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Keywords:

  • acute myeloid leukaemia;
  • minimal residual disease;
  • remission;
  • flow cytometry;
  • prognosis

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Prevalence of aberrant AML immunophenotypes and sensitivity of residual disease studies
  6. Relationship between measurements of residual disease by flow cytometry versus morphology, cytogenetics and RT-PCR
  7. Relationship of flow cytometric detection of residual disease to presenting features and to treatment outcome
  8. Prognostic significance of flow cytometric detection of residual disease
  9. Discussion
  10. Acknowledgments
  11. References

Summary. In children with acute myeloid leukaemia (AML), morphological and karyotypic studies cannot precisely assess response to treatment, and less than one-third of patients have genetic markers for molecular studies of residual disease. We determined the usefulness of a four-colour flow cytometric strategy developed in our laboratory to study residual disease. We first compared the immunophenotypes of AML cells obtained from 54 children at diagnosis with those of cells from 59 normal or regenerating bone marrow samples. Forty-six of the 54 AML cases (85·2%) had immunophenotypes that allowed detection of 0·1–0·01% residual leukaemic cells. Of 230 bone marrow samples obtained from those 46 patients during and off treatment, 61 (26·5%) had ≥ 0·1% AML cells by flow cytometry. We found that core binding factor-associated AML had a significantly better early treatment response. Mean (± standard error) 2-year survival estimate was 33·1 ± 19·1% for patients with ≥ 0·1% AML cells by flow cytometry after induction therapy, but 72·1 ± 11·5% for those with < 0·1% AML cells (P = 0·022); overt recurrence of AML within the subsequent 6 months was significantly more likely in the former group. The assay described here holds promise for guiding the choice of post-remission treatment options in children with AML.

More than 85% of children with acute myeloid leukaemia (AML) enter morphological remission after one or two courses of chemotherapy, but approximately half of these patients have persistent occult disease that leads to overt relapse (Woods et al, 2001; Arceci, 2002). Slow clearance of AML cells by remission induction therapy is associated with a poor treatment outcome (Wheatley et al, 1999; Estey et al, 2000; Kern et al, 2003). However, the assessment of treatment response by morphological examination of the bone marrow can be subjective and lacks sensitivity.

Studies of residual disease by reverse transcriptase polymerase chain reaction (RT-PCR) in adult patients have indicated that quantification of PML-RARA, AML1-ETO and CBFB-MYH11 transcripts helps to predict outcome (Grimwade, 1999; Lo Coco et al, 1999; Tobal et al, 2000; Buonamici et al, 2002; Guerrasio et al, 2002), but genetic markers suitable for RT-PCR studies are found in less than one-third of these patients (Liu & Grimwade, 2002). Aberrant immunophenotypes defined by flow cytometry have potentially wider applicability. In adults with AML, three-colour flow cytometry can identify abnormal immunophenotypes at diagnosis in 65–75% of patients; detection of residual cells with identical immunophenotypes during treatment was associated with a poorer outcome (San Miguel et al, 1997; Venditti et al, 2000; San Miguel et al, 2001). In children with AML, observation of abnormal profiles by three-colour flow cytometry in bone marrow samples obtained during treatment was associated with an increased risk of treatment failure (Sievers et al, 1996, 2003).

In this study, we used four-colour flow cytometric techniques that enabled minimal residual disease (MRD) monitoring in > 95% of children with acute lymphoblastic leukaemia (ALL) with 0·01% sensitivity (Campana & Coustan-Smith, 2002). In ALL, this method has been shown to provide reliable prognostic information in a clinical setting (Coustan-Smith et al, 1998, 2000, 2002a,b). The purpose of the study was fourfold: (1) to establish the prevalence of abnormal immunophenotypes in childhood AML by systematically comparing leukaemic cells and normal haematopoietic progenitors, including cells obtained from recovering bone marrow; (2) to determine the sensitivity of the method by testing mixtures of normal and leukaemic cells; (3) to assay residual leukaemic cells in bone marrow samples from children receiving treatment for AML and compare the results with those of morphological, karyotypic and molecular studies; and (4) to determine how residual disease detected by flow cytometry is related to the clinical and biological features of the disease and to the outcome of treatment.

