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© 2002 American Society for Clinical Oncology Minimal Residual Disease Tests Provide an Independent Predictor of Clinical Outcome in Adult Acute Lymphoblastic LeukemiaByFrom the Department of Hematology, Royal Free and University College School of Medicine, London, United Kingdom. Address reprint requests to Letizia Foroni, MD, PhD, Department of Haematology, Royal Free and University College School of Medicine, Pond St, London NW3 2QG, United Kingdom; email: letizia{at}rfc.ucl.ac.uk
PURPOSE: Investigation of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) using molecular markers has proven superior to other standard criteria (age, sex, and WBC) in distinguishing patients at high, intermediate, and low risk of relapse. The aim of our study was to determine whether MRD investigation is valuable in predicting outcome in Philadelphia-negative adult patients with ALL. PATIENTS AND METHODS: MRD was assessed in 85 adult patients with B-lineage ALL by semiquantitative immunoglobulin H gene analysis on bone marrow samples collected during four time bands in the first 24 months of treatment. Fifty patients received chemotherapy only and 35 patients received allogeneic (n = 19) or autologous (n = 16) bone marrow transplantation (BMT) in first clinical remission. The relationship between MRD status and clinical outcome was investigated and compared with age, sex, immunophenotype, and presenting WBC count. RESULTS: Fisher’s exact test established a statistically significant concordance between MRD results and clinical outcome at all times. Disease-free survival (DFS) rates for MRD-positive and -negative patients and log-rank testing established that MRD positivity was associated with increased relapse rates at all times (P < .05) but was most significant at 3 to 5 months after induction and beyond. MRD status after allogeneic BMT rather than before was found to be an important predictor of outcome in 19 adult patients with ALL tested. In patients receiving autologous BMT (n = 16), the MRD status before BMT was more significant (P = .005). CONCLUSION: The association of MRD test results and DFS was independent of and greater than other standard predictors of outcome and is therefore important in determining treatment for individual patients.
PATIENTS WITH ACUTE lymphoblastic leukemia (ALL) can carry a burden of up to 1012 malignant lymphoblasts at presentation. Chemotherapy achieves clinical remission (CR) within 1 month in most children (> 95%) and adults (> 70%). Presently, the aim of all postinduction treatment regimens is to achieve and sustain continuous clinical remission (CCR). At 5 years, 70% of children and 35% of adults survive.1-6 Improvement in survival in adults is modest, especially in older patients,7 60% to 65% relapsing mainly in the first 5 years. Disease, undetected by light microscopy (up to 1010 leukemic cells), may expand at any time, leading to relapse. The value of detecting residual disease with greater sensitivity than light microscopy using molecular or immunologic techniques has been extensively evaluated in childhood ALL8-14 but less in adult ALL.2,14-17 Minimal residual disease (MRD) is defined as the lowest level of disease detectable in patients in CR by the methods available. In childhood ALL, MRD status is now established as an independent predictor of outcome, with the newly developed real-time quantitative polymerase chain reaction (PCR) techniques promising to revolutionize MRD investigation and patient management. Most studies in children have concluded that conversion to MRD negativity shortly after induction therapy and maintenance of MRD negativity are prerequisites for long-term disease-free survival and that MRD positivity often precedes clinical relapse. Furthermore, predictions of outcome on the basis of MRD analysis in children are more accurate than predictions on the basis of other prognostic indicators, such as age, sex, immunophenotype, presenting WBC count, and karyotype.10,13 Studies to date indicate that MRD results may be less reliable for predicting clinical outcome in adults than in children.14,16,17 With the exception of the t(9;22) translocation that occurs in 11% to 30% of adult patients with ALL,18-20 a relatively small proportion of patients carry known chromosomal translocations.19 The aim of the present study was to determine the value of molecular MRD monitoring in predicting outcome in adult B-lineage ALL using immunoglobulin antigen receptor genes as targets.
