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Keywords:

  • minimal residual disease (MRD);
  • flow cytometry;
  • T-cell acute lymphoblastic leukaemia (T-ALL)

Abstract

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

Summary. Flow-cytometric detection of minimal residual disease (MRD) identifies patients with high relapse risk in childhood acute lymphoblastic leukaemia (ALL). We studied the efficacy of this method in adult T-ALL treated with the Italian co-operative GIMEMA (Gruppo Italiano Malattie Ematologiche dell'Adulto) LAL0496 protocol. Bone marrow samples from 53 patients were taken at fixed treatment time points and MRD was analysed using a leukaemia-specific immunophenotype (cytoplasmic-CD3/nuclear-terminal desoxynucleotidyl transferase). The median follow-up was 17 months (range 3–61) and a median of 4·5 analyses/patient was performed (range 3–12). Six out of 53 (11·3%) patients were refractory to treatment, 30/53 (56·6%) relapsed and 17/53 (32·1%) remain in continuous complete remission. The probability of relapse at 2 years for MRD-positive patients at preconsolidation was 81·5%vs 38·9% for MRD-negative patients (P = 0·00078). This risk was still 54·5% for MRD-positive vs 15·8% for MRD-negative patients pre-third reinduction (P = 0·0098) and 50·0% for MRD-positive vs 16·4% for MRD-negative patients pre-sixth reinduction (P = 0·032). The relapse-predicting value of MRD did not depend on features at diagnosis such as age, sex and leucocyte count. Our data suggest that immunophenotypic MRD monitoring in the first year of treatment is a useful outcome predictor for adult T-ALL patients.

Minimal residual disease (MRD) in acute leukaemia may persist after treatment and eventually lead to clinical relapse, despite the achievement of a morphologically documented complete remission (CR). Thus, the early detection of MRD in the bone marrow (BM) during CR may identify high-risk patients who could benefit from more intensified treatment strategies (Campana & Pui, 1995; Bear, 1998; Pui & Campana, 2000; Radich, 2000; Sievers & Radich, 2000).

Several methods are available for MRD detection in acute lymphoblastic leukaemia (ALL). Molecular approaches based on the polymerase chain reaction (PCR) can identify residual clones bearing gene fusions or antigen-receptor gene rearrangements. At present, these are the most sensitive techniques for MRD detection (one leukaemic cell/105−106 normal cells), although molecular abnormalities are detectable only in a proportion of ALL patients (Biondi et al, 1992; Campana & Pui, 1995; Gibson et al, 1996; Heid et al, 1996; van Dongen et al, 1999; Foroni et al, 1999; Freeman et al, 1999; Hosler et al, 1999; Martuza et al, 2002). MRD detection by flow cytometry is based on the identification of leukaemia-associated marker combinations, which are either not expressed or expressed at different levels of intensity by normal BM cells (Syrjäläet al, 1994; Campana & Pui, 1995; Jennings & Foon, 1997; Ciudad et al, 1998a; Campana & Coustan-Smith, 1999; Griesinger et al, 1999; San Miguel et al, 1999; Garcia Vela et al, 2000; Porwit-McDonald et al, 2000). A number of studies, using either flow cytometric or molecular approaches, showed that the presence of detectable MRD at any time point during the treatment course can predict relapse in childhood ALL (Biondi et al, 1992; Campana & Pui, 1995; Bear, 1998; Campana & Coustan-Smith, 1999; van Dongen et al, 1999; Foroni et al, 1999; Griesinger et al, 1999; San Miguel et al, 1999; Coustan-Smith et al, 2000; Pui & Campana, 2000; Radich, 2000; Sievers & Radich, 2000; Dworzak et al, 2002) and consequently current multicentre protocols have been designed on the basis of MRD monitoring.

Few studies have been reported to date on the clinical impact of immunophenotypic detection of MRD in adult ALL (Ciudad et al, 1998a,b; Griesinger et al, 1999; San Miguel et al, 1999; Porwit-McDonald et al, 2000; Krampera et al, 2001). The small number of suitable patients in each centre may be an obstacle in assessing the effectiveness of this method in adults, especially for relatively rare diseases such as T-ALL, which represents about 20% of adult ALL (Rivera & Crist, 1995). We carried out a multicentric study of immunophenotypic MRD detection in adult T-ALL patients who underwent the same therapeutic protocol. We quantified the amount of residual BM leukaemic cells bearing the cytoplasmic CD3 (cyCD3)/nuclear terminal desoxynucleotidyl transferase (TdT) marker combination, which is expressed in > 90% of T-ALL patients and in a small proportion of T cells in the normal thymus, but not in normal BM (Campana & Pui, 1995; Jennings & Foon, 1997; Coustan-Smith et al, 1998, 2000; Campana & Coustan-Smith, 1999). MRD monitoring was focused especially on the first year of treatment, when relapse is more likely to occur, in order to establish the treatment time points that were most informative with regard to clinical relapse. Our data suggest that in adult T-ALL the flow-cytometric approach is useful for the early identification of patients with high relapse risk and poor outcome.

