Introduction

Minimal residual disease (MRD) is a predictor of outcome in patients with acute lymphoblastic leukemia (ALL).^{1, 2, 3, 4, 5, 6} Tumor load reduction during and after induction treatment provides crucial information about the response to treatment and risk of relapse. This allows the identification of low-risk and high-risk patients, who may profit from therapy reduction or therapy intensification, respectively.^{3, 7, 8} Currently, three different techniques are broadly applicable for MRD detection in ALL.^{9, 10, 11, 12} Real-time quantitative polymerase chain reaction (RQ-PCR) analyses of junctional regions of rearranged immunoglobulin (IG) and T-cell receptor (TCR) genes can be applied in approximately 95% of patients with ALL. Using IG/TCR gene rearrangements as MRD-PCR targets, sensitivities of 10^-4 can generally be obtained, depending on the type of IG/TCR rearrangement and on the characteristics of the junctional region.^{10, 13} RQ-PCR analyses of fusion gene transcripts resulting from chromosomal translocations such as t(4;11), t(9;22), and t(12;21) have also been used as MRD-PCR targets. This method can only be applied in approximately 30% of ALL patients, but has the advantages of high target stability and sensitivity at 10^-4–10^-6.^{14, 15, 16} The third method is multiparameter flow cytometric analysis. This approach is based on the presence of aberrant immunophenotypic features that allow discrimination of ALL blasts from normal B and T cells. Flow cytometry can be applied to approximately 90% of ALL patients and can reach sensitivities of 10^-3–10^-4.^{6, 11, 17, 18}

A few studies compared molecular methods and flow cytometry for MRD detection in ALL patients.^{12, 19, 20, 21, 22} Previously, these comparisons were hampered by the lack of sufficiently quantitative molecular methods.

In the present study, we analyzed MRD levels in 71 follow-up bone marrow (BM) samples from 22 children with ALL. We used flow cytometry and RQ-PCR using TCRD, TCRG, Ig heavy chain (IGH), and Ig kappa light chain (IGK) gene rearrangements as molecular markers.

Design and methods

Patients

A total of 93 BM samples (22 obtained at diagnosis and 71 during follow-up) from 22 children with ALL were studied (Table 1). In all, 11 children were treated according to the Nordic Society of Pediatric Hematology and Oncology – 1992 (NOPHO-92) protocol;²³ their archival material was selected to ensure that MRD-positive samples (as assessed by earlier performed flow cytometry and ASO-PCR) were included. We also prospectively studied 11 of 20 consecutive patients treated according to the NOPHO-2000 protocol. Inclusion was based on the availability of sufficient cell material. Patients had at least one IG/TCR gene rearrangement that could be used as MRD-PCR target with a sensitivity of at least 10^-4. The morphological diagnosis of ALL was established according to WHO classification²⁴ and EGIL criteria:²⁵ two patients presented with T-ALL and 20 with B-precursor ALL (Table 1). One patient (no. 9) was followed after relapse of the disease. This study was approved by the Ethics Committee at Karolinska Hospital, Stockholm, Sweden.

Table 1 - Characteristics of the ALL patients included in this study.

Full table

Sample processing

BM cell suspensions were collected in heparinized tubes, immediately diluted at a ratio of 1:1 (v/v) in sterile physiological saline, and maintained at room temperature (RT) until processed. Flow cytometry was performed within 24 h using whole, nonseparated BM. For molecular analysis, mononuclear cells from BM were separated by Ficoll–Isopaque (Pharmacia Amersham, Uppsala, Sweden) gradient centrifugation, washed twice, re-suspended in PBS supplemented with 50% human AB serum and 10% DMSO, and frozen in liquid nitrogen. Three samples with low numbers of cells (<1.5 10⁶, from pt. 1 at 1 month and pt. 16 at 1 and 12 months) were separated from plasma by centrifugation. Pellets were frozen and stored at -80°C until use.

