On the other hand, BM chimerism values were similar in all measurements prior to relapse (Figure 2b). When examined individually, the overall prediction of relapse was 88.2% (15 of 17) and the median time between the appearance of increasing MC and relapse was 58 days (range: 9–167). Comparison between the percentage of relapsed and nonrelapsed patients with increasing qrt-PCR chimerism (88.2 vs 16.7%) was statistically significant (Po0.001, w2 test). Table 3 details the characteristics of the qrt-PCR chimerism in all the relapsed patients analyzed. Prediction by qrt-PCR failed in two patients (UPNs 2133 and 2169), both sharing an inconsistent collection of samples: in the first case, no samples were obtained for more than 2 months before a late relapse, and in the second patient, with an early relapse, only one PB sample was collected during the first 3 months after SCT.
Determination before relapse Patients
BM PB
7
6 10
8 13
14 16
14 16
14 16
Figure 2 Determination of chimerism by qrt-PCR in nonrelapsed and relapsed patients. (a) Bone marrow and peripheral blood chimerism quantification found at different intervals (1, 2, 3, 4, 6, 9, 12, 18, 24, 36 and 48 months) after SCT in nonrelapsed patients. Filled squares, BM; empty squares, PB. (b) Increasing qrt-PCR MC in relapsed patients. Chimerism quantification of samples prior to relapse in BM and PB. Points represent the mean chimerism7standard error of the mean of all patients available at each determination. A significant increase in MC was observed in PB, but not in BM, in the sample prior to relapse (Wilcoxon paired test). Filled squares, BM; empty squares, PB. The arrow indicates the relapse sample (REL).
group, minimal residual disease could be followed simultaneously by rearrangements detected with standardized molecular methods (two patients with BCR-ABL, two with AML1-ETO and one with TEL-AML1). All five showed undetectable BM levels of the corresponding rearrangement with effect from the first sample after SCT, despite the high level of host chimerism sometimes observed. By contrast, PB chimerism, which always remained at levels below those of BM, decreased from values around 1.5% to undetectable levels (below 0.01% in most patients) after the first year, usually maintaining a stable CC later. Leukemia
Comparison between VNTR and qrt-PCR chimerism In our series, 19 of 20 relapsed patients (95%) with an informative locus could be evaluated by VNTR chimerism, although one other patient had to be excluded later because of failure in DNA amplification of one sample. Only eight of these 18 relapsed patients (44.4%) showed increasing MC in PB or BM, which enabled prediction of relapse (Table 3). This percentage was significantly lower than that reported above for qrt-PCR (88.2%) (P ¼ 0.006, w2 test). As expected, no MC could be detected by conventional PCR in samples with qrt-PCR values lower than 0.1% and just a few samples showed VNTR MC with 0.1–1% qrt-PCR host chimerism. The median time at which relapse was predicted by VNTR was 37.5 days (14–154), slightly shorter but not different in comparison to the same period obtained with qrt-PCR (58 days) (P ¼ 0.91, Mann– Whitney test).
Discussion Our results show for the first time that determination of null alleles and indels by LightCycler qrt-PCR is a very sensitive, reliable and accurate method to detect residual host cells postSCT in patients with acute leukemia. Most of the polymorphisms used attained a sensitivity of 104 (0.01%), with some detecting
Real-time PCR and indels chimerism A Jime´nez-Velasco et al
341 Table 3 UPN
Sex
Chimerism status previous to relapse in PB and BM, determined by qrt-PCR and VNTR, and survival data of relapsed patients Age Leukemia type
Stem cell Status pre-SCT Conditioning source
Qrt-PCR BM
2080 2081 2100 2107 2122 2123 2126 2133 2138 2147 2161 2165 2167 2169 2182 2184 2185 2186 2192 2198
F F F F F M F M M F F M F F M M M M F M
38 24 28 48 7 26 33 4 8 5 17 21 49 7 35 5 27 25 11 10
Myeloid Lymphoid Myeloid Myeloid Lymphoid Lymphoid Lymphoid Lymphoid Myeloid Myeloid Lymphoid Lymphoid Myeloid Myeloid Myeloid Lymphoid Lymphoid Lymphoid Lymphoid Lymphoid
PBb BMe PB BM PB PB PB BM UCBl BM PB BM PB PB PB PB BM PB PB PB
No remission No remission No remission No remission CRi CR CR CR CR CR CR CR No remission CR CR CR CR CR No remission CR
BUCYc IMCd BUCY+ VP16 NIf BUCY NEh BUCY NI BUCY IMC IMC TBIj TBI IMC BUCY No-iMCk BUCY+ VP16 IMC BUCY IMC BUCY NE BUCY No-iMC BUCY NI BUCY No-iMC BUCY IMC BUCY NE TBI No-iMC TBI IMC TBI IMC TBI IMC
VNTR
PB
BM
PB
IMC NI IMC NI IMC IMC IMC NE IMC IMC IMC IMC NI CC IMC IMC IMC IMC IMC IMC
IMC CCg NV CC CC IMC CC CC IMC IMC NE IMC No-iMC CC CC NE NI CC CC CC
IMC IMC IMC CC CC IMC CC NE IMC IMC IMC CC CC CC CC NE NI CC CC CC
Relapse OSa Status (months) (months) 14.7 2.6 2.0 15.4 32.9 8.4 34.3 1.4 4.7 2.9 3.3 6.7 4.7 24.4 12.7 2.8 16.3 5.3 3.5 8.4
35.7 4.3 3.2 17.1 36.1 30.4 55.3 5.8 20.2 3.9 5.6 7.1 6.4 29.8 14.2 15.2 19.6 16.6 4.7 9.0
Dead Dead Dead Dead Dead Dead Alive-CR Dead Dead Dead Dead Dead Dead Alive-NRm Dead Dead Alive-NR Dead Dead Dead
a
OS, overall survival. PB, peripheral blood. BUCY, busulphan+cyclophosphamide. d IMC, increasing mixed chimerism before relapse. e BM, bone marrow. f NI, no informative marker. g CC, donor complete chimerism before relapse. h NE, not evaluable (not enough samples or no PCR amplification). i CR, complete remission. j TBI, total body irradiation-based conditioning. k No-iMC, no increasing mixed chimerism. l UCB, umbilical cord blood. m NR, no remission. b c