During the normal course of early B-cell development, one each of three immunoglobulin H gene (IGH) segment types, ie, variable (VH), diversity (DH), and joining (JH), is selected randomly to undergo a somatic VH-(DH)-JH recombination event.21,22 Because the resultant complementarity-determining region 3 (CDR3) is unique (in size and sequence) for each B cell and its clonal progeny, CDR3 is a valuable leukemia-specific target for tracking MRD. Molecular PCR identification of leukemic-specific CDR3 is based on the principle that a monoclonal population of CDR3-bearing leukemia cells will exhibit overamplification of one particular size of CDR3 at presentation (known as a fingerprint) relative to the much smaller or invisible background population of heterogeneously sized CDR3-bearing normal B cells.17,23-25 In contrast, PCR amplification of normal BM cells results in evenly distributed DNA bands or ladders. An MRD-positive fingerprint pattern during CCR indicates that the patient has a burden of one to five (or higher) leukemic cells in 103 normal cells. The leukemic CDR3 region can be additionally analyzed for DNA sequence, and an allele-specific oligonucleotide (ASO) can be generated for an additional MRD PCR test, with 10-fold higher sensitivity ( In the present study, we have prospectively tested 85 adult B-lineage Philadelphia-negative ALL patients for MRD at various time points in their disease by fingerprinting and ASO, expanding on our previous study.17 The relationship between MRD status and clinical outcome was investigated. This was compared with other prognostic indicators, such as age, sex, immunophenotype, and presenting WBC count, with the aim of establishing the best overall predictor of clinical outcome.
Patient Samples Bone marrow samples were requested from adult patients (15 to 55 years of age) with B-lineage acute lymphoblastic leukemia who had either been entered into the Medical Research Council United Kingdom Acute Lymphoblastic Leukemia (UKALL) XII trial or had been treated according to the trial protocol. The trial was designed to evaluate the efficacy of bone marrow transplantation (BMT) and, later, peripheral-blood stem-cell transplantation (PBSCT) in adult patients aged 15 to 55 years. All patients with matched related donors received allogeneic BMT. Patients with no donor were randomly selected to receive myeloablative chemotherapy (etoposide + total-body irradiation) with autologous BMT or PBSCT or intensive consolidation and maintenance chemotherapy.26 Patients were diagnosed at the Royal Free Hospital and other participating centres in the United Kingdom, and all participating patients had consented to the investigation of MRD as part of the trial. Patients with mixed immunophenotype, Philadelphia-positive patients, and patients who failed to achieve clinical remission were excluded from the study because of the poor response to treatment and to avoid skewing data toward a high incidence of poor performers. Only samples from patients with complete CR defined by less than 5% blasts in the bone marrow (BM) at time of MRD assessment were included. The study aimed at evaluating the overall ability of an MRD test to predict outcome; the hematologic status was the parameter against which all tests were measured, even in patients with short-term follow-up because of death. Bone marrow samples (fresh, frozen, or archived) from diagnostic presentation and paired follow-up samples were received from 110 adult patients with ALL. A clonal IGH gene marker was identified in 85 (77%) of the patients, and these were processed for MRD analysis. The group investigated for MRD analysis was comparable (in age, sex, and total WBC) to the group of patients for whom no material was received (data not shown).
Patient Data Patients were all treated with the UKALL XII protocol.26 Fifty (59%) of the 85 patients received chemotherapy only, 16 (19%) received autologous BMT (of which only one had a PBSCT), and 19 (22%) received an alloBMT after chemotherapy treatment in first CR. Routine cytogenetic analysis of diagnostic materials was carried out as part of ongoing data collection by the Leukemia Research Funded Cytogenetics group at the Royal Free Hospital (under Dr C.J. Harrison), and data have been kindly made available to us. Samples were processed using standard techniques and karyotypes described according to the International System for Human Cytogenetic Nomenclature.
DNA Extraction Procedures
Identification of Clonal VH-(DH)-JH Marker in Presentation DNA
Molecular MRD Monitoring of Follow-Up BM DNA
Statistical Analysis
Clonality of VH-(DH)-JH Rearrangements at Diagnosis One hundred ten samples of diagnostic BM DNA from adult patients with B-lineage ALL were tested for the presence of unique clonal VH-(DH)-JH rearrangements, of which 85 (77%) were found to have at least one discrete product. Fifty (59%) of these 85 had only one clone, 18 (21%) had two clones, and 17 (20%) had three or more clones. In 25 patients (23%), no VDJ IGH clonal marker was identified. For MRD analysis, all available IGH clones were followed in patients with more than one clone.