Identification of MRD-PCR targets

IGH gene rearrangements were identified with VH family-specific framework region-1 (FR1) primers in combination with a consensus joining primer JH.²⁶ To identify IGK-Kde rearrangements, a reverse Kde primer was used in combination with one of four family-specific primers, or with an RSS primer located in the intron between the J-C gene segments.²⁷ To identify TCRG and TCRD, consensus primers were used as described.²⁸

PCR amplification, heteroduplex analysis, and sequencing were performed as described in Malec et al.²¹ The junctional region of each IGH, IGK-Kde, TCRG or TCRD gene rearrangement was sequenced twice, starting from two independent PCR reactions and in opposing sequence orientations. Sequences were identified using DNAPLOT software (V Muller and H-H Althaus, University of Cologne, Germany) by searching for homology with all known human germline sequences obtained from both the VBASE directory of human Ig genes and the BLAST databases.

Detection of MRD by RQ-PCR analysis of IG/TCR gene rearrangements

All TaqMan probes and consensus primers used have been described previously.^{13, 27, 29, 30} ASO primers were designed with Primer Express 1.0 software (Applied Biosystems) and Oligo 6.1 software (W Rychlik, Molecular Biology Insights, Cascade, CO, USA) so that at least part of each ASO primer was complementary to the junctional region and could be used in combination with the germline TaqMan probes and germline reverse primer. At least one pair of specific primers was designed for each patient and tested for sensitivity. The ABI 7700 Prism Sequence Detection System (Applied Biosystems) with a 96-well thermal cycler was used at a standard annealing temperature of 60°C. To determine the efficiency of amplification and sensitivity of the target, diagnostic DNA was serially diluted in 10-fold steps into control DNA from mononuclear cells (MNC) of a pool of 10 healthy donors, from 10^-1 to 10^-6. The serial dilutions of diagnostic DNA were subjected to RQ-PCR together with negative controls (H₂O in duplicate and control DNA in six-fold). Serial dilutions of diagnostic samples were analyzed in duplicate, and follow-up samples were analyzed in triplicate. To correct for the quantity and quality of DNA, we used RQ-PCR analysis of the albumin gene.²⁹ The quantitative range of probe and primer combinations was defined as the dilution step with a maximal difference in cycle threshold value of 1.5 between the duplicate dilution samples and with a maximal Ct value of 40 cycles. In this range, the standard curve should have a correlation coefficient of at least 0.98 for precise quantification. Furthermore, the Ct values with reproducible sensitivity had to be at least three cycles lower than those obtained from any nonspecific amplification of normal MNC DNA. The sensitivity was defined as the lowest dilution of the standard curve showing specific amplification in at least one well and with a Ct value at least one lower than the nonspecific amplification.¹⁰ In all but one patient (no. 14), at least one MRD-PCR target had a sensitivity of 10^-4.

Flow cytometry

The flow cytometric method used has been described in Bjorklund et al.⁶ A stain and then lyse/wash technique was used. At diagnosis, leukemic blasts were immunophenotyped with triple or four-color immunofluorescence using fluorochrome-conjugated antibodies. Based on the results obtained at diagnosis, the individual follow-up protocols were tailored and used to determine the levels of remaining leukemic cells in remission samples. All antibody combinations used for follow-up were chosen based on aberrant marker expression, by comparison to the normal bone marrow patterns determined during BIOMED-1 Concerted Action 'Investigation of Minimal Residual Disease in Acute Leukemia: International Standardization and Clinical Evaluation'.^{31, 32} Cells (10⁵–1.5 10⁶) were analyzed using a 'live gate' acquisition method, giving sensitivity for MRD detection of at least 10^-4.

Statistical analysis

We used Pearson correlation test and Cohen's kappa test in the SPSS statistical package v.9.0 to estimate the relation between results obtained with the two techniques.

Results

Identification of MRD-PCR targets

At diagnosis, 48 IG/TCR gene rearrangements were found in the 22 ALL patients studied: 19 IGH, 12 IGK-Kde, 11 TCRG, and six TCRD gene rearrangements. In all, 17 patients had two or more targets and five patients had one target. Due to the low sensitivity (quantitative range 10^-3) afforded by four targets (in pts. 7, 12, 15, and 20), limited junctional region in two targets (in pts. 9 and 19) and insufficient material of follow-up samples in five patients (no. 1, 3, 13, 16, and 17), 16 (73%) patients were monitored with one RQ-PCR target, and six patients (27%) were monitored with two targets (Table 1). When MRD was studied by two MRD-PCR targets, the highest MRD level was used for statistical analysis.