Patients.  Between November 1998 and July 2002, 64 patients were enrolled in the St Jude AML97 study, which accepted all patients with newly diagnosed AML except those with acute promyelocytic leukaemia with the t(15;17)/PML-RARA (Crews et al, 2002). For 10 of these 64 patients, no diagnostic material was available to determine the presence of leukaemia-associated immunophenotypes; one patient's diagnostic bone marrow sample was not forwarded to our laboratory; for the remaining nine patients [four with French–American–British (FAB) subtype M7, two FAB M4, one FAB M5, two unclassifiable by FAB] insufficient material was available. To define leukaemia-associated immunophenotypes, we compared diagnostic AML samples with bone marrow samples from healthy individuals (n = 21) and from patients with leukaemia (other than AML) or lymphoma undergoing therapy (n = 38). A total of 230 bone marrow samples obtained from children with AML during treatment were also studied. All samples were processed within 4 h of collection. These studies were approved by the St Jude institutional review board, with informed consent obtained from the parents or guardians of each child.

Treatment protocol.  The upfront ‘window’ before induction therapy consisted of cytarabine and 2-chlorodeoxyadenosine daily for 5 d (Crews et al, 2002). Each of the two cycles of remission induction therapy comprised daunorubicin, cytarabine and etoposide. Patients with ‘favourable’ cytogenetic abnormalities [t(8;21), inv(16), t(9;11)] were treated with two courses of post-remission consolidation chemotherapy, the first consisting of cytarabine and lasparaginase, and the second consisting of mitoxantrone and cytarabine. Haematopoietic stem cell transplantation was considered for patients with karyotypes associated with poorer outcome or AML that was not in clinical remission after one course of induction chemotherapy, and for patients who did not have either favourable cytogenetics or high-risk features if a related human leucocyte antigen (HLA)-matched donor was available. Central nervous system treatment consisted of one dose of intrathecal cytarabine at diagnosis and triple intrathecal chemotherapy with methotrexate, hydrocortisone and cytarabine subsequently.

Flow cytometric detection of residual disease.  Bone marrow aspirates were placed in preservative-free heparin, and mononuclear cells were separated by centrifugation on a density step (AccuPrep; Nycomed, Oslo, Norway). Mononuclear cells were labelled with various combinations of monoclonal antibodies conjugated to fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein and allophycocyanin (Campana & Coustan-Smith, 2002). Matched non-reactive fluorochrome-conjugated antibodies served as controls. The staining procedure has been described previously (Campana & Coustan-Smith, 2002). The resulting four-colour cell staining was analysed with a dual-laser FACSCalibur flow cytometer with cell quest software (Becton Dickinson, San Jose, CA, USA). Side-by-side comparisons of the results obtained in diagnostic AML samples and in reference control samples were performed to define leukaemia-associated immunophenotypes (expressed on leukaemic cells but not on normal bone marrow cells).

To monitor residual disease, marker combinations that allowed the identification of one leukaemic cell per 103 or more normal mononucleated bone marrow cells or greater were selected at diagnosis in each case and then applied during clinical remission (Table I). The flow cytometry protocol used for residual disease detection has been described in detail previously (Campana & Coustan-Smith, 2002). In all samples, 0·5–1 × 106 mononuclear cells were labelled with each antibody combination and with isotype-matched control antibodies; we acquired data from all mononuclear cells in each test tube. To determine the proportion of mononuclear cells within each sample and to distinguish them from residual erythrocytes, platelet aggregates and debris, cells in one tube were stained with SYTO-13 (50 nmol/l; Molecular Probes, Eugene, OR, USA), a green fluorescent nucleic acid stain that is cell permeable. (Fornas et al, 2000) Results of residual disease analysis were expressed as percentage of nucleated cells with the abnormal immunophenotype. To ensure objective and consistent estimates, these percentages were calculated using fixed templates for each antibody combination; the templates divided dot-plot areas occupied by normal bone marrow cells from areas that are invariably empty in analyses of normal bone marrow cells (Campana & Coustan-Smith, 2002). Flow cytometric data were recorded with no knowledge of patient clinical status or diagnostic features other than immunophenotype.

