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Keywords:

  • acute lymphoblastic leukaemia;
  • allogeneic transplantation;
  • MRD

Abstract

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Summary. In this study, we used multiparameter flow cytometry to quantify minimal residual disease (MRD) in 165 serial bone marrow samples from 40 patients diagnosed with acute lymphoblastic leukaemia (ALL) who underwent allogeneic stem cell transplantation (allo-SCT) from siblings (n = 34) or unrelated donors (n = 6). Samples were prospectively taken from 24 patients before starting the conditioning regimen, at days +30, +60 and +90 and subsequently every 2–3 months. Samples from 16 patients in complete remission (CR) after allo-SCT were taken at least twice. Six of 24 patients harboured MRD (0·2–10% of mononuclear cells) at transplant and 18 were negative. Estimated disease-free survival for the MRD+ and MRD– groups at transplant was 33·3% and 73·5% respectively (P = 0·03). During follow-up, increasing MRD levels were detected in nine patients, a finding that preceded marrow relapse by 1–6 months. Two patients with stable low MRD levels remained in CR. When we used flow cytometry to test the effect of donor leucocyte infusions (DLI) in six patients, we observed that the only sustained remission was achieved when DLI was applied prior to overt relapse. We conclude that MRD by flow cytometry can rapidly assess tumoral burden before transplant to predict outcome, and can be clinically useful for the timing of DLI for increasing levels of leukaemia after transplant.

Chemotherapy cures a substantial proportion of patients with acute lymphoblastic leukaemia (ALL) (Copelan & McGuire, 1995; Pui & Evans, 1998). However, patients with early bone marrow relapses (Torres et al, 1999) and those with poor risk features (i.e. BCR–ABL, MLL–AF4 rearrangements) (Marks et al, 1998) require allogeneic haematopoietic stem cell transplantation (allo-SCT). The main cause of treatment failure is leukaemia recurrence, which occurs in up to 60% of patients depending on the phase of disease, age at the time of transplant and on the incidence of graft-versus-host disease (GVHD) (Barrett et al, 1989). Once patients have relapsed, their prognosis is dismal (Kumar, 1994). Additional chemotherapy generally results in few long-term disease-free survivors and only a small proportion of patients benefits from second transplants (Michallet et al, 2000). Therefore, immune modulators (e.g. interleukin 2 or α2b-interferon) (Mehta et al, 1997) or donor leucocyte infusion (DLI) are often used as salvage treatment strategy (Collins et al, 1997; Porter et al, 2000). The strikingly poor outcomes of DLI for relapsed ALL (Collins et al, 2000), especially when compared with patients with chronic myeloid leukaemia, might be partly due to the fact that, in the latter group of patients, DLI is administered while the tumoral burden is still low (Serrano et al, 2000). Indeed, some reports support the importance of the graft-versus-leukaemia (GVL) effect in mediating a clinically useful antileukaemic responses in ALL when it is applied early (Keil et al, 1997; Yazaki et al, 1997).

To date, attempts to monitor small persistent amounts of ALL cells after allo-SCT have relied on non-quantitative polymerase chain reaction (PCR) amplification of BCR–ABL fusion transcripts (Miyamura et al, 1992; Radich et al, 1997) or rearranged immune receptors (Knechtli et al, 1998a,b). In either case, PCR-positive patients after transplant are at a higher risk of relapse than negative cases, but quantitative techniques are increasingly needed to make clinical decisions in individual patients (Mitterbauer et al, 1999). Detection of mixed chimaerism is also used, but its predictive value is less clear (Serrano et al, 1999; Choi et al, 2000).

Flow cytometric detection of minimal residual disease (MRD) provides reliable quantification of leukaemia cells, and strongly correlates with treatment outcome in patients with ALL treated with standard chemotherapy regimens (Ciudad et al, 1998; Coustan-Smith et al, 1998, 2000). However, its application to test the rate of leukaemia elimination achieved by myeloablative regimen, the impact of residual tumoral cells on clinical outcome and the effectiveness of DLI has not been reported to date.