MRD Tests
Sixty-four patients tested negative by fingerprinting (ie, MRD Results of MRD were also analyzed according to the three major treatment groups, chemotherapy (50 patients), autologous BMT (16 patients), and allogeneic BMT (19 patients; Figs 1, 2, and 3, respectively), and this analysis is briefly discussed below.
Chemotherapy Patients Each of the 50 patients was monitored for 24 months or longer from time of referral to establish how long CCR had been maintained or until relapse event or death (in CR) occurred (Fig 1). Patients who relapsed showed a persistently higher incidence of MRD-positive tests than did patients who stayed in long-term CCR. This pattern was consistent throughout from early time points (0 to 2 and 3 to 5 months) and became more marked later (6 to 9 and 10 to 24 months). At 0 to 2 months, 35.7% (five of 14) of CCR patients tested were MRD positive, whereas 64% (nine of 14) of patients who relapsed were MRD positive. At 3 to 5 months, 10.5% (two of 19) of CCR patients were MRD positive, whereas 75% (12 of 16) of relapse patients were MRD positive. At 6 to 9 months, zero of nine patients in CCR tested positive, whereas 80% (eight of 10) of relapse patients were MRD positive. At 10 to 24 months, 6.6% (one of 15) of CCR patients were MRD positive, whereas 55.5% (five of nine) of relapse patients were MRD positive. A conversion from MRD-positive to MRD-negative test was observed in three long-term CCR patients (nos. 57, 392, and 40, Fig 1), with evidence of a gradual decline of leukemia burden (for instance, in patient no. 40). Patient no. 240 was MRD positive at later time points and yet has maintained CCR for 82 months. Among 27 patients who later relapsed, eight (29.6%) were MRD negative at the last time point taken before relapse. One of these patients had an extramedullary relapse with no evidence of BM involvement (patient no. 396), five had intervals of longer than 10 months between the last MRD test and relapse, and two tested negative at five (patient no. 224) and seven (patient no. 243) months before relapse. In one patient (no. 224), no ASO test was performed. In this group of patients, relapses occurred predominantly within the first 24 months, whereas five (18.5%) patients relapsed between 28 and 59 months from presentation (Fig 1).
Autologous BMT Patients
Allogeneic BMT Patients By contrast, there was statistically significant correlation between MRD test after BMT and clinical outcome. Patients in long-term CCR showed no MRD after BMT, and the two patients who were MRD positive either relapsed or died with detectable MRD. The association between MRD-positive tests after allogeneic BMT and relapse will require a larger cohort of patients to confirm the preliminary observation presented here.
Concordance of MRD Test Results and Clinical Outcome Among patients who received either chemotherapy or autologous BMT and remained in CCR (32 patients), there was a progressive decrease in the total number of MRD-positive tests during the 24 months from presentation (Table 1), as follows: 27.7% (five of 18) at 0 to 2 months; 12% (three of 25) at 3 to 5 months; 0% (zero of 13) at 6 to 9 months, and 6.2% (one of 16) at 10 to 24 months. Among relapse patients (34 patients), the total number of MRD-positive tests remained consistent over the course of 24 months, ie, 66.5% (12 of 18) at 0 to 2 months; 69.5% (16 of 23) at 3 to 5 months; 78.5% (11 of 14) at 6 to 9 months; and 50% (five of 10) at 10 to 24 months. The correlation between a positive test and relapse and a negative test and CCR was statistically significant at all time bands (P < .05 to P < .0001). The same analysis performed on allogeneic BMT patients confirmed that the relationship was only significant for MRD tests performed after BMT (P = .039) and not for tests carried out before BMT (P = .58).
DFS Curves The DFS curves of combined chemotherapy and autologous BMT patients are summarized in Fig 4. Censorship events were time beyond when data were no longer available or death in CR. Open or closed circles mark all events. The DFS curves were generated for MRD data taken at four time periods (0 to 2, 3 to 5, 6 to 9, and 10 to 24 months), and each graph compares DFS for MRD-positive patients with MRD-negative patients, with the log-rank test result displayed for each. The differences between the two groups are statistically significant at all time points and clearly increase with time, becoming more pronounced with later MRD tests, up to 9 months. For instance, at 0 to 2 months, MRD-negative DFS stabilized at approximately 65% and MRD-positive DFS stabilized at 22%, whereas at 3 to 5 months, MRD-negative DFS stabilized at 74% and MRD-positive DFS stabilized at 11%. The pattern continues at 6 to 9 months, when the MRD-negative DFS stabilized at 80% and the MRD-positive DFS continued to drop to 0%. From analysis of DFS in this group, we confirm that there is a significant difference in DFS between MRD-positive and MRD-negative patients, but only for tests carried out after BMT (P = .003) and not before BMT (P = .39), although patient numbers are small in both comparisons and larger studies will be required to additionally confirm these data, as previously mentioned.