Table I.  Marker combinations used to study MRD in AML.
Marker combination*No. of patients studied (%)†
  • *

    The order of the individual markers in each set corresponds to the fluorochrome to which they were conjugated, as follows: fluorescein isothiocyanate/phycoerythrin/peridinin chlorophyll protein/allophycocyanin.

  • Number of patients in whom the marker combination listed afforded a sensitivity of detection of at least 0·1% among the 46 patients with aberrant immunophenotypes. Aberrant immunophenotypes were identified with at least four marker combinations in 11 patients, with three combinations in 12 patients, with two combinations in 16 patients, and with one combination in seven patients.

CD13/CD117/CD34/CD3319 (41)
CD15/CD117/CD34/CD3319 (41)
CD13/CD133/CD34/CD3314 (30)
CD13/CD56/CD34/CD3313 (28)
HLA-DR/CD117/CD34/CD3311 (24)
CD11b/CD13/CD34/CD33 8 (17)
CD38/CD13/CD34/CD33 8 (17)
CD15/CD13/CD34/CD33 6 (13)
CD7/CD13/CD34/CD33 5 (11)
CD45/CD13/CD34/CD33 5 (11)
CD19/CD13/CD34/CD33 4 (9)
CD11b/CD117/CD34/CD33 4 (9)
HLA-DR/CD13/CD34/CD33 3 (7)
CD13/anti-7·1/CD34/CD33 3 (7)

Cytogenetics and RT-PCR.  Karyotype at diagnosis and during treatment was examined by standard cytogenetics on metaphase spreads (Raimondi et al, 1998). For RT-PCR analysis of residual disease, total RNA was extracted using a commercial kit (Gentra, Minneapolis, MN, USA), and cDNA was synthesized with random hexamers and Superscribe II (Invitrogen, Carlsbad, CA, USA). PCR was performed using oligonucleotide primers to MLL-AF9, MLL-AF10, AML1-ETO or CBFB-MYH11 as described previously (Downing et al, 1993; Shurtleff et al, 1995; Rubnitz et al, 2002). On each run, extracted RNA from a cell line containing one of the above translocations or an in vitro-transcribed RNA was 10-fold serially diluted and included in the run to generate a relative standard curve. PCR products were separated on 1·2% agarose gel, transferred to nylon membranes (Hybond-N; Amersham, Arlington Heights, IL, USA), and Southern blot analysis was performed with transcript-specific probes. Autoradiography was performed using XAR-5 film (Kodak, Rochester, NY, USA).

Statistical analysis.  Overall survival and leukaemia-free survival were examined for all patients tested for residual disease at the end of each phase of therapy. Overall survival was defined as the time that elapsed between the date of the test and the date of death; any patient still alive at the last follow-up date was considered censored for all overall survival analyses. Leukaemia-free survival was defined as the time between the date of the test and the date of relapse; any surviving patient who had not experienced relapse at the time of last follow-up and patients who were removed from the study for reasons other than relapse were censored in these analyses. The Kaplan–Meier method was used to compute estimates of overall survival and leukaemia-free survival. The exact log-rank test was used to compare survival curve estimates. Multiple Cox regression analyses of survival included residual disease detected by flow cytometry, age, leucocyte count and karyotype. Fisher's exact test was used to examine the association between residual disease status and clinical and biological presenting features, and with 6-month leukaemia-free survival.

Prevalence of aberrant AML immunophenotypes and sensitivity of residual disease studies

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Prevalence of aberrant AML immunophenotypes and sensitivity of residual disease studies
  6. Relationship between measurements of residual disease by flow cytometry versus morphology, cytogenetics and RT-PCR
  7. Relationship of flow cytometric detection of residual disease to presenting features and to treatment outcome
  8. Prognostic significance of flow cytometric detection of residual disease
  9. Discussion
  10. Acknowledgments
  11. References