Patients, materials and methods

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients.  Forty patients were eligible for this study on the basis of both the presence of leukaemia-associated immunophenotypes at diagnosis or last relapse, and the availability of serial bone marrow specimens. The serial collection and analysis of marrow samples began in January 1999. Thus, from 37 patients transplanted between January 1994 and December 1998, we tested only post-transplant follow-up samples in 16 long-term survivors (43·2%). From 33 patients transplanted between January 1999 and January 2001 at two centres, we were able to obtain marrow samples throughout scheduled intervals from 24 patients (72·7%); clinical characteristics are listed in Table I. Median age at transplant was 18 years (range 3–49 years), median white blood cell (WBC) count at diagnosis was 12·1 × 109/l (1–215 × 109/l). BCR–ABL fusion transcript or specific translocation t(9;22) was observed in seven cases. Seventeen patients underwent allo-SCT in first complete remission (CR), 17 were in second CR and six cases had two or more marrow relapses.

Table I.  Clinical characteristics of patients included in this study.
ParametersN
  • MRD, minimal residual disease; SCT, stem cell transplantation; WBC, white blood cell; CR, complete remission; TBI, total body irradiation; CY, cyclophosphamide; TT, thiotepa; FLU, fludarabine; CsA, cyclosporin; MTX, methotrexate; GVHD, graft-versus-host disease.

  • *

    T-cell depletion was used in three cases.

  • Extramedullar relapses [testes and central nervous system (CNS)] were seen in two patients.

Number of total patients40
Median age (years) at SCT (range)18 (3–49)
Lineage: B/T30/10
Sex (male/female)28/12
Median WBC count (× 109/l) at diagnosis (range)12·1 (1–215)
Cytogenetics
 Normal15
 t(9; 22)/Bcr–Abl  7
 Other abnormal  9
 Failed  9
Status at SCT
 1CR17 (42·5%)
 2CR17 (42·5%)
 > 2CR  6 (15%)
Donor type
 HLA identical sibling31 (77·5%)
 Matched Unrelated  6 (15%)
 Haploidentical related  3 (7·5%)
Graft source
 Bone marrow30 (75%)
 Peripheral blood  8 (20%)
 Umbilical cord  2 (5%)
Preparative regimen
 TBI + CY31 (77·5%)
 BU + CY  6 (15%)
 TBI + FLU + TT  3 (7·5%)
GVHD prophylaxis
 CsA  7 (17·5%)
 CsA + MTX30* (75%)
 CD34+  3 (7·5%)
Acute GVHD, grade II–IV17 (42·5%)
Chronic GVHD (extensive/limited)  4/4
Bone marrow relapse post-SCT  9
Median follow-up (Range), months29·5 (3–91)

Allogeneic stem cell transplantation.  Allogeneic procedures were performed in two centres: University Hospital ‘Reina Sofía’, Cordoba, and Hospital ‘Niño Jesus’, Madrid, Spain. Procedure details are given in Table I. At the time of transplant, all cases harboured less than 5% lymphoblasts by light microscopy. Conditioning regimens used included cyclophosphamide (120 mg/kg) plus either total body irradiation (TBI; 13 Gy) or busulphan (16 mg/kg). Donors were human leucocyte antigen (HLA)-identical siblings in 31 cases, HLA identical unrelated in six cases and HLA haploidentical sibling donor for three patients in whom ALL identical unrelated donors were not found. In these latter cases, the conditioning regimen included TBI, fludarabine (200 mg/m2) and thiotepa (10 mg/kg), the haematopoietic stem cell source was isolated CD34 cells from mobilized peripheral blood and no drugs were used as GVHD prophylaxis. For all remaining patients, conditioning consisted of cyclosporin A (CsA) (3 mg/kg) starting at day −1 with or without methotrexate (15 mg/m2). CsA was withdrawn at a rate of 20% weekly starting on day +100. All procedures were approved by appropriate institutional ethic committees. Acute graft-versus-host disease was staged according to Przepiorka et al (1995) and treated with prednisone 1 mg/kg.

Bone marrow samples and detection of MRD.  After informed consent from patients or parents, bone marrow aspirates were obtained prior to starting the conditioning regimen, at day +30 or at the time of neutrophil recovery, whichever occurred first, and at days +60 and +90. Subsequent samples were taken every 2 months during the first year, every 3 months in the second year and twice a year thereafter.

