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Keywords:

  • AML M0;
  • clinical characteristics;
  • immunophenotype;
  • cytogenetics

Abstract

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

Haematological, immunophenotypic and cytogenetic characteristics were analysed in 241 patients with acute myeloid leukaemia (AML) M0, including 58 children. Children < 3 years and adults between 60 and 70 years of age were most frequently affected. Immunophenotyping showed a heterogeneous phenotype. Anti-myeloperoxidase was positive in about half of the patients. Cytogenetic data were available from 129 (54%) patients. A normal karyotype was found in only 24%. Most of the abnormalities were unbalanced and the chromosomes 5, 7, 8 and 11 were the most frequently affected. Survival data were available from 152 treated patients (63%). The median overall survival for all patients was 10 months, 20 months for children (n = 36), 10 months for the young adult group (n = 50) and 7 months for the elderly patients (n = 66) (P = 0·09). Karyotype was not a prognostic factor influencing survival. AML M0 shows the immunological characteristics of early progenitor cells, but the expression of the different markers and cytogenetic abnormalities is heterogeneous. The prognosis is poor compared with other de novo AML and similar to that of AML with multilineage dysplasia or AML following myelodysplastic syndromes.

In the diagnosis of acute leukaemias, the differentiation between myeloid and lymphoid blasts, based on morphological features only, has been shown to be inaccurate (Bennett & Reed, 1975). As myeloperoxidase (MPO) was regarded as an indicator for the presence of primary neutrophilic granulation (Ackerman, 1968), a positive cytochemical reaction for this enzyme became the hallmark for myeloid differentiation. Later on, this was confirmed by ultrastructural (Bessis & Maigné, 1970) and immunophenotypic (van der Schoot et al, 1990) studies, the two latter techniques being more sensitive than light microscopy cytochemistry. According to these techniques, some undifferentiated leukaemia's could be assigned to the myeloid lineage (Marie et al, 1982; Lee et al, 1987; Imamura & Kuramoto, 1988; Le Maistre et al, 1988; Matutes et al, 1988; Campos et al, 1989; Campana et al, 1990; van Wering et al, 1990). In addition, culture techniques have been used for differentiation (Lemež, 1985). The French–American–British (FAB) Cooperative Group classified leukaemias with a negative cytochemical MPO reaction at the light microscopic level but expressing one or more myeloid markers and/or ultrastructural MPO and absence of expression of lymphoid markers as AML M0 (Bennett et al, 1991), a term used for the first time by Galton et al (1975). More recently, the World Health Organization (WHO) grouped these minimally differentiated acute myeloid leukaemias in their classification of neoplastic diseases of the haematopoietic and lymphoid tissue under the heading of AMLs not otherwise categorized (Harris et al, 1999).

AML M0 is a rare disease comprising about 5% of AML in adults (Cuneo et al, 1995; Segeren et al, 1995). This subtype has also been reported to occur in infancy and childhood, but is also rare (Creutzig et al, 1995; Huang et al, 1999). Several reports have described the morphological, immunological and cytogenetic features of this subtype of AML (Sempere et al, 1993; Stasi et al, 1994; Venditti et al, 1994, 1997; Creutzig et al, 1995; Cuneo et al, 1995; Segeren et al, 1995; Cohen et al, 1998; Villamor et al, 1998; Costello et al, 1999; Huang et al, 1999; Mori et al 2000). However, analysis of the literature shows that these papers include only a relatively small number of patients and the findings are controversial with respect to the expression of myeloid and non-myeloid antigens and the prognostic significance of cytogenetic abnormalities. Overall, there is agreement to regard AML M0 as having a poor prognosis.

To date, there has been no comprehensive study of AML M0. Here, we describe the analysis of the clinical, cytological, immunological and cytogenetic characteristics at diagnosis of 241 patients, including 58 children, and correlate these data with the clinical outcome.

Patients and methods

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

Patient population Data from 241 patients, diagnosed between 1986 and 1999 in eight different European countries, were analysed. Morphology, immunophenotype and cytogenetics were processed and reviewed by national reference centres (France, Germany, The Netherlands) or single hospitals (Ferrara, Jihlava, London, Salamanca). The classification of AML M0 was made according to the FAB criteria: > 30% blasts in the bone marrow, < 3% of the blasts positive by light microscopy for MPO or Sudan Black-B (SBB), a positive reaction with at least one myeloid marker (Bennett et al, 1991). Expression of lymphoid markers was allowed as long as the leukaemia did not meet the criteria for acute lymphoblastic leukaemia (ALL) or biphenotypic acute leukaemia (BAL) as proposed by the European Group for the Immunological Characterization of Leukaemias (EGIL) (Bénéet al, 1995) Cytoplasmic (c) CD3- and cCD22-positive leukaemias were excluded from this analysis.