Other Prognostic Factors The correlation of other independent prognostic variables with clinical outcome was examined in several ways and compared with correlation between MRD status and outcome. There were no overall differences between patients who remained in CCR and those who relapsed for the variables age at presentation and WBC count (log) (P = .77 to .99). The Cox regression multivariant model was used to determine the most significant independent prognostic variable. This involved comparing MRD status at each time period, with the continuous variables above, for effects on DFS rates (Table 2). The only covariable to have any significant independent effects on DFS was MRD status (at all four time periods); ie, Wald statistics were the highest within each comparison, eB deviated the most from numerical one, and P values were significant (.039 to .001). In contrast, age and WBC count did not correlate with DFS for each comparison, ie, Wald statistics were relatively low, exp(B) was mainly close to numerical one, and P values were not significant (.06 to .991). The model confirmed that the impact of MRD status on DFS became stronger with the time when the test was taken; ie, exp(B) values decreased additionally below numerical one with time (0.238, 0.199, 0.113, and 0.064 at 0 to 2, 3 to 5, 6 to 9, and 10 to 24 months, respectively). Other prognostic variables examined were sex and immunophenotype. Contingency table analyses of sex and immunophenotype versus clinical outcome revealed no statistically significant deviations from the normal (P = .22 and P = .75, respectively; Table 3). Detailed analyses with patient karyotype data were not carried out, because the proportion of patients with known abnormal karyotype markers were low, and subgrouping additionally reduced the numbers within each group to levels too low for statistical study (not shown).
Relative Risk of Relapse The relative risk of relapse (ratio between observed and expected number of relapses) was calculated for each prognostic subgroup, whereby deviation from numerical one gave a measure of predictive value. A relative risk of relapse of greater than one would indicate a potential predictor of relapse, and a relative risk of less than one would indicate a potential predictor of CCR. For simplicity, the variables age and WBC count (log) were stratified into broad categorical groups. It was observed that the DFS of subgroups was not significantly different when patients were split by age, WBC count (log), sex, or immunophenotype (P = .78 to .10; DFS curves not shown). In addition, the relative risks of relapse for these groups were all close to numerical one with one exception. Patients with an intermediate WBC count were less likely to relapse (.64) than those with high or low WBC levels (1.10 and 1.15, respectively), but differences were not statistically significant. The largest deviations from numerical one were found when patients were divided by MRD status at the time periods 0 to 2, 3 to 5, 6 to 9, and 10 to 24 months, with risks of relapse for MRD-positive patients at 1.36, 1.62, 1.92, and 1.6, respectively, and risks of relapse for MRD-negative patients at 0.61, 0.47, 0.36, and 0.48, respectively.
The results of our study have demonstrated that MRD monitoring in adult B-lineage ALL, using rearranged IGH gene targets, is a valuable approach to identifying patients with either a high or low risk of relapse. We studied 110 patients who received treatment according to the Medical Research Council UKALL XII trial protocol. The patients examined had similar demographics to the larger population of adult B-lineage ALL (who satisfied the same exclusion criteria and received the same treatment protocol) for the following factors: clinical outcome, disease-free survival, age at presentation, WBC count (log), sex, immunophenotype, and karyotype (data not shown). Therefore, we believe that our conclusions may reasonably be applied to all adult patients with B-lineage ALL receiving similar treatment.