Patients. Between April 1996 and February 2002, 93 adult T-ALL patients consecutively admitted to 34 different Italian Haematology centres were treated with the multicentric co-operative GIMEMA (Gruppo Italiano Malattie Ematologiche dell'Adulto) LAL0496 protocol. The therapy schedule consisted of an intensive treatment in the first 3 months (induction and consolidation) followed by chemo/radioprophylaxis on the central nervous system and maintenance/reinduction cycles for up to 3·5 years from diagnosis (Fig 1). Patients who underwent bone marrow transplantation (BMT) were censored from this study.

image

Figure 1. GIMEMA LAL 0496 protocol. VCR, vincristine; DNM, daunorubicin; PDN, prednisone, L-ASE, L-Asparaginase; Ara-C, cytarabine; VP-16, etoposide; MTX, methotrexate; *IT-MTX, intrathecal methotrexate; 6-MP, 6-mercaptopurine; CTX, cyclophosphamide; CNS-RT, central nervous system radiotherapy; G-CSF, granulocyte-colony stimulating factor; sm, square metre; i.v., intravenously; i.m., intramuscular; Gy, Grays.

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Methods.  BM assessments were approved by the institutional ethical committee and were carried out according to informed consent guidelines. BM samples were collected in each referral centre at diagnosis and, regardless of clinical status, during the clinical follow-up, i.e. before consolidation (median 2 months from diagnosis), and before the third (median 6 months from diagnosis) and the sixth reinduction cycles (median 9 months after diagnosis). Additional MRD assessments were established before each reinduction cycle from the eighth to the 12th cycles (Fig 1). Fresh heparinized BM samples were sent overnight to the Sample Collection Centre in Rome, where mononuclear cells (MNC) were separated by density gradient centrifugation and cryopreserved in liquid nitrogen. Flow-cytometric analyses were performed at the MRD Analysis Centre in Verona. No information about the MRD status of the patients was given to the centres until the end of the study.

Thawed BM MNC (106 cells/tube) were incubated with Ortho-Permeafix (2 ml at room temperature for 40 min) for cell fixation and permeabilization (Pizzolo et al, 1994). After washing with a solution containing 1% phosphate-buffered saline, 1% bovine serum albumin and 0·1% sodium azide, samples were incubated for 60 min at 4°C with a fluorescein isothiocyanate (FITC)-conjugated anti-TdT (Dako, Glostrup, Denmark) monoclonal antibody (mAb). After 30 min, an R-phycoerythrin (R-PE)-conjugated anti-CD3 mAb (Becton Dickinson, San Jose, CA, USA) was added (0·1 µg/sample). Isotype controls were run in parallel (FITC-conjugated anti-IgG1 for the anti-TdT mAb and R-PE-conjugated anti-IgG1 for the anti-CD3 mAb; both reagents were purchased from Becton Dickinson). Samples were then washed twice and resuspended. At least 100 000 events were acquired by a flow cytometer equipped with an argon–ion laser (488 nm, FACScan; Becton Dickinson), using the cell quest software. CyCD3+/TdT+ cell clusters were expressed as absolute number of positive cells on the entire MNC population. As CyCD3+/TdT+ cell clusters were not found in quiescent or regenerating normal BM, we considered these cells as an expression of MRD when more than 10 clusterized cells/100 000 acquired MNC were detected. This was consistent with the flow-cytometric 10−4 sensitivity we found in preliminary experiments using T-cell blast-limiting dilution, as well as with that reported in literature (Campana & Pui, 1995; Jennings & Foon, 1997; Coustan-Smith et al, 1998, 2000; Campana & Coustan-Smith, 1999). The sensitivity of this method could be actually higher, as we expressed the MRD on the BM MNC population only, excluding granulocytes by density gradient centrifugation.

Statistical analysis.  Distributions of presenting features at diagnosis according to the degree of MRD during clinical remission were compared by the Fisher's exact test. The probability of relapse following MRD detection was estimated using the cumulative incidence procedure, applied to the follow-up time up to the end of February 2002. Relapse probabilities were compared using the log-rank test. All quoted P-values are two sided and all confidence intervals refer to 95% boundaries (95% CI).