To determine the applicability of four-colour flow cytometry to studying residual disease in childhood AML, we compared the immunophenotypes of the leukaemic cells from 54 children with newly diagnosed AML with those of bone marrow mononuclear cells obtained from 21 healthy donors and 38 patients undergoing treatment for ALL or lymphoma. Samples were collected from the latter patients 1–2 weeks after remission induction therapy or at the end of therapy when patients were in complete remission by clinical, morphological and flow cytometric criteria and had active haematopoietic regeneration. Abnormal immunophenotypic features were detected in 46 out of 54 (85·2%) patients (Table I). The leukaemic immunophenotypes of 26 patients (48·1%) were sufficiently distinct from those of normal and regenerating bone marrow cells to allow a sensitivity of detection of one leukaemic cell among 10 000 or more normal bone marrow mononuclear cells (i.e. a sensitivity of ≥ 0·01%); in the other 20 patients (37·0%), partial overlap between the immunophenotypes of regenerating marrow cells and leukaemic cells limited the sensitivity to 1 in 1000 (i.e. 0·1%). The eight patients lacking leukaemia-associated immunophenotypes had AML M1 (n = 1), M2 (n = 4), M5 (n = 2) and M7 (n = 1); three cases had t(9;11)/MLL-AF9, and one had t(8;21)/AML1-ETO. There was no significant difference in gender, age, leucocyte count, karyotype, overall survival and leukaemia-free survival between the 46 patients with leukaemia-associated immunophenotypes and the patients who could not be studied because of lack of available diagnostic sample (n = 10) or lack of suitable immunophenotype (n = 8).

To ensure that the sensitivity of 0·01% calculated by comparing the immunophenotype of leukaemic and normal cells corresponded in practice to the detection of residual leukaemic cells, we tested prepared mixtures of primary AML cells and normal bone marrow cells. The cell mixtures contained leukaemic cells in various proportions between 10% and 0·001%. The cases of AML used for these studies expressed aberrant immunophenotypes with the following five sets of markers among those listed in Table I: (a) CD13, CD117, CD34 and CD33; (b) CD13, CD133, CD34 and CD33; (c) CD13, CD56, CD34 and CD33; (d) CD11b, CD13, CD34 and CD33; and (e) CD38, CD13, CD34 and CD33. In mixtures containing 0·01% AML cells, leukaemic cells could be detected clearly with all these combinations (Fig 1).

image

Figure 1. Sensitivity of flow cytometry for the detection of residual disease. Primary AML cells were mixed with normal bone marrow cells at the proportions indicated. Flow cytometric dot plots depict the results of CD33 and CD38 staining on selectively analysed CD34+ CD13 cells. The dashed line encloses the leukaemia-specific area of the dot plot. Cells expressing the leukaemia immunophenotype are clearly distinguishable in mixtures containing 0·01% AML cells.

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Relationship between measurements of residual disease by flow cytometry versus morphology, cytogenetics and RT-PCR

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Prevalence of aberrant AML immunophenotypes and sensitivity of residual disease studies
  6. Relationship between measurements of residual disease by flow cytometry versus morphology, cytogenetics and RT-PCR
  7. Relationship of flow cytometric detection of residual disease to presenting features and to treatment outcome
  8. Prognostic significance of flow cytometric detection of residual disease
  9. Discussion
  10. Acknowledgments
  11. References

Next, we used the aberrant immunophenotypes identified in the above studies to determine the presence of residual leukaemia in 230 bone marrow samples obtained from the 46 patients at various time points during treatment (n = 164) and off therapy (n = 66). To ensure a definition of residual disease positivity applicable to all 46 patients, we used a threshold of 0·1%; patients whose values were lower than this threshold were considered to be residual disease-negative. Of the 230 bone marrow samples studied, 61 (26·5%) were residual disease-positive by flow cytometry (Fig 2).

image

Figure 2. Detection of residual disease by flow cytometry in patients with AML during treatment. Flow cytometric dot plots depict the results of CD13 and CD33 staining on selectively analysed CD45+ CD34 cells (top row), of CD56 and CD13 staining on CD33+ CD34+ cells (middle row) and of CD117 and anti-HLA-DR (HLA-Dr) staining on CD33+ CD34+ cells (bottom row). For each antibody combination, the staining of two normal bone marrow samples (left), of one AML sample collected at diagnosis (middle right) and of one bone marrow sample collected from the corresponding patient during treatment (right) is shown. Leukaemia-specific areas in each set of dot plots are enclosed by a dashed line; the percentage of residual disease in the follow-up samples is indicated. The follow-up sample in the top row was obtained after window therapy, whereas the follow-up samples shown in the middle and bottom rows were collected after induction therapy; in all three follow-up samples, morphological and karyotypic analysis failed to detect evidence of leukaemia.