Bone marrow aspirates (n = 165) were collected in preservative-free heparin or in ethylene diamine tetraacetic acid (EDTA) at the intervals indicated above. Mononuclear cells were isolated by Ficoll–Hypaque (Lymphoprep, Nycomed, Oslo, Norway) density gradient centrifugation and washed three times in phosphate-buffered saline (PBS). Combinations of monoclonal antibodies against surface, cytoplasmic or nuclear leucocyte antigens defining leukaemia-associated immunophenotypes and staining techniques were as reported previously (Coustan-Smith et al, 1998). Mononuclear bone marrow cells were washed twice in PBS containing 0·2% bovine serum albumin and 0·2% sodium azide (PBSA). Human immunoglobulins (Flebogamma, Grifolls, Barcelona, Spain) were added to saturate Fc receptors. Cells were incubated for 10 min in the dark with optimally titrated antibodies against surface antigens. Cells were washed twice in PBSA and resuspended in PBS containing 0·5% paraformaldehyde for subsequent acquisition. For intracellular staining we used Permeafix (Ortho, Raritan, NJ, USA) according to the manufacturer's instructions.

Definition of leukaemia-associated immunophenotypes.  Combinations always included one or two markers that identify immature lymphoid progenitors (i.e. CD19, CD34 or CD10) and in addition one marker not expressed on normal lymphoid progenitors such as a myeloid antigen. Simultaneous expression of markers normally present at discrete stages of differentiation was also exploited, and the intensity of antigen expression, especially for CD10 and CD34, also differentiated ALL blast cells from their normal counterparts. For T-ALL the presence of double-positive cells for CD3 and TdT was restricted to blast cells since their normal counterparts are only found in thymus.

Commercially available reagents were used that were mostly mouse anti-human antibodies labelled with fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein and allophycocyanin. They were used to define leukaemia-associated immunophenotypes by multiparameter flow cytometry: CD38 (IgG1, clone TI16), CD10 (IgG1, clone ALB1), KOR-SA3544 (IgG1, clone KOR-SA3544), CD21 (IgG1, clone BL13) (from Immunotech, Marseille, France), CD45 (clone HI30, Pharmingen), anti-TdT (clone HT-6), CD66 (IgG1, clone Kat4c), CD3 (IgG1, clone UCHT1), CD5 (clone DK23), anti-μ-chain [polyclonal rabbit anti-human F(ab)2], CD19 (IgG1, clone HD37), CD13 (IgG1, clone WM-47), CD33 (IgG1, clone WM-54) (from Dako, Denmark), CD15 (IgM, clone MMA), CD34 (IgG1, clone HPCA2) and anti-human HLA-DR (IgG2a, clone L243) (from Becton Dickinson, San Jose, CA, USA). Combinations of these reagents are listed in Table II. Informative leukaemia-associated immunophenotypes were present in 100% of T-lineage ALL and 87·5% of B-lineage ALL.

Table II.  Immunophenotypic combinations used to assign leukaemia-associated immunophenotype.
ALL lineageCombinationPercentage*
  • ALL, acute lymphoblastic leukaemia.

  • *

    Percentage of patients who displayed these specific leukaemia-associated immunophenotype combinations based on differences of antigen intensity expression or aberrant expression compared with normal lymphoblast. All patients were traced with at least two combinations.

T-ALLTdT/CD3cyt/DRdim100
CD34/CD3cyt/DRdim  62
TdT/CD5/DRdim  62
B-ALLCD45/CD19/CD34–CD10  40
CD38/CD19/CD34–CD10  33
CD13/CD19/CD34–CD10  29
CD33/CD19/CD34–CD10  22
TdT/μcyt/CD34  22
CD66/CD19/CD34–CD10  18
CD21/CD19/CD34–CD10    7
CD15/CD19/CD34–CD10    3

Flow cytometry.  A dual-laser FACScalibur flow cytometer with cellquest software (Becton Dickinson) was used. The acquisition protocol included a first record of light-scattering and fluorescence signals of 10 000 events which enabled us to draw one gate around lymphoid cells and a second gate to define immature features such as CD19–CD34. Using Boolean instruction, electronic events falling within these two gates were selectively recorded, until all cells run through the cytometer or 10 000 events with immature lymphoid features were stored. To record results, captured data were reanalysed to assess the expression of leukaemia-associated immunophenotypes with a back-gating in the light-scattered dot plot. Events comprising a homogeneous cluster were expressed as a percentage of total run cells (Fig 1).

image

Figure 1. Gating strategy for minimal residual disease detection by flow cytometry. Electronic events falling within R1 (lymphoid cells by light-scattering properties) and R2 (defining immature lymphoid markers as CD19–CD34) were selectively recorded. Captured data were reanalysed to assess the expression of leukaemia-associated immunophenotypes (R3) in the events falling in the R1 and R2 regions. Finally, a back-gating in the light-scattered dot plot gathered all leukaemia cells (R1 + R2 + R3). Events comprising a homogeneous cluster were expressed as percentage of total run cells.