Comparison of the results obtained by flow cytometry and RQ-PCR

We compared MRD levels determined by flow cytometry or RQ-PCR analysis in 71 follow-up BM samples; 34 out of 71 (47%) samples were taken during the first 3 months of treatment. The results are presented in Figure 1 and Table 2. Flow cytometry detected cells with a leukemia-related aberrant phenotype in 24 (34%) follow-up samples; MRD levels were 0.006–30%. RQ-PCR analysis allowed the detection of leukemic targets in 38 (54%) follow-up samples. In 13 of the 38 samples the target level was outside the quantitative range (ie, the exact MRD level could not be quantified); in the remaining 25 samples, target levels were 0.0024–68%. In 19 follow-up samples from six patients, MRD was analyzed with two MRD-PCR targets. Concordant results (qualitative MRD data) between both MRD-PCR targets were obtained in 16 out of 19 (84%) samples; in the three discordant samples with a quantitative range of 10^-4 and 10^-5 MRD levels were 0.001–0.02%. Furthermore, in three of six MRD-positive samples, MRD levels differed more than 10-fold between the two MRD-PCR targets.

Figure 1.

Comparison of quantitative MRD results, as obtained by RQ-PCR analysis and flow cytometry. Group A represents 30 samples that were negative by both methods. Group B (n=19) represents samples for which MRD levels were quantifiable by both methods; the Pearson correlation coefficient for this group was r=0.793 (P<0.01). The gray dots represent those samples in which the MRD levels as determined by each method differed more than five-fold. Groups C and F represent samples for which MRD was only detectable by flow cytometry, or RQ-PCR, respectively. Group D represents samples that were positive by flow cytometry, but in which MRD was detected at only low levels by RQ-PCR. Group E represents 11 samples in which MRD was detected below the quantifiable level of the RQ-PCR analysis and which were negative by flow cytometry.

Full figure and legend (53K)

Table 2 - Concordance between flow cytometry (F) and RQ-PCR (M) MRD detection results depending on applied cutoff level and time point in treatment.

Full table

Concordance between the flow cytometric and RQ-PCR results depended on the applied cutoff level (Table 2). At a cutoff level of 0.1%, 94% of samples gave concordant results and at a cutoff level of 0.01%, 89% of samples gave concordant results. However, there was only 72% concordance when the 0.001% cutoff level was applied: no MRD was detected using both methods in 30 samples (42%), whereas in 21 samples (30%) both methods could detect MRD. There was no difference in concordance between samples analyzed with three-color (n=38) and four-color (n=33) flow cytometry, or between samples taken early (first 3 months of therapy) and later during treatment (Table 2). There was a significant positive correlation between the two techniques (Cohen's kappa test =0.449, P<0.001) when results were grouped into positive and negative categories at the lowest cutoff level possible. In the 19 samples in which MRD levels could be quantified by flow cytometry and RQ-PCR, there was a significant positive correlation between results obtained by the two techniques (P<0.01, Pearson correlation r=0.793) (Figure 1).

Samples with discordant results

In 20 samples (28%), flow cytometry and RQ-PCR showed discordant results: in 17 samples (from 11 patients), MRD was detected by RQ-PCR but not by flow cytometry, whereas in three samples (from two patients) MRD was detected by flow cytometry but not by RQ-PCR. In most samples with discordant results (11 samples, 10 patients), MRD could not be detected by flow cytometry, but very low levels (under the limit of the quantitative range) could be detected by RQ-PCR. In all 10 patients, sensitivities of at least 0.01% were reached by RQ-PCR analysis. Therefore, MRD levels were probably below 0.01% and thus below the detection limit of the applied flow cytometry method.

Six samples (pt. 2 at 1 month, pt. 5 at 1 month, pt. 7 at 6 months, pt. 16 at 1 month, and pt. 17 at 2 and 4 months) were clearly positive by RQ-PCR with MRD levels within the quantitative range, but negative by flow cytometry. In four of those patients (pts. 2, 5, 7, and 16), the MRD-PCR targets allowed a sensitivity of at least 0.01%, and detected MRD levels ranged from 0.0035 to 0.085%. Probably, these relatively low levels were also below the detection limit of flow cytometry. In the two follow-up samples of pt. 17, relatively high MRD levels were detected by RQ-PCR analysis (0.17 and 0.73%), whereas flow cytometry could no longer detect cells with the aberrant CD2+CD56+CD19+CD34+ immunophenotype observed at diagnosis, suggesting an immunophenotypic shift of the leukemic cells.