Results

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

Forty of the 93 patients (43·0%) could not be evaluated because of the lack of one or more MRD assessments. Analysis of data on MRD and clinical outcome was therefore performed in 53/93 (57·0%) patients: 6/53 (11·3%) were resistant to induction, 30/53 (56·6%) relapsed and 17/53 (32·1%) remain in continuous CR (CCR, Table I). The median follow-up was 17 months (range 3–61) and a median of 4·5 immunophenotypic analyses/patient was carried out (range 3–12). All patients were cyCD3+/TdT+ at diagnosis and the majority of T-ALL blasts expressed this immunophenotype (median 84·9%, range 54·1–98%). CyCD3+/TdT+ cell clusters persisted in all of the refractory patients.

Table I.  Flow cytometric detection of MRD in T-ALL patients: assessable patients and clinical results.
Enrolled patients (n = 93) 
  1. CCR: continuous complete remission.

Non assessable patients (no MRD samples)40/93 (43·0%)
Assessable patients53/93 (57·0%)
 Relapsed30/53 (56·6%)
 No response6/53 (11·3%)
 CCR17/53 (32·1%)

At the first MRD evaluation (preconsolidation, median 2 months from diagnosis), 18/47 (38·3%) non-refractory patients were MRD positive (median percentage of cyCD3+/TdT+ cells: 0·075%, range 0·013–17·8) and 29/47 (61·7%) were MRD negative. The probability of relapse at 2 years from diagnosis was 81·5% for the MRD-positive patients (95% CI 57·5–93·5%) and 38·9% for the MRD negative patients (95% CI 23·1–57·4%) (Fig 2). The difference between the two groups was statistically significant (P = 0·00078) (Table II).

image

Figure 2. Cumulative incidence of relapse, according to preconsolidation MRD assessment. Comparison between MRD-positive and MRD-negative patients in terms of relapse risk.

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Table II.  Cumulative relapse according to MRD detection at preconsolidation, pre-third and pre-sixth reinduction assessments.
MRD assessments Patients (n)Median percentage of cyCD3+/TdT + cellsProbability of relapse at 2 years95% CIP-value
Preconsolidation
 MRD+180·075% (0·013–17·8)81·557·5–93·50·00078
 MRD2938·923·1–57·4 
Pre-third reinduction
 MRD+110·043% (0·014–4·02)54·528·0–78·70·0098
 MRD2115·85·5–37·7 
Pre-sixth reinduction
 MRD+ 40·03% (0·017–0·14)50·015·0–85·00·032
 MRD2016·45·5–39·7 

At the following MRD evaluation (pre-third reinduction, median 6 months from diagnosis), 11/32 (34·4%) patients were MRD positive (median percentage of cyCD3+/TdT+ cells: 0·043%, range 0·014–4·02%) and 21/32 (65·6%) were MRD negative. The probability of relapse at 2 years from diagnosis was 54·5% for the MRD-positive patients (95% CI 28·0–78·7%) and 15·8% for the MRD-negative patients (95% CI 5·5–37·7%) (Fig 3). The difference between the two groups was statistically significant (P = 0·0098) (Table II).

image

Figure 3. Cumulative incidence of relapse according to pre-third reinduction MRD assessment. Comparison between MRD-positive and MRD-negative patients in terms of relapse risk.

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Among the subsequent follow-up MRD evaluations, the one carried out before the sixth reinduction cycle (median 9 months from diagnosis) predicted relapse: 20/24 (83·3%) patients still in CCR were MRD negative and 4/24 (16·7%) were MRD positive (median percentage of cyCD3+/TdT+ cells: 0·03%, range 0·017–0·14%). The probability of relapse at 2 years from diagnosis was 50·0% for the MRD-positive patients pre-sixth reinduction (95% CI 15·0–85·0%) and 16·4% for the MRD-negative patients (95% CI 5·5–39·7%) (Fig 4). The difference between the two groups was statistically significant (P = 0·032) (Table II). The remaining MRD assessments did not prove to be statistically significant, probably as a result of the small number of patients studied. The comparison of the MRD status at preconsolidation (available in all the assessable patients) with different clinical and biological features at diagnosis, such as age (< or > 30 years), sex and leucocyte count (< or > 100 × 109/l) showed no statistical association (Table III). Ploidy and cytogenetic analysis was not carried out in all the patients and was not included in the study. Table IV summarizes the clinical outcome of the 47 non-refractory patients, according to the results of the MRD testing at various times.

image

Figure 4. Cumulative incidence of relapse according to pre-sixth reinduction MRD assessment. Comparison between MRD-positive and MRD-negative patients in terms of relapse risk.