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Figure 3 summarizes the comparison between results obtained by flow cytometry and those obtained by morphology, cytogenetics and RT-PCR. Of the 195 samples that had no morphological evidence of AML, 34 contained AML cells identifiable by flow cytometry (Fig 3A, left). In 24 other samples, cells with suspicious but unclear morphology for leukaemic myeloblasts were seen; 17 of these samples had AML cells by flow cytometry (Fig 3A, centre). Overall, 51 of the 219 (23·3%) samples that did not have definite leukaemia blast cells by morphology contained leukaemic cells by flow cytometry. The remaining 11 samples contained AML blast cells on the basis of morphology; 10 of these had 9% or more AML cells by flow cytometry but one sample included no cells with distinctive leukaemic immunophenotypic abnormalities (Fig 3A, right). This sample, obtained after ‘window’ therapy from a patient with M4, contained 11% blast cells that were deemed to represent persistent disease. In view of the fact that the sample also lacked the t(8;21) karyotypic abnormality identified at diagnosis, it is possible that these cells were normal regenerating myeloid cells.

image

Figure 3. Measurement of residual disease by flow cytometry versus morphology (A), cytogenetics (B) and RT-PCR (C). The y-axes indicate the percentage of residual disease by flow cytometry in samples collected during treatment. In (C), white dots indicate samples with leukaemia transcripts corresponding to < 0·1% residual disease.

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Of the 230 samples studied by flow cytometry, 103 (from 35 patients) were also analysed for the presence of leukaemic karyotypic abnormalities established at diagnosis (Fig 3B). These were the t(8;21) in six patients, inv(16) in five patients, t(9;11) in five patients, other 11q23 translocations in four patients and other abnormalities in 15 patients. Of the 103 follow-up samples studied, 94 had no detectable leukaemic karyotypes, but 18 of these had leukaemic cells detectable by flow cytometry (Fig 3B, left). The remaining nine of the 103 samples had abnormal karyotypes similar to those determined at diagnosis, and eight also had residual leukaemic cells detectable by flow cytometry (Fig 3B, right). In contrast, one sample, collected after window therapy from a patient with AML-M4e, did not. In this sample, inv(16) was noted in two out of 20 metaphases. There were no AML blast cells in the sample by morphology, and molecular studies were not done. The reason for the discrepancy between flow cytometry and cytogenetics in this sample remains unclear.

Of the 230 samples studied by flow cytometry, 95 were analysed for the presence of leukaemia-associated transcripts identified at diagnosis (Fig 3C). These 95 samples were from 20 patients with the following molecular abnormalities established at diagnosis: AML1-ETO (n = 6), CBFB-MYH11 (n = 4), MLL-AF9 (n = 6) and MLL-AF10 (n = 4). In 89 samples, leukaemic transcripts were either undetectable (n = 70) or yielded weak signals corresponding to estimated levels of residual disease < 0·1% (n = 19). However, 10 of these 89 samples had ≥ 0·1% residual disease by flow cytometry (Fig 3C, left). In the remaining six of the 95 samples, transcripts indicating residual disease ≥ 0·1% were detected. Three of these samples also had residual cells detectable by flow cytometry, whereas three samples (collected from two patients with AML1-ETO) did not (Fig 3C, right). These samples had 0·1–1% estimated levels of residual disease by RT-PCR. Considering that quantification of residual disease by the RT-PCR method used in this study may lack accuracy, we speculate that levels of residual disease in these samples may have been lower than 0·1%.

Relationship of flow cytometric detection of residual disease to presenting features and to treatment outcome

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Prevalence of aberrant AML immunophenotypes and sensitivity of residual disease studies
  6. Relationship between measurements of residual disease by flow cytometry versus morphology, cytogenetics and RT-PCR
  7. Relationship of flow cytometric detection of residual disease to presenting features and to treatment outcome
  8. Prognostic significance of flow cytometric detection of residual disease
  9. Discussion
  10. Acknowledgments
  11. References

Table II summarizes the detection of residual disease determined by flow cytometry after induction 1 and induction 2, according to presenting features. There was no significant correlation between residual disease detected by flow cytometry at each time point and gender, age, leucocyte count or FAB subtype. Among the various karyotypic and molecular subtypes examined, patients with core binding factor-AML, i.e. those with t(8;21)/AML1-ETO or inv(16)/CBFB-MYH11, had a particularly good early response to treatment. After window therapy, residual disease was detectable in only two out of 11 such patients compared with 18 of the 30 patients with other karyotypes (P = 0·033). After induction 1, residual disease was detected in one out of 11 of the former group and in 16 out of 33 of the latter group (P = 0·031).