Eighty-five (77%) of the 110 patients had at least one identifiable IGH gene marker, which was monitored for MRD. This observation is in broad agreement with others.15,16,30 One study demonstrated that detectable IGH clonality decreases with increasing age of patients with ALL.30 Patients with no IGH markers can potentially be monitored for MRD by alternative targets (eg, incomplete TCR- For assessment of MRD, all patients included in this study were tested using the fingerprinting method in a radiolabeled assay after induction therapy. All tests were carried out at time of clinical and morphologic complete remission. The value of using a combination of fingerprinting and ASO techniques is highlighted in our study. Twenty-one patients were fingerprinting positive (ie, level of disease between 1:102 and 103), and 18 of these patients relapsed, indicating that in patients who are considered in complete clinical remission, fingerprinting is capable of identifying a group of patients at high risk of relapse. This is comparable with the ability of other less-sensitive techniques, such as gene scanning,33 to rapidly identify patients with large leukemic burden. Both of these techniques are therefore useful for rapid screening of this group of patients. Even in the 32 patients negative by fingerprinting that we were unable to assess by the ASO technique (because of lack of material or background signal), 25 remained in CCR. In the remaining 32 patients, the ASO technique was applied, and in nine of them, a level of disease between 1:104 and 105 was detected and six later relapsed. An overall evaluation of the techniques used in this study would suggest that, albeit relatively less sensitive, techniques such as fingerprinting (and gene scanning)33 may be informative in identifying a group of patients with high level of MRD at high risk of relapse. Similarly, a level of MRD less than 1:103 (fingerprinting negative) identifies a low-risk group of patients. However, it is paramount that MRD studies include a technique with a level of detection of 1:104 or higher, such as the ASO technique to identify patients with low risk of relapse, as with the six patients identified in our study. The future application of real-time PCR may facilitate this assessment, as recently demonstrated in some pilot studies. However, MRD evaluation in ALL patients using antigen receptor genes will always have to be more labor intensive than any of the translocation-based studies because of the requirement for the identification of patient-specific markers involving direct sequencing and ASO generation. Irrespective of the use of the ASO technique, in 12 of our patients, relapse was preceded by an MRD-negative test at the last time point measured. At least five of these patients had been tested by the ASO technique. This could be interpreted as false negatives or associated with change of clonality, as previously described.34-36 Most of these results, however, could be accounted for in this study. Two patients (patient no. 134, Fig 1, and patient no. 95, Fig 2) suffered extramedullary (CNS) relapse with no BM involvement, and eight patients had long time gaps (> 5 months) between MRD test and relapse event, leaving only two potential false negatives (patient no. 389, tested by ASO 3 months previously, and patient no. 149, Fig 3). Three recommendations can be made from these observations. First, the method can only predict relapse involving the BM. This point is highlighted by patient no. 95 (Fig 2), in whom the BM harvest is MRD positive (associated with a high risk of future relapse) but then converts to MRD negativity 4 months before CNS relapse. The occurrence of isolated CNS relapses in allogeneic BMT patients has been described37 and linked to a less-profound graft versus leukemia effect in the CNS than in the BM environment. Optimal treatment for extramedullary relapse remains uncertain. Second and more importantly, patients should be regularly monitored, with intervals between MRD assessment not greater than 3 months. Finally, all protocols for MRD investigation should guarantee a level of sensitivity equal or greater than 1:104, and all subclones should be followed. The use of multiple markers (such as TCRD and TCRG in addition to IGH) should also be mandatory in clinical studies, where treatment may be changed according to MRD results. It is likely that the introduction of real-time PCR for these studies (as discussed above) will facilitate and provide qualitative and quantitative MRD measurements in the future. Our data have highlighted differences between patients receiving autologous and allogeneic BMT. Autologous BMT patients seem to show that clinical outcome is linked to the MRD status before BMT in much the same way that MRD status at similar time periods and outcome are linked in chemotherapy patients. In both groups, the deciding factor in overall outcome seems to be the MRD status (before autologous BMT or the equivalent times in chemotherapy patients). Consequently, we suggest that where autologous BMT is to be carried out, the harvested BM cells should be regularly tested for MRD status. It is likely that purging procedures could be applied to patients with residual disease before reinfusion. This was shown to have great impact on outcome in patients transplanted for chronic lymphoid malignancies38 and in patients with high-risk acute lymphoid malignancies,39 but not in all studies.40 Conversely, our study would suggest that pretransplant MRD status