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Sensitivity and quality controls.  Leukaemia blast cells from patients with B-lineage ALL and T-lineage ALL were mixed with normal bone marrow cells at serial 10-fold dilution. Then, staining procedures were carried out. Acquisition and analysis of 2–5 × 105 cells per tube generated a clearly detectable cluster at the 0·01% level. Normal bone marrow cells from transplant donors as well as from patients diagnosed with malignancies other than ALL recovering from myeloablative regimen and allogeneic rescues were assessed with a complete panel of antibody combinations, with no detectable abnormal clusters when running up to 5 × 105 cells. For each assay, isotype-matched non-reactive antibodies were run as internal control.

Statistical analysis.  Cumulative actuarial probabilities (plus or minor standard errors) of disease-free survival (DFS) were calculated using Kaplan–Meier plots. Differences between time-to-relapse distribution function were compared by the log-rank test. Results have been analysed up to April 2001, which allows a minimum follow-up of 3 months for all patients. Multivariate analysis of variables affecting DFS was performed by Cox regression model.

Results

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Quantification of tumour burden before transplant

Twenty-four patients were tested for the presence of MRD in morphological remission bone marrow specimens, taken 1–21 d (median 7 d) before the beginning of conditioning regimen (Fig 2). Six patients (25%) harboured detectable levels of residual disease ranging from 0·2% to 10%. They were in first CR (n = 1), second CR (2CR, n = 2) and more than 2CR (n = 3). One patient maintained stable low levels during follow-up, but five patients had increasing amounts of leukaemia cells in subsequent tests and four of them eventually had an overt relapse 2·5–5 months after transplant. Based on these observations, UPN 376 underwent DLI at day +210, when blast cells increased up to 7%, achieving a prompt CR.

image

Figure 2. Clinical course and immunological MRD results in 24 patients tested before and after allo-SCT. Open squares represent marrow aspirates testing negative and shaded squares indicate positive determination. Numbers above positive determinations represent the percentage of blast cells among mononuclear marrow cells. R, relapse; asterisk, patient died; HAP, HLA haploidentical donor; UD, HLA identical unrelated donor; UPN, unique patient number; DLI, donor leucocyte infusion.

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By contrast, only three out of 18 patients (1CR = 6, 2CR = 8 and more than 2CR = 4), who were MRD negative prior to conditioning regimen, eventually relapsed. There was no difference in clinical status when comparing MRD-negative and MRD-positive groups. Two-year DFS for pretransplant MRD-positive patients was 33·3 ± 19·2% compared with 73·5 ± 13·5% for the group of patients testing MRD negative (P = 0·03 by log-rank test). Other statistically significant adverse clinical variables included age greater than 14 years (P = 0·002), advanced status at transplant (P = 0·0049), conditioning regimen without radiotherapy (P = 0·001), absence of acute GVHD (P = 0·03) and MRD positive after SCT (P = 0·001). In the multivariate analysis only MRD positive after SCT and advanced status at SCT remained statistically significant.

Sequential determinations of residual disease during post-transplant follow-up and clinical outcome

We studied MRD in serial post-transplant bone marrow specimens obtained from 40 patients. Median follow-up was 29·5 months (range 3–91). Median number of follow-up marrow aspirates performed per patient was three (range 2–10). At the time of closing the study, 11 patients had relapsed (nine in the marrow, two in extramedullar sites).

Nine patients showed increasing levels of leukaemic cells during follow-up and eight eventually relapsed (Figs 2 and 3). The time elapsed between detection of MRD and clinical relapse ranged between 1 and 6 months (median 2·5 months). Strikingly, a transient decrease of blast cells was observed in two patients (UPNs 376 and 365), one at 5 months and one at 7 months after transplant, coincident with the onset of skin GVHD when CsA had been reduced. Additionally, two patients have shown stable low MRD levels and both still remain in CR.

image

Figure 3. Clinical course and immunological MRD results of consecutive marrow aspirates after SCT. Open squares represent marrow aspirates testing negative and shaded squares indicate positive determination. Numbers above positive determinations represent the percentage of blast cells among mononuclear marrow cells. R, relapse; asterisk, patient died; UD, HLA identical unrelated donor; UPN, unique patient number; DLI, donor leucocyte infusion.