Table II.  Residual disease after remission induction chemotherapy in childhood AML according to presenting clinical and cellular features.
Presenting featureResidual disease after induction 1Residual disease after induction 2
++
  1. FAB, French–American–British classification; RAEBt, refractory anaemia with excess blasts in transformation.

Gender
 Female14 8154
 Male13 9116
Age (years)
 < 1 3 0 30
 1–914 6135
 ≥ 101011105
Leucocyte count
 ≤ 50 × 109/l1812187
 > 50 × 109/l 9 5 83
FAB
 M1 7 3 72
 M2 6 3 71
 M4 5 5 53
 M4e 3 0 20
 M5 5 3 52
 M7 1 2 02
 RAEBt 0 1 00
Karyotype
 t(8;21) 5 1 51
 inv(16) 5 0 40
 t(9;11) 4 3 42
 11q23 3 1 20
 Other1012117

A summary of the sequential results of flow cytometric assessment of residual disease in the 46 patients monitored and their outcome is presented in Fig 4. In 29 patients, all assays performed during therapy, after completion of induction 1, were negative (Fig 4A). Nineteen of these patients were alive and in complete remission at the time of this analysis, and three others had died while in remission. The other seven patients had relapsed; in four out of the seven, relapse was preceded by the appearance of cells with aberrant immunophenotypes while in morphological remission. In the remaining 17 patients, flow cytometry detected residual AML cells after induction 1 and in subsequent assays (Fig 4B). Five of these patients relapsed, one died of infection with persistent disease, three had refractory leukaemia, and three patients died in remission. Five patients in this group were still in remission at the time of this analysis, three of whom had a follow-up of less than 1 year. Of the remaining two patients, one (no. 31, Fig 4B) was diagnosed with AML M2 and t(8;21), she had persistent residual disease by flow cytometry during treatment, a result that was consistently corroborated by RT-PCR. This patient is in clinical remission 24 months after diagnosis; flow cytometry was consistently negative off therapy, but positive signals were detectable by RT-PCR off therapy and at the time of this report. The other patient (no. 30; Fig 4B) has Down's syndrome and was diagnosed with AML M7; she is still in remission 46 months from diagnosis. The relationship between cells with aberrant immunophenotypes and the course of the disease in this patient is unclear, but it could be speculated that the phenotypically aberrant cells detected lacked clonogenic potential.

image

Figure 4. Summary of sequential results of residual disease studies by flow cytometry in the 46 patients with leukaemia-associated immunophenotypes at diagnosis and their outcome. (A) Patients with negative flow cytometric assays during therapy, after completion of induction 1. (B) Patients with detectable residual disease during therapy, after completion of induction 1. Ind., induction; Con, consolidation; mts., months; CR, complete remission.

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Prognostic significance of flow cytometric detection of residual disease

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Prevalence of aberrant AML immunophenotypes and sensitivity of residual disease studies
  6. Relationship between measurements of residual disease by flow cytometry versus morphology, cytogenetics and RT-PCR
  7. Relationship of flow cytometric detection of residual disease to presenting features and to treatment outcome
  8. Prognostic significance of flow cytometric detection of residual disease
  9. Discussion
  10. Acknowledgments
  11. References

We determined the prognostic significance of residual disease detected in the bone marrow samples taken immediately after remission induction therapy. The mean (± SE) 2-year overall survival estimate was 33·1 ± 19·1% for patients who had residual disease ≥ 0·1% in the after induction 1 sample and 72·1 ± 11·5% for those who had fewer or no detectable leukaemic cells (P = 0·022). Even after three patients who had morphologically detectable leukaemic cells were excluded, the difference in outcome remained significant: 30·0 ± 17·7% versus 72·1 ± 11·5% (P = 0·044; Fig 5). Similarly, significant differences were observed for after induction 2 samples: the 2-year survival estimate was 31·7 ± 18·5% for patients with ≥ 0·1% residual disease-positive cells (all of whom were in morphological remission) and 80·0 ± 10·3% for patients with fewer or no detectable leukaemic cells (P = 0·048). A further subdivision of patients according to levels of residual disease, i.e. ≥ 1% versus < 1%, was not informative in this series.

image

Figure 5. Probability of overall survival according to residual disease detected by flow cytometry after induction 1. All patients included in this analysis were in morphological remission.