For most analyses, the patients were stratified into three age groups: 0–15, 16–60 and > 60 years. Patients that were treated underwent protocols used in the different centres and the response was recorded according to local or protocol registry data.

Morphological analysis Bone marrow and peripheral blood slides were stained with May–Grünwald–Giemsa and by cytochemical reactions for MPO, SBB and alpha-naphtyl acetate esterase with and without sodium fluoride inhibition.

Immunophenotyping Flow cytometric analysis of bone marrow and/or the peripheral blood cells was performed with an extensive panel of monoclonal antibodies according to (national) protocols currently in use in the different centres. The reactivity with the following markers was analysed: CD34, TdT, HLA DR and CD7, referred to as early precursor markers; anti MPO, CD11c, CD13, CD14, CD15, CD33, CD65s and CD117, referred to as myeloid markers; CD2, CD3, CD4, CD5, CD8, CD10, CD19 and CD22, referred to as lymphoid markers; the natural killer (NK) cell-associated markers CD16 and CD56; the platelet-associated antigens CD41 and CD61, and glycophorin-A as an erythroid marker. A case was considered to be positive for a marker if ≥ 20% of the blasts were positive, except for CD34 and TdT for which the threshold was 10%, according to EGIL guidelines (Bénéet al, 1995). In this analysis, we scored leukaemias positive for anti-MPO if > 10% of the blasts showed expression, and in another analysis, analogous to the FAB criteria, if > 3% of the blasts were positive.

Cytogenetic analysis Cytogenetic analysis was performed by local or national reference centres. Chromosomal abnormalities were described according to the International System for Human Cytogenetic Nomenclature (Mitelman, 1995). Complex karyotypes were defined by the presence of three or more events in the same clone.

Statistical analysis Patient characteristics in the three age groups were compared using Pearson's chi-squared test in case of discrete variables or using the Kruskal–Wallis test in case of continuous variables. Overall survival was measured from diagnosis until death from any cause. Patients still alive at the date of last contact were censored at the last follow-up date. Overall survival was estimated using the Kaplan–Meier method. The following variables were included as prognostic factors for survival: age, sex, haematological parameters (logarithms of leucocyte, blast and platelet count), cytogenetics and immunological markers (negative versus positive). Univariate survival analysis was performed using the log-rank test to see whether there was a difference in survival between subgroups, and univariate Cox regression was used to determine whether the relationship was monotonous. All reported P-values are two-sided and a significance level α = 0·05 was used.

Results

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

Patients

This study comprises 241 patients with the diagnosis AML M0. For the presentation of the data, they were stratified into three groups according to age: children (0–15 years) (n = 58), young adults (16–60 years) (n = 79) and elderly patients (> 60 years) (n = 104). The age distribution of the whole group is shown in Fig 1, which shows that AML M0 affects all ages, but the highest frequency is seen in infants (0–3 years) and in adults between 60 and 70 years.

image

Figure 1. Age distribution in 241 AML M0 patients at diagnosis. The number of patients is given in 5 year groups and the insert shows the distribution for ages 0–5 years.

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The main clinical characteristics for the three age groups are shown in Table I. There was a female predominance in children (M/F ratio 0·87), while it was more common in young adults and elderly males (M/F ratio 1·6 for both groups). Organomegaly (enlarged lymph nodes, spleen or liver) at presentation was seen more frequently in children than in adults.

Table I.  Clinical and haematological characteristics of 241 patients with AML M0.
 0–15 years16–60 years> 60 yearsAll
  1. Hb, haemoglobin; WBC, white blood cell count; PB, peripheral blood; BM, bone marrow. The median values are given with the range in brackets.

n5879104241
Male/Female27/3149/3063/41 
Hb (g/dl)8·98·88·58·8
(4·0–16·9)(4·3–14·9)(1·4–15·6)(1·4–16·9)
WBC × 109/l23·86·49·89·8
(0·6–436)(0·2–350)(0·5–294)(0·2–436)
Platelets × 109/l50825160
(11–417)(7–400)(2–1210)(2–1210)
% blasts (PB)29214837
(0–93)(0–97)(0–99)(0–99)
% blasts (BM)80808280
(58–90)(33–98)(30–100)(30–100)
Lymph nodes45%12%13%22%
Spleen50%15%15%24%
Liver54%35%17%33%

Haematological values

Laboratory values are given in Table I. The haemoglobin level was not different between the three age groups. A significantly higher white blood cell count was found in children than in adults (young adults plus elderly patients) (P = 0·01). The platelet count was not different between children and adults. The proportion of bone marrow blasts was high in all age groups with very reduced haemopoietic reserve, which made assessment of dysplastic features difficult. The cytochemical reaction for MPO or SBB in the bone marrow blasts was negative in 75% and positive (but in less than 3% blasts) in 25% of the patients.