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By contrast, only one patient with marrow relapse (UPN 324) occurred among 29 patients with no previous MRD detection. She underwent allogeneic transplant from an HLA unrelated donor for BCR–ABL-positive ALL and showed a late marrow relapse in a scheduled bone marrow aspirate 26 months after transplant. The last scheduled MRD assay had been performed 6 months earlier and was negative.

Quantification of lymphoblastic leukaemia cells after DLI

We used flow cytometry to test the efficiency of DLI in six patients. Clinical features of DLI are described in Table III. UPNs 330, 334 and 354 had not been included previously in the study group and were only tested post-relapse after allo-SCT. Five patients were initially treated with standard induction chemotherapy regimens and an additional chemotherapy course prior to DLI, which consisted of 1 × 107−8 CD3-positive cells from HLA identical donors. Clinical outcomes and MRD results are summarized in Fig 4. Only one patient tested negative prior to DLI, whereas the remaining patients still harbour detectable MRD levels (0·05–40%). UPN 354 did not show any response and died of leukaemia progression. Morphological complete remission was achieved in four patients, but blast cells were detected by flow cytometry in UPN 382, and in UPN 330, who eventually died from bacterial infections. UPN 324 and 334 relapsed at day +110 and +30 respectively. A transient decrease in blast count was observed after treatment with tyrosine kinase inhibitor (STI571) in UPN 324, but eventually she died from leukaemia progression.

Table III.  Clinical characteristics of patients receiving DLI.
UPN Type Cytogenetic molecular studies Age at DLI Chemotherapy pre-DLI Interval relapse/DLI Type of donor MRD level pre-DLI CD3+ cells/kg CD34+ cells/kgAcute/chronic GVHD after DLI DFS/OS after DLI (d)
  • Rel, relapse; T, T-ALL; B, B-ALL;DLI, donor leucocyte infusion; MTX, high-dose methotrexate (1 g/m2); ARA-C, cytosine arabinoside; DN, daunorubicin; PDR, prednisone; VC, vincristine; CY, cyclophosphamide; HLA id. sib., HLA identical sibling donor; UD, unrelated donor; DFS, disease-free survival; OS, overall survival.

  • *

    Patient died.

334TBCR–ABL38MTX (× 2)90 dHLA id. sib.14%1 × 1070/–  15/60*
330BBCR–ABL36ARA-C/MTX45 dHLA id. sib.0·05%1 × 1072·1 × 106 – / –    7/7*
376BBCR–ABL5030 dHLA id. sib.7%1 × 108I skin/I360/360
382B46XX33ARA-C/MTX30 dHLA id. sib.0·14%1 × 1083·4 × 106II skin/–  90/90*
324BBCR–ABL22DN/PDR/VC30 dHLA id. UD0%1 × 1080/–110/270*
354B46XY7DN/PDR/VC GY/MTX60 dHLA id. sib.40%1 × 1081·27 × 1060/–  15/30*
image

Figure 4. Clinical course and immunological MRD results of six patients who underwent donor leucocyte infusion after SCT. Open squares represent marrow aspirates testing negative and shaded squares indicate positive determination. Numbers above positive determinations represent the percentage of blast cells among mononuclear marrow cells. R, relapse; asterisk, patient died; UPN, unique patient number; DLI, donor leucocyte infusion; STI571, tyrosine kinase inhibitor.

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As described above, UPN 376 was infused for increasing levels of MRD up to 7%, prior to overt relapse. He did not receive chemotherapy and was infused with 1 × 108 CD3/kg. The patient is alive and in complete remission 12 months after DLI. The serial determinations of this patient are shown in Fig 5.

image

Figure 5. Representative dot plots of MRD analysis of marrow samples from patient UPN 376. Displayed events are gated on CD19- and CD34-positive cells. The x-axis displays expression of aberrant antigen CD66c (KORSA) and y-axis displays CD19 expression. MRD was detectable prior to conditioning regimen. A transient decrease in the leukaemic population was observed at day +150 coincident with GVHD. Re-emergence of blast was seen at day +210. At that time the patient underwent DLI with a prompt disappearance of leukaemic cells. At last follow-up the MRD was still negative.