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The multiple Cox regression method was used simultaneously to account for the effects of age at diagnosis (treated as a continuous variable), residual disease status by flow cytometry, leucocyte count at diagnosis and karyotype on overall survival (Table III). Among patients tested by flow cytometry after induction 1, after adjusting for covariates, leucocyte count and karyotype were not found to be significantly associated with overall survival. Older patients were more likely to die: a 1-year increase in age increased the hazard rate by a factor of 1·15 (P = 0·019). Patients in remission with detectable AML cells by flow cytometry were 3·79 times more likely to die than those with undetectable disease (P = 0·037). Similar observations were made for tests after induction 2: patients with detectable AML cells by flow cytometry while in remission were 6·15 times more likely to die than those with undetectable disease (P = 0·028).

Table III.  Effect of presenting features and flow cytometric detection of AML cells on overall survival by multiple Cox regression analysis.
Risk factor*After induction 1After induction 2
UnivariateMultivariateUnivariateMultivariate
P-valueP-valueHazard ratioP-valueP-valueHazard ratio
  • *

    Patients who were in morphological remission and were tested for residual disease by flow cytometry after induction 1 (n = 41) and after induction 2 (n = 36) were included in the analysis.

Age0·0280·0191·150·0750·0281·17
(continuous variable)
Leucocyte count0·3140·7431·210·2450·7451·26
(> 50 × 109/l vs.
≤ 50 × 109/l)
Karyotype0·0540·1612·390·1480·3062·08
(unfavourable
vs. favourable)
Residual disease0·0290·0373·790·0350·0226·15
(present vs. absent)

To determine the relationship between detection of residual disease by flow cytometry and relapse of AML, we analysed the leukaemia-free survival of only those patients who were in morphological remission at the time of the test and who then either relapsed or continued on the study for at least 6 months. Patients who had residual disease ≥ 0·1% by flow cytometry after induction 1 or induction 2 had a significantly higher rate of relapse within 6 months after the test than patients with lower levels or undetectable disease. Of the nine patients included in this analysis who had residual disease ≥ 0·1% after induction 1, four had a morphological relapse within the subsequent 6-months, whereas none of the 22 patients with a residual disease < 0·1% or a negative test experienced relapse (P = 0·004). Three of the six patients with residual disease ≥ 0·1% but only one of the 21 with < 0·1% after induction 2 had relapses within 6 months (P = 0·022).

In 10 patients, immunophenotypic studies were also performed at relapse. At least one of the leukaemia-associated immunophenotypes determined at diagnosis and used to monitor residual disease was retained. However, in one of the 10 patients, two out of three immunophenotypic abnormalities seen at diagnosis were not detectable at relapse and, in another two patients, one of three abnormalities disappeared, underscoring the benefit of using multiple marker combinations.

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Prevalence of aberrant AML immunophenotypes and sensitivity of residual disease studies
  6. Relationship between measurements of residual disease by flow cytometry versus morphology, cytogenetics and RT-PCR
  7. Relationship of flow cytometric detection of residual disease to presenting features and to treatment outcome
  8. Prognostic significance of flow cytometric detection of residual disease
  9. Discussion
  10. Acknowledgments
  11. References

The main purpose of this study was to establish and validate a method to monitor residual disease in children with AML. We found that immunophenotypes suitable for the detection of residual disease can be identified by four-colour flow cytometry in 85% of children and that such immunophenotypes allow a sensitivity of detection as much as 100 times that afforded by morphological examination. Unlike ALL, AML offers few suitable targets for PCR-based studies of residual disease. For example, Boeckx et al (2002) found immunoglobulin and/or T-cell receptor gene rearrangements (a common target for residual disease studies in ALL) in only nine out of 95 AML cases studied. The same authors found AML1-ETO, CBFB-MYH11, PML-RARA, BCR-ABL or MLL-AF4 fusion transcripts associated with non-random karyotypic abnormalities in 17 out of 105 AML cases (Boeckx et al, 2002). Studies of a potentially more widely applicable molecular marker, Wilm's tumour gene (WT1), are somewhat contradictory (Inoue et al, 1996; Gaiger et al, 1998), and its usefulness awaits confirmation.