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Table III.  Distributions of presenting features at diagnosis, according to MRD status at preconsolidation assessment, analysed by the Fisher's exact test.
Presenting features (at diagnosis)MRD status at pre-consolidation (n = 47)
MRD+MRDP-value
Age (years)
 14–3013210·83
 31–60 5 8 
Sex
 male14240·61
 female 4 5 
WBC (109/l)
 ≤10013160·25
 > 100 513 
Table IV.  Clinical outcome of the non-refractory patients, according to the different results of the main MRD assessments.
Pre-consolidationPre-third reinductionPre-sixth reinduction Patients (n)Relapsed patients (n)
  • *

    relapsed at < 6 months from diagnosis.

MRD+**1010/10
MRD+MRD+1 1/1
MRD+MRD2 1/2
MRD+MRD+MRD+1 1/1
MRD+MRDMRD2 1/2
MRD+MRD+MRD2 1/2
MRD+MRDMRD+0 
MRD**5 5/5
MRDMRD1 0/1
MRDMRD+4 4/4
MRDMRDMRD13 3/13
MRDMRD+MRD+0 
MRDMRDMRD+3 2/3
MRDMRD+MRD3 1/3
Total47 

Discussion

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

The role of immunophenotypic MRD detection in predicting clinical relapse has been documented in childhood ALL (Campana & Coustan-Smith, 1999; Griesinger et al, 1999; San Miguel et al, 1999; Coustan-Smith et al, 2000; Dworzak et al, 2002). The aim of our study was to assess the impact of this approach in adult T-ALL. Blasts from this form of ALL consistently express a leukaemia-specific marker combination (cytoplasmic CD3 and nuclear TdT) that is never observed in normal BM and may be easily monitored by flow cytometry (Campana & Pui, 1995; Campana & Coustan-Smith, 1999).

An adequate number of suitable patients (53 adult patients with > 90% of cyCD3+/TdT+ blasts at diagnosis) was studied during the treatment course, although they accounted for 57·0% of all those enrolled in the LAL0496 protocol. Lack of adequate sample collection by some centres was found to be the main problem. We chose the time of BM assessment as the treatment time point for MRD detection within the year 1 of treatment, when relapse is more likely to occur, to statistically correlate the clinical status of patients at that time to MRD findings.

The most informative data were obtained at the time of preconsolidation (median 2 months from diagnosis), pre-third reinduction (median 6 months from diagnosis) and pre-sixth reinduction (median 9 months from diagnosis). The persistence of cyCD3+/TdT+ cell clusters (≥ 0·01%, i.e. ≥ 10 clusterized leukaemic cells/100 000 acquired events) at these assessments was associated with a higher relapse risk. This finding was a relapse-predicting factor independent of other clinical-biological features, such as age (< or > 30 years), sex and leucocyte count (< or > 100 × 109/l). The most informative MRD assessment seemed to be at preconsolidation, following the intensive induction treatment. Ten/15 patients who relapsed early (< 6 months from diagnosis) were MRD+ at this point. Patients with detectable MRD at preconsolidation who were still MRD+ in the following assessments had a high relapse rate. Similarly, the appearance of MRD pre-third and/or pre-sixth reinduction, following a negative MRD assessment at preconsolidation, was associated with a significantly increased incidence of relapse.

It is difficult to speculate on whether these MRD assessments identify some biologically different situations. Pre-consolidation (and probably pre-third reinduction) assessment could identify patients with an early selection of refractory clones. Conversely, pre-sixth reinduction assessment could identify leukaemic clones, which are still responsive to chemotherapy, but will expand as soon as the dose-intensity of treatment decreases after the sixth reinduction. Nevertheless, the high relapse risk associated with MRD detection strongly suggests that these treatment time points may be chosen to recognize patients who need a more intensive and potentially more toxic therapy (including BMT) from those who can be cured with a conventional and less intensive therapy. Unfortunately, MRD monitoring failed to predict the relapse in a minority of patients. In these patients, the combined use of immunophenotype with PCR could lower the risk of MRD detection failure due to immunophenotypic changes during the course of the disease, as previously suggested for childhood ALL (Neale et al, 1999).

In conclusion, adult T-ALL patients with detectable MRD at the end of induction and during the following reinduction cycles must be considered at high risk of leukaemic reappearance and, therefore, candidates for alternative treatments.

Acknowledgments

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

Grants from Istituto Superiore di Sanità (Italy Usa Project ‘Terapia dei Tumori’), Rome, Ministero dell'Università e della Ricerca Scientifica (MURST), Rome, and Fondazione Cariverona, Verona, Italy supported this work.

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