carries little prognostic significance in the group receiving allogeneic BMT, whereas posttransplant MRD assessment seems of greater value in predicting outcome, particularly in the CCR group, where a larger cohort of patients was analyzed in our study. It is well established that allogeneic BMT improves prognosis in adult ALL,26,41,42 and our data show that the procedure can successfully clear residual disease. These data differ from those reported for children,43 where outcome is closely linked with the pre-BMT status in patients undergoing allogeneic BMT. Additional studies will be required to investigate the difference between the two age groups. MRD investigation of adult ALL may provide clues about the kinetics of disease. MRD studies to date have revealed that children tend to clear disease at early time points from the start of treatment (eg, day 21).9-13 Our results16 and those of others17 suggest that adults do not respond to treatment as rapidly and often remain MRD positive up to 2 months from the start of treatment. In children, MRD status at 21 days from start of treatment is highly predictive of outcome,10,11,13 whereas in adults, later times are progressively more predictive. Our study, as the first of its kind in terms of size and time of follow-up, suggests that a continuous monitoring of adult patients during the first 6 to 9 months of treatment provides useful information on the dynamic of treatment response and helps to identify patients with higher risk of relapse or CCR. It also provides useful information in both the autologous and allogeneic BMT groups. Our data suggest that MRD-negative patients have a good chance of sustaining CCR, whatever their treatment, whereas MRD-positive patients do poorly unless given an allogeneic BMT. These findings may be important in designing future trials. Continuation of chemotherapy rather than BMT procedures (particularly allogeneic with the higher mortality rate) could be reserved for patients who are consistently MRD negative or have converted to PCR negativity early after induction. Autologous BMT seems to have little impact, at present, in patients with detectable disease in the BM harvest. In these patients, BM purging or delaying BM collection could be applied to reduce the incidence of relapse. In our experience, even a low level of residual disease (equal to 1 in 104) is associated with a poor outcome in autologous BMT. Allogeneic BMT (for patients with an available donor) could be reserved for patients with residual disease beyond 4 to 6 months. For patients with no available donors who are still MRD positive, alternative chemotherapy strategies may need to be applied, such as idarubicin, fludarabine, cytarabine, and filgrastim (Ida-FLAG), which is presently reserved for patients with resistant disease or Philadelphia-positive disease.44,45 Alternatively, matched unrelated donor transplantation could be considered, particularly in younger patients. In summary, our data provide the strongest evidence to date that the molecular MRD status of BM is a strong predictor of outcome in adults with B-lineage ALL. MRD status becomes progressively more predictive with the passage of time, particularly during the first year of treatment. There is good agreement between MRD status and clinical outcome in patients receiving chemotherapy or autologous BMT, and, therefore, these patients should be monitored repeatedly. Patients reinfused with MRD-positive harvested cells have a high chance of clinical failure, so all harvested material should be screened before transplant. For patients receiving allogeneic BMT, at present, test results are mainly predictive when carried out after BMT. Finally, MRD status in Philadelphia-negative ALL patient has been shown to be a better predictor of outcome than the other commonly described prognostic indicators, such as age, presenting WBC count, sex, and immunophenotype. Keeping these results in mind, restratification of patients based on MRD status could in the future modify management of adult B lineage ALL. Large randomized studies are underway in some European countries, which allow tailoring of treatment on the basis of MRD results. We propose that this should be made available on a large scale to all children and adults with ALL in randomized studies.
Supported by the Kay Kendall Leukemia Fund and Leukemia Research Fund. We thank Richard Morris, Julie Burrett, Georgina Harrison, Sue Richards, Christine Harrison, Magda Jabbar, the clinicians in the United Kingdom who supplied bone marrow samples, Professor I. Franklin, and Professor A. Burnett.
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Copyright © 2002 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
1 in 104). 
2 contingency tests, and parametric and nonparametric t tests) using statistics programs GraphPad, Prism, and SPSS (Software Inc, San Diego, CA). Disease-free survival (DFS) curves were generated using the Kaplan-Meier method
1:103). In 32 of these patients, no additional analysis was carried out because of lack of available allele-specific primer (ASO) or cross reactivity of the ASO with normal bone marrow cells. In this group, 25 patients remained in CCR and seven subsequently relapsed. The remaining 32 patients (also fingerprinting negative) were additionally tested by the more sensitive ASO method to assess for residual disease at levels of 1:104 to 105.
rearrangements or TCRG gene rearrangements), because ALL of B cells often exhibits cross-lineage rearrangements.