Immunophenotyping

The results are summarized in Fig 2 for each of the three age groups. The number of patients evaluable for each marker is indicated. Anti-MPO was positive in 55% of the cases, if the cut-off point for positivity was ≥ 3%. If the cut-off point was set to ≥ 10%, the marker was positive in 19% of the children and in 40% of the adults. Expression of CD34 and HLA DR, markers associated with early precursors, was high in the three age groups. CD117, TdT and CD7, other markers associated with early precursor cells, were found to be positive in 55%, 46% and 41% of the cases respectively. Expression of CD117, a marker preferably expressed on immature myeloid leukaemic cells (Bénéet al, 1998), was positive in the absence of the myeloid markers CD13 and/or CD33 in 5 out of 177 (3%) of the leukaemia cases. In the children's group, CD13 was less often expressed than CD33 (56% versus 79%), both markers being positive in 42% of patients, CD13 alone in 14% and CD33 alone in 37%. In the young adults and elderly patients, expression of CD13 and CD33 was seen in 83% and 73%, respectively, co-expression of both markers in 61%, CD13 alone in 23% and CD33 alone in 13%. The myeloid marker CD65s was less often positive: 39% in children, 33% in the young adults group and 22% in the elderly. CD15 was rarely tested in the children and was positive in 25% of the adults. CD14 was consistently negative in the children's group and was positive in 6% of the adults. CD11c was not tested in children and was positive in 50% of the adults. The NK-cell marker CD56 was positive in 11 out of 21 (52%) children, in 9 out of 28 (32%) adults and in 1 out of 31 (3%) elderly patients. CD16 was only positive in 2 out of 67 patients. The erythroid marker glycophorin-A was not tested in the children's group, but was positive in 3% of the 68 adults tested. Out of lymphoid-associated but not specific markers, CD2 was positive in 35% of children and in 16% of adults, CD4 in 35% of children and in 26% of adults, and CD19 in 19% of children and in 8% of adults. Other lymphoid markers were rarely expressed as follows: CD5 was positive in 4% of adults, CD10 in 5% of adults and CD8 was negative.

image

Figure 2. Immunological marker expression in AML M0. The percentages of cases positive for a marker and the total number tested per marker are stratified for children, young adults and elderly patients. For anti-MPO, the percentages are given with a cut-off point of 10% and 3% positive blasts.

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Special attention was paid in cases that expressed lymphoid-associated markers to exclude the diagnosis of BAL by applying the scoring system for the definition of BAL (Bénéet al, 1995). In this system MPO scores two points, CD13, CD33, CD65s and CD117 score one point each, and CD14 and CD15 score 0·5 points each for myeloid differentiation; BAL is defined as a myeloid acute leukaemia scoring over two points for lymphatic marker expression and a lymphatic acute leukaemia scoring over two points for myeloid marker expression. Although not all leukaemias were tested for all these myeloid markers, CD19-positive leukaemias scored more than two points for myeloid expression in 87% of cases. As cCD22 (positivity was a reason for exclusion from this study and counts for two points of the B lineage) was not tested in 15 of the CD19-positive (one point of the B lineage) leukaemias with more than two myeloid points (n = 24), the diagnosis of BAL could not ruled out in these cases. Of these CD19-positive and CD22 not tested cases, eight were TdT (0·5 points) positive. CD10-, CD2- or CD5-positive leukaemias scored more than two myeloid points in 100%, 84% and 100% of the cases respectively.