up to 105 (0.001%). Even more importantly though, and as shown in cell mixture experiments, this procedure not only enables detection, but also reliable quantification at these levels. Additionally, intra- and interassay variability studies confirmed the reproducibility of qrt-PCR measurements. Two other qrt-PCR approaches have been described previously for chimerism determination: chromosome Y-derived genes and SNP-based methods. The former has proved to be reliable and extremely sensitive, both with conventionalPCR5,33 and with qrt-PCR technology,16 with the known limitation of the requirement for sex mismatch, thus preventing its use as a routine procedure alone. We have partly shared this approach, considering the SRY gene as a null allele present only in males. The SNP method, which is sex-independent, is based on a rather frequent polymorphism in the human genome (around 1/1000 bp),34 in which a single-nucleotide difference may distinguish donor and recipient alleles. With this approach, all donor/recipient pairs can be analyzed and up to 103 host DNA detection has been described.18 Alizadeh et al,16 using a similar method but with short-sized polymorphisms, reported a sensitivity of 104. The main concern in both cases derives from the similarity between both alleles, producing a certain amount of nonspecific amplification, which may reduce the accuracy of quantification. Our approach differs from SNP because of the larger size of the sequences inserted and their complete lack, instead of replacement, in the donor, with the consequent exclusion of nonspecific amplification. Determination of chimerism by conventional PCR does not reach qrt-PCR sensitiv-
ity,9,35,36 except in some cases in which cell subpopulations have been isolated by immunomagnetic techniques.6,37 With respect to informativeness, more than 80% of the patients in our series could be analyzed using an initial panel of 10 markers. However, the increasing number of indels identified,19 and the specific databases already available (http://research.marshfieldclinic.org/genetics/indels), will allow testing of new sequences to increase the percentage of informativeness and recruit the optimal group of polymorphisms for each population. The results obtained with determination of chimerism in patients support an improvement in the prediction of relapse by qrt-PCR in comparison with conventional PCR. Quantification by qrt-PCR has confirmed that chimerism variability, either individual or temporal, does not enable establishment of a common threshold for the risk of relapse in all patients. In fact, prediction of relapse has been found at chimerism levels ranging from 0.1 to 10%, depending on the post-transplantation time and the individual kinetics. For this reason, an increase in individual values at any chimerism level has to be considered the best criterion for relapse prediction, in agreement with previously reported results.4–8 Amplification by qrt-PCR not only anticipated a significantly higher percentage of relapses compared to VNTR, but, in addition, patients without increasing MC determined by qrt-PCR rarely relapsed (two of 22) in comparison with VNTR (10 of 34). Results approaching those of qrt-PCR (83% sensitivity) have only been reported when VNTR or STR chimerism was analyzed in lineage-specific cells,6,37 Leukemia
Real-time PCR and indels chimerism A Jime´nez-Velasco et al
342 supporting the idea that more sensitive techniques enable better relapse prediction. Persistence of recipient BM chimerism in our nonrelapsed patients has been observed more than 4 years post-SCT. A long persistence of MC of several hematopoietic lineages has been described, mainly associated with T-cell-depleted grafts,38 but rarely for such a long time after unmanipulated SCT.39,40 One possible explanation could be that chimerism levels under 1%, as found with qrt-PCR, are difficult to be noticed by lesssensitive methods. The persistence of high and more variable levels of chimerism in BM may contribute to poorer detection of small increments in chimerism, which would easily be found in PB. In our patients, their nonleukemic character was confirmed by study of the minimal residual disease, showing high levels of MC in BM associated with undetectable levels of sensitive molecular leukemic rearrangements (BCR-ABL, AML1-ETO, TEL-AML1). After the detection of rising numbers of host cells, relapses in our series usually developed within the next 2 months (median: 58 days, range: 9–167 days). Similar periods have been found by Mattson et al6 and by our own group;7 and these intervals may be enough to implement therapeutic measures that improve SCT outcome, as shown by Bader et al.3,8 Other authors report a shorter interval (0.5 months; range: 0.5–10.5),33 and yet others fail to detect MC, despite very close collection of PB samples (even weekly).10,41,42 Although low sensitivity could account for these failings, our study also supports that relapse in acute leukemia develops over a very short period in some patients. Systematic studies to clarify this point should be designed, but our results suggest that the sample collection schedule in acute leukemia has to be prioritized, irrespective of the sensitivity of the method used. In conclusion, we consider that qrt-PCR approaches are becoming the best option for the follow-up of chimerism after SCT, and that procedures based on null alleles and indels may constitute one of the most useful variants.
Acknowledgements We would like to thank the nursing and clinical staff, the medical residents of the Hematology Department, and the Transplantation Unit for taking care of our patients and helping us to collect the present data. We are grateful to Beli Torroba for her useful technical assistance and to Ian Johnstone for help with the English editing of the manuscript. This work was supported by grants 02/ 1299 and 03/0141 from the Fondo de Investigaciones Sanitarias (FIS) and grants 143/03 and 144/03 from Junta de Andalucı´a. GN holds fellowship Cajamar-Fundacio´n Hospital Carlos Haya and MB FIS 01/F018
Supplementary Information Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu).
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