Because of its wide applicability, flow cytometry is a promising tool to monitor residual disease in AML (Campana, 2003). However, residual disease studies in AML present some specific technical difficulties, which may affect the sensitivity of residual disease detection (Campana & Coustan-Smith, 2002). San Miguel et al (2001) and Venditti et al (2000) identified immunophenotypes that allowed detection of AML cells with a sensitivity of 0·01% in 75% and 68% of patients respectively. Although we found aberrant phenotypes in 85% of patients, a sensitivity of 0·01% could be reliably achieved in only 48% of cases; in the remaining 37%, a level of 0·1% was the maximum sensitivity that could be reliably achieved, after comparing the phenotype of leukaemia cells with a large number of normal and regenerating bone marrow samples. Therefore, we used the 0·1% threshold for our studies of residual disease in follow-up samples. It is noteworthy that, in our hands, the same methodological approach used in this study can consistently achieve a sensitivity of 0·01% in more than 95% of children with ALL. The difference in sensitivity between our study and that of the adult series may be ascribed to age-related differences in phenotypic make-up, consistent with the different genetic composition of AML in adults and children (Mrozek et al, 2001). Another possibility is that, in children, normal haematopoietic cells regenerating after chemotherapy express immunophenotypes that are rarely seen in adults, thus increasing normal background and decreasing sensitivity.

In our series, flow cytometric detection of AML cells after induction therapy was an independent predictor of treatment outcome. These results are consistent with those reported in adult AML patients (Venditti et al, 2000; San Miguel et al, 2001) and in a recently reported study of children with AML (Sievers et al, 2003). Perhaps because of the limited sensitivity of detection in the latter study (0·5%), the proportion of patients with responsive disease (defined by the authors as those with < 30% blasts after one course of therapy) found to have occult disease was only 13%. In contrast, the proportion of patients with no identifiable leukaemic cells by morphology who had residual disease by flow cytometry in our series was 34·1% and 27·8% after induction 1 and induction 2 respectively.

After remission induction therapy, the current therapeutic options for children with AML comprise additional intensive chemotherapy, antibody therapy or allogeneic or autologous haematopoietic cell transplantation (Sievers et al, 1999; Burnett et al, 2000; Woods et al, 2001; Arceci, 2002). The method described here should provide a more rational means of selecting therapeutic options, and a more accurate estimate of the extent of leukaemic cytoreduction achieved with different therapies. Thus, residual disease assessment with this method is now used for risk assignment in our recently initiated multicentre AML2002 study.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Prevalence of aberrant AML immunophenotypes and sensitivity of residual disease studies
  6. Relationship between measurements of residual disease by flow cytometry versus morphology, cytogenetics and RT-PCR
  7. Relationship of flow cytometric detection of residual disease to presenting features and to treatment outcome
  8. Prognostic significance of flow cytometric detection of residual disease
  9. Discussion
  10. Acknowledgments
  11. References

We thank Peixin Liu and Mo Mehrpooya for assistance with flow cytometric studies, Zhe Zhang for statistical analysis, and James Boyett for statistical advice. This work was supported by grants CA60419 and CA21765 from the National Cancer Institute and by the American Lebanese Syrian Associated Charities (ALSAC). C.-H. Pui is supported by the American Cancer Society F.M. Kirby Clinical Research Professorship.

References

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Prevalence of aberrant AML immunophenotypes and sensitivity of residual disease studies
  6. Relationship between measurements of residual disease by flow cytometry versus morphology, cytogenetics and RT-PCR
  7. Relationship of flow cytometric detection of residual disease to presenting features and to treatment outcome
  8. Prognostic significance of flow cytometric detection of residual disease
  9. Discussion
  10. Acknowledgments
  11. References
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