Cytogenetic analysis

Data on cytogenetic analysis were available in 129 patients: 30 children, 49 young adult patients and 50 elderly patients. Because there were no statistically significant differences in frequencies of chromosomal abnormalities between the three groups data are given for the whole group unless otherwise indicated. A normal karyotype was found in 31 patients (24%), complex abnormalities were detected in 23 patients (18%) and a nearly tetraploid karyotype was present in eight patients (6%). The remaining 67 (52%) patients had one or two clonal abnormalities. Unbalanced aberrations were the most common and chromosomes 5, 7, 8 and 11 were the most frequently affected, respectively, in 23 (21%), 21 (21%), 17 (14%) and 19 (16%) patients. The most common abnormalities were −5 or 5q–, −7 and +8, but they rarely occurred as a single event. Chromosome 11 abnormalities included seven patients, three children and four adults, with 11q23 translocations being the sole abnormality in two of them. Chromosome 13 abnormalities were detected in 11 (9%) patients, mainly as trisomy 13. The most common balanced abnormality was t(9;22), found in eight patients. In six of these patients it was the sole event and in one patient it was part of a complex karyotype. As three of the eight t(9;22)-positive patients were not tested for CD22, but were positive for CD19 and TdT, BAL cannot be excluded in these cases.

Clinical outcome

Data on survival from diagnosis were available in 152 treated patients, which included 36 children, 50 young adults and 66 elderly patients. The median overall survival for all patients was 10 months, with differences according to age: 20 months for children, 10 months for young adult patients and 7 months for the elderly (P = 0·09). The survival curves are shown in Fig 3. These curves held true throughout the period of study. A higher leucocyte count as a single variable was correlated with a shorter survival (P = 0·02). A higher platelet count was associated with a longer survival time (P < 0·01). There was no association between leucocyte count and platelet count. Expression of the immunological markers tested showed no correlation with prognosis except for CD10 and CD14, both associated with a shorter survival (P < 0·01), and CD15 (P < 0·05). However, as the number of cases tested for these markers were small and cytogenetic data were not available in all these cases, no definite conclusions can be made. The impact of cytogenetic abnormalities on overall survival from diagnosis is shown in Fig 4. There were no differences in survival between patients with normal or abnormal karyotypes or between the different groups of abnormalities (P > 0·6). Of the 143 treated patients from whom a response and follow-up was available, 48% reached complete remission (CR): children 61% (n = 33), young adult group 55% (n = 49) and elderly 34% (n = 61) (P = 0·03). Data about myeloablative therapy followed by stem cell rescue, either allogeneic or autologous, were insufficient and not reliable for analysis.

image

Figure 3. Overall survival from diagnosis and age 0–15 years, 16–60 years and > 60 years.

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image

Figure 4. Overall survival from diagnosis and cytogenetic analysis in AML M0: normal karyotype, complex (≥ 3) clonal abnormalities, nearly tetraploid karyotype and one or two clonal abnormalities.

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Discussion

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

The diagnosis

As the diagnosis of AML M0 is made by the lack of MPO or SBB expression at the light microscopy level and ultrastructural analysis is by no means practical, the immunophenotype is essential. The most specific myeloid marker is MPO, but this marker, except for one study (Venditti et al, 1997) which documented expression in 100% of the patients, was positive in no more than half of our cases, even if the cut-off point for positivity was set very low. The use of CD13 and CD33 is helpful as, in our experience, these markers are expressed in about 80% of the patients. However, because they are not strictly myeloid-specific, diagnosis based on the reactivity with these markers poses problems with myeloid antigen-positive ALL and BAL. Because immunological criteria are defined for the latter types of acute leukaemias (Bénéet al, 1995), we also included cases who had expression of lymphoid markers as long as they were not strictly lymphoid-specific and ruling out the diagnosis of ALL or BAL in our series of AML M0.

The immature nature of the blasts in AML M0 was confirmed by the high incidence of expression of markers associated with haematopoietic progenitor cells, such as CD34, HLA DR and CD117. The latter marker recognizes an epitope of the c-kit protein, a ligand for the stem cell factor and also highly associated with early myeloid differentiation and more specific than CD13 or CD33 for the myeloid lineage (Bene et al, 1998). As a more recent addition to the diagnostic panel, CD117 was tested in a lower number of patients. In a previous study, we found 70% of positive cases in 106 patients. The lower percentage of CD117-positive patients in this study could partly be explained by a lower expression in the elderly group, which is more strongly represented. There were subtle differences in the expression of CD13 and CD33 between children and adults, CD13 being expressed in a lower frequency in children than in adults.

The patients

This largest and most comprehensive study in AML M0 patients confirmed that, in adults, the highest incidence of this AML was in patients aged 60–70 years (Cuneo et al, 1995; Segeren et al, 1995), but it is also seen in children. In children under 16 years a prevalence was seen in 0–3-year-old infants. This was in agreement with a study restricted to nine children with AML M0 (Huang et al, 1999). In adults, males are more frequently affected than females, while there is a predominance for girls in children.