Three samples (pt. 4 at 16 months and 24 months; pt. 14 at 2 months) were MRD positive by flow cytometry (0.008, 0.01, and 0.05%, respectively), but MRD negative by RQ-PCR. In two additional samples (pt. 4 at 6.5 months and pt. 11 at 6 months) MRD was detected by flow cytometry and RQ-PCR, but MRD levels could not be quantified by the latter technique. Pt. 4 was monitored using two MRD-PCR targets (V2-D3 and VH4-JH4b) with a quantitative range of 10^-3, and pt. 14 using a single VII-Kde MRD-PCR target with the same quantitative range. Therefore, the negative MRD results obtained by RQ-PCR may be due to the limited quantitative range of the applied MRD-PCR targets. Pt. 11 had two potential MRD-PCR targets (VH2.26-JH4b and V2-J1.3). The IGH target appeared to represent a subclone, but due to limited material, the patient's 6-month follow-up sample could only be analyzed using this MRD-PCR target. Consequently, the MRD level as determined using RQ-PCR analysis was underestimated.

Four of the 19 samples in which MRD could be quantified by both RQ-PCR and flow cytometry had MRD estimates by either technique that differed more than five-fold. In two samples (pt. 11 at 24 months and pt. 9 at 3 months) MRD levels by flow cytometry were considerably higher than MRD levels by RQ-PCR analysis (0.04 vs 0.0024% and 0.8 vs 0.02%). As indicated above, the difference in the sample from pt. 11 could have been due to the use of the IGH target alone. In the sample from pt. 9, the IGH target was stable, as shown by high MRD levels in a subsequent sample; the reason for the discrepancy in MRD levels remains unclear. In two instances (pt. 1 at 1 month and pt. 22 at 4 months) higher MRD levels were detected by RQ-PCR.

Discussion

RQ-PCR with patient-specific junctional regions of rearranged IG/TCR genes as targets, and flow cytometry based on the detection of aberrant leukemia-related phenotypes, can yield clinically relevant MRD information. Prospective MRD studies identified a significant proportion of ALL patients with good clinical outcome (relapse rate: 2–15%), who had undetectable MRD during the early phase of treatment using techniques with a sensitivity of at least 10^-4.^{2, 3, 4, 5, 33, 34} MRD levels 10^-3 during any phase of remission were related to higher risk of relapse.

We compared MRD results obtained by flow cytometry and RQ-PCR in 71 follow-up samples from 22 children with ALL. MRD results were comparable but not identical. The discordance may be due to differences between the methods. Flow cytometry quantifies viable cells expressing an aberrant immunophenotype within the background of normal (regenerating) bone marrow cells. Its sensitivity (typically 10^-4) depends on the numbers of cells analyzed and the type of aberrant phenotype.^{34, 35} RQ-PCR quantifies rearranged IG/TCR genes, and generally reaches a sensitivity of 10^-5 and a quantitative range of 10^-4.^{10, 26, 27} Therefore, in practice, the sensitivity of RQ-PCR is generally higher than that of flow cytometry.

In line with that, the concordance of the MRD results in our study depended on the applied cutoff level (Table 2). At the cutoff level of 0.01%, used in several (particularly flow cytometry-based) MRD studies,^{4, 12} 87% of samples gave concordant results. If a cutoff level of 0.001% was applied (suggested as relevant by some molecular method-based studies^{9, 10}), the concordance dropped to 72%. In most cases, discordant results were due to negative flow cytometric MRD results. The sensitivity level of flow cytometry depends on the number of analyzed cells: for a sensitivity of 0.01% at least 200.000–500.000 cells should be analyzed. Comparable to our previous study,⁶ this was possible in over 80% of samples, implying that in over 80% of samples a sensitivity level of 10^-4 was achieved. The MRD levels detected by RQ-PCR in most (12/17=70%) discordant samples were below or just at the level of the assumed detection limit of flow cytometry. Only in one patient (no. 17) lack of sensitivity of flow cytometry seemed due to immunophenotypic changes, which have been reported in 6% of ALL patients.³⁶ Application of multiple antibody combinations and application of six-color flow cytometry may reduce the risk of false-negative flow cytometry results and may further improve its sensitivity.