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Discussion

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In the present study we used a quantitative technique to monitor MRD in ALL, focusing on the impact of allogeneic transplant on the course of the disease. Some authors have attempted to detect aberrant lymphoid phenotypes in marrow aspirates (Wells et al, 1998) or peripheral blood (Shulman et al, 1999) after transplant in patients with ALL, based on a CD45 expression gating strategy. This approach yielded a sensitivity level of 0·3% and positive results seemed to be associated with a higher risk of relapse, but serial quantitative analysis was not performed and studied aberrant phenotypes did not strictly correspond to those at diagnosis.

In our study, many follow-up marrow samples were collected to gather insight into the kinetics of tumoral clearance by conditioning regimens and the re-emergence of disease when it escapes surveillance mechanisms. For patients with follow-up throughout the transplant procedure, we found that those with detectable levels of residual disease before conditioning regimen were more likely to relapse. Thus, MRD detection before transplant enabled us to quantify the quality of clinical remission regardless of the number of previous relapses. Additionally, this finding suggests that the capability of high-dose radiochemotherapy regimens to eliminate successfully the leukaemia clone might be limited in patients bearing a large tumoral burden. This is in agreement with previous studies in the autologous transplant setting (Uckun et al, 1993) or in allogeneic procedures that used semiquantitative molecular techniques (Knechtli et al, 1998a), which found a strong correlation of a positive pretransplant test with subsequent relapses. Large series are needed to assess this measurement as an independent prediction factor and to determine whether these poor prognosis patients could benefit from additional chemotherapy courses before transplant in chemosensitive cases, intensified conditioning regimens, non-T-depleted grafts or continuing treatment after transplant.

Patients testing negative at all points that were checked were unlikely to relapse. By contrast, a single positive determination, followed by increasing subsequent amounts, seems to carry a dismal prognosis, evolving to overt relapse in most instances. Our data and those obtained by molecular techniques (Radich et al, 1997; Knechtli et al, 1998b) suggest that possible pre-emptive therapeutic interventions (withdrawal of immunosuppression, interferon or even DLI) might be useful to prevent an overt relapse when applied within the window provided by serial follow-up samples. It is noteworthy that flow cytometry is a rapid assay which allows precise quantification of leukaemic cells and discriminates between viable and dead or dying cells. By contrast, molecular techniques are extremely useful because clonal antigen-receptor gene rearrangements occur in virtually all cases, but the complexity of the quantification protocols may hamper the suitability for routine clinical studies in most centres. When possible, tandem application of both techniques is highly recommended (Neale et al, 1999).

Intrinsic mechanisms to keep control of a residual amount of ALL cells, undetectable by morphological assessment, remain poorly understood. Several clinical factors seem to be involved such as the presence of GVHD, steroid treatment and CsA toxicity against ALL cells (Ito et al, 1998). Moreover, the existence of an immunological surveillance exerted in vivo by donor T cells targeted against specific tumoral antigens (i.e. WT1 antigen or p190) (Ohminami et al, 2000; Tanaka et al, 2000) or minor HLA antigens (Mutis et al, 1999) may be responsible for durable remission reported in some patients after either DLI or the induction of GVHD disease. Indeed, in our series we observed a reduction in leukaemia cell percentage after the onset of GVHD in two cases, and a complete response of a BCR–ABL-positive ALL after DLI applied before a frank relapse occurred. However, the mechanisms whereby ALL cells escape this immunological surveillance remain to be unravelled.

We conclude that MRD studies by flow cytometry provide new insights into the kinetics of ALL cells after allo-SCT and may be clinically useful in three main aspects: (1) assessment of the leukaemic burden before transplant to predict outcome, (2) detection of increasing amounts of leukaemia after transplant to determine the necessity of DLI in a timely fashion and (3) measurement of the efficacy of therapeutic interventions after transplant. However, larger series of DLI applied at a time of low tumoral burden are needed to assess whether responses could also be related to intrinsic leukaemic immunological features.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The authors thank Dr Dario Campana for the critical reading of the manuscript and providing helpful suggestions. We are specially indebted to Elaine Coustan-Smith for the technical training. Thanks to M. C. Acedo for help in the collection of serial samples. This work was supported by grant FIJC-98/ESP-Glaxo from the International Foundation José Carreras, Barcelona, Spain.

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  1. Top of page
  2. Abstract
  3. Patients, materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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