The morphology

In nearly all patients, the bone marrow was heavily infiltrated by blasts. Maturation along the myeloid lineage was absent and the number of non-myeloid cells markedly decreased. This makes assessment of the presence of dysplastic features difficult. Multilineage dysplasia in AML M0, however, has been described (Cuneo et al, 1995; Lemežet al, 1998), which may suggest a stem cell dysfunction in AML M0.

The cytogenetics

A high incidence of chromosomal aberrations were very common in AML M0, mainly unbalanced and similar to those seen in myelodysplastic syndromes or acute leukaemias secondary to alkylating agents. This may suggest a similar causative process with stem cells or very early progenitor cells as a target. The incidence of complex and nearly tetraploid karyotypes was high, indicating that the nearly tetraploid karyotype is preferentially seen in AML M0 compared with other AML subtypes (Lemežet al, 1998). The same holds for the presence of t(9;22), the Philadelphia chromosome, with or without additional abnormalities, in acute leukaemias fulfilling the FAB criteria for AML (Paietta et al, 1998).

The clinical outcome

Reports on therapy response and clinical outcome in AML M0 are limited and concerned only small numbers of patients. Conventional chemotherapy, albeit heterogeneous in the different studies and mainly AML-like, has yielded a low response rate and a high incidence of relapse (Creutzig et al, 1995; Cuneo et al, 1995; Segeren et al, 1995; Huang et al, 1999). In our series, we have shown that age, leucocyte count and platelet count were predictive prognostic factors in treated patients. No conclusions can be drawn regarding the prognostic impact of immunological markers, as the antigens that appear to influence survival, CD14, CD10 and CD15, were positive and/or tested in only a relatively small number of patients. CD14 as an adverse prognostic factor has been described in other subtypes of AML (Bénéet al, 1999). In contrast to findings reported in other subtypes of AML, chromosomal aberrations in general or a specific abnormality in particular were not found to be an independent factor for prognosis, although all aberrations found are associated overall with a poor prognosis in AML (Bitter et al, 1987). A normal karyotype in our series of AML M0 was not associated with a more favourable clinical outcome. This suggests that the subtype AML M0, as defined by morphological and immunophenotypic criteria per se confers a poor prognosis. Most patients were treated only with chemotherapy in conventional doses. From our data, it is unclear how far stem cell transplantation may contribute to a longer remission and prolongation of the survival.

The reason for the poor prognosis of AML M0 is unclear at present, but several factors may be responsible for this. First, this minimally differentiated leukaemia shows many features of stem cells and such leukaemias are relatively insensitive to chemotherapy (Visser & van Bekkum, 1990). Second, the p-170, a protein that mediates multidrug resistance and which is expressed in normal stem cells, is frequently positive in AML M0 blasts (Campos et al, 1992; Wuchter et al, 1999). Further, the high incidence of unfavourable chromosomal abnormalities may account for a poor prognosis. Although we did not find that an abnormal karyotype was a prognostic factor, the high incidence found may indicate that other more subtle abnormalities or mutations that may possibly be revealed using other techniques, such as fluorescence in situ hybridization (FISH) and DNA sequencing, are critical for these secondary changes and are also present in hitherto normal karyotypes. Escape from immune recognition by poor antigen presentation and a low expression of co-stimulatory molecules have also been suggested as unfavourable factors in controlling minimal disease in AML M0 (Costello et al, 1999).

The classification

Our findings indicate that AML M0 defined by morphology, cytochemistry and immunophenotype is heterogeneous in marker expression and cytogenetic abnormalities. In the recent WHO classification of neoplastic diseases of the haematopoietic and lymphoid tissues (Harris et al, 1999), AML M0 is included under the heading AML not otherwise categorized as AML minimally differentiated. Minimal myeloid differentiation, also in the cytochemical MPO-negative cases, is suggested by the expression of the specific myeloid marker MPO in a substantial number of cases using immunological or ultrastructural techniques, but early differentiation into other lineages such as erythroid cannot be excluded, as early differentiation antigens, specific for the erythroid lineage, are lacking. The cytogenetic abnormalities in this subtype of AML are similar to those in other stem cell disorders such as BAL, secondary leukaemias and myelodysplastic syndromes, and it has a common relative insensitivity to chemotherapy. Future biological studies may reveal causative mechanisms underlying these characteristics and lead to a more appropriate classification and the development of more adequate treatment strategies.

Acknowledgment

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

We thank Professor F. Schmalzl for the helpful discussion on cytochemistry.

References

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