Negative results by RQ-PCR but detectable MRD by flow cytometry were obtained in only three samples that were analyzed by a single MRD-PCR target only. One of the causes for false-negative RQ-PCR results may be instability of IG/TCR gene rearrangements due to secondary and/or ongoing IG/TCR gene rearrangements resulting in loss of the leukemia-specific MRD-PCR targets. A different clonal pattern between diagnosis and relapse has been reported in 30% of targets.^{37, 38, 39, 40} However, in most patients at least one IG/TCR gene rearrangement is preserved throughout the disease course.^{37, 38, 39, 40, 41} To reduce the risk of false-negative results, preferably two IG/TCR gene rearrangements should be used as MRD-PCR target.⁸ This should be facilitated by supplementing the classical IGH, IGK-Kde, TCRD, and TCRG targets with 'new' MRD-PCR targets, such as V2-J and TCRB gene rearrangements.^{42, 43, 44} A further cause for lower MRD levels detected by RQ-PCR may be caused by monitoring of a subclone, as potentially happened in pt. 9.

In 14 (79%) samples that showed quantifiable levels of MRD, similar levels were found by both techniques. In the remaining four (21%) samples, the levels differed more than five-fold with the RQ-PCR levels being higher. One of these four samples was taken during the early phase of treatment. The differences in MRD levels detected by flow cytometry and RQ-PCR observed in these samples can in part be attributed to the inability of PCR-based methods to distinguish between viable and nonviable leukemic cells, whereas flow cytometry routinely excludes dead cells from analysis as a result of light-scatter gating.^{19, 45, 46} Some differences could also be due to the fact that the flow cytometry method used in this study was performed on whole BM, whereas density gradient-isolated mononuclear cells were used to obtain DNA for subsequent RQ-PCR analysis, which can result in an enrichment of leukemic cells. In a recently published study, Neale et al used mononuclear cells for both four-color flow cytometry and quantitative PCR (analysis of IGH only) in order to compare both methods in assessing MRD in a large series of precursor-B-ALL patients. An excellent qualitative concordance (96% concordance) was found.⁴⁷ However, even in that study a considerable fraction of samples (approximately 10%) showed a difference in quantitative MRD levels between flow cytometry and RQ-PCR of more than five-fold. We performed flow cytometry on whole, unseparated bone marrow samples (obtained from both precursor-B-ALL and T-ALL patients), since this procedure provides the possibility of reliable enumeration of cell populations present in the sample, and minimizes the chances of modifications of antigen expression.⁴⁸ On the other hand, the use of mononuclear cells rather than whole BM for flow cytometric immunophenotyping may contribute to increased sensitivities, due to loss of nonlysed red cells, debris, and dead cells.

We conclude that both flow cytometry and RQ-PCR can be used for detection of MRD in childhood ALL, as has also been indicated by Neale et al.⁴⁷ However, both methods can yield false-negative results, due to immunophenotypic shifts during treatment or due to loss of MRD-PCR targets because of clonal evolution.^{49, 50} Also, variance in MRD quantification can lead to difference in risk stratification by either method. Tandem use of both methods according to a common risk algorithm seems therefore not warranted. The flow cytometric method has the advantage of being fast and being able to distinguish between viable and nonviable leukemic cells, but is somewhat less sensitive than the RQ-PCR method. Improvements in both methods for MRD detection can be achieved either by increasing the number of marker combinations and fluorochromes used in flow cytometric analysis, or by using at least two targets per patient in the case of RQ-PCR, which will reduce the chance of false-negative results. Therefore, additional efforts are needed for further optimization and standardization in order to obtain reliable MRD measurements in multi-center clinical studies and evaluate the prognostic value of both methods in their ability to segregate risk categories. To that end, we need to await the outcome observations from the recently started studies where both methods are used in parallel.

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Acknowledgements

This study was supported by grants from The Swedish Children's Cancer Foundation and Stockhols County Council. The excellent technical assistance of Marianne Lestrin, Britt Lundh, Shalah Tarahumi, Margareta Söderqvist and Margareta Waern is gratefully acknowledged. We thank Professor Dr Jacques JM van Dongen for his continuous support and for critically reviewing the manuscript.