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Flow Cytometric Immunophenotypic Characterization of Pediatric and Adult
Minimally Differentiated Acute Myeloid Leukemia (AML-M0)
Article in American Journal of Clinical Pathology · March 2000
DOI: 10.1309/2FYJ-00BE-R8N0-HMPQ · Source: PubMed
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Hematopathology / PEDIATRIC AND ADULT AML-M0
Flow Cytometric Immunophenotypic
Characterization of Pediatric and Adult Minimally
Differentiated Acute Myeloid Leukemia (AML-M0)
Patricia K. Kotylo, MD,1 In-Sook Seo, MD,1 Franklin O. Smith, MD,1
Nyla A. Heerema, PhD,1 Naomi S. Fineberg, PhD,1 Kathy Miller, MD,1
Marianne E. Greene, MD,2 Pauline Chou, MD,2 and Attilio Orazi, MD3
Key Words: Minimally differentiated acute myeloid leukemia; Pediatric; Adult; Immunophenotyping
Abstract
We reviewed the clinicopathologic and
immunophenotypic profiles of 7 pediatric and 11 adult
minimally differentiated acute myelogenous leukemias
(AML-M0). We also compared and evaluated
myeloperoxidase in leukemic blasts using standard
cytochemical and polyclonal antibody immunohistochemical stains. No distinctive clinical findings were
noted in either patient group; however, thrombocytopenia typically was more prominent in adults. Adult
AML-M0 also was associated with an immature
myeloid profile (CD34+, terminal deoxynucleotidyl
transferase positive, CD13+, and CD33+), in contrast
with pediatric AML-M0, which usually lacked terminal
deoxynucleotidyl transferase or CD34 but expressed
bright CD33 with weak or negative CD13.
Coexpression of the T-cell–associated antigen CD7 was
observed in both groups. Antibody immunohistochemical stains were more sensitive than cytochemical
stains for detection of myeloperoxidase activity and a
useful adjunct for establishing a diagnosis of myeloid
leukemia in paraffin-embedded marrow tissues.
Minimally differentiated acute myeloid leukemia (AMLM0) is a rare form of acute myeloid leukemia constituting
approximately 2% to 3% of acute myelogenous leukemias.
The 1991 French-American-British (FAB) group criteria for
AML-M0 include1 less than 3% blasts that are positive for
Sudan black B (SBB), myeloperoxidase, or both and expression of myeloid associated markers, such as CD13 or CD33,
with absence of lymphoid antigens. AML-M0 blasts have
been described as immature and agranular and frequently
lack morphologic features of a specific cell lineage.2 Confirmation of the myeloid origin of AML-M0 may be accomplished with immunophenotyping and immunologic or
ultrastructural peroxidase studies.
The FAB study group acknowledged that some lymphoid
associated antigens, such as CD2 or CD7, may be expressed
in a proportion of AML-M0.1 However, no study to date has
evaluated and compared complete immunophenotypic profiles
in pediatric and adult AML-M0. Previous studies also lack
correlation of routine cytochemical and immunoperoxidase
stains for myeloperoxidase with flow cytometric immunophenotyping in well-characterized AML-M0.
We reviewed and compared the immunophenotypic and
cytogenetic profiles of 7 pediatric and 11 adult cases of
AML-M0. We also evaluated standard cytochemical
myeloperoxidase stains of marrow aspirate smears and an
immunohistochemical stain for myeloperoxidase (aMPX)
applied to the bone marrow samples in these same cases.
Materials and Methods
We reviewed 243 cases of adult AML at University
Hospital (Indianapolis, IN) during a 5-year period and found
© American Society of Clinical Pathologists
Am J Clin Pathol 2000;113:193-200 193
Kotylo et al / PEDIATRIC AND ADULT AML-M0
❚Table 1❚
Clinicopathologic Findings at Diagnosis for Patients With Acute Myeloid Leukemia (M-0)*
Case No./
Sex/Age
Adult
1/M/80
2/M/84
3/M/58
4/M/55
5/F/58
6/F/31
7/M/60
8/M/71
9/M/60
10/M/63
11/M/79
Pediatric
1/M/14
2/M/12
3/M/6 mo
4/F/6 mo
5/F/8 mo
6/F/2 mo
7/M/15
WBC Count
× 109/L/
(×
Hemoglobin
% Blasts)
(g/L)
Platelet
Count
× 109/L)
(×
Bone
Marrow/
% Blasts
Cytochemical
Stains
aMPX (%)
Cytogenetics
7.9/60
6.7/14
35.6/75
19.3/70
1.0/2
9.5/54
9.4/42
1.8/1
18.6/50
100
74
95
86
102
92
96
90
83
19
48
52
16.7
157
72
20
52
65
50
95
100
100
60
90
95
80
90
–
–
–
–
–
–
2% MPX+
–
2% SBB+
2
–
3
ND
7
10
3
ND
15
–Y,del(6)
Normal chromosomes
Normal chromosomes
Normal chromosomes
+8
Multiple anomalies
Dicentric (7;20)(p11;p11)
+8
+13
38.3/79
1.2/24
66
95
58
59
95
71
PAS+
PAS+
10
–
+13
+13
19.6/43
105
476
65
–
10
+13
1.9/2
19.9/30
6.5/1
10.0/3
40.0/40
28.6/87
62
103
74
62
82
54
155
204
211
364
50
53
94
92
62
32
60
85
PAS+
3% MPX+
–
–
–
PAS+
ND
ND
–
2
–
–
Normal
Multiple anomalies
t(10;11)(p11.2;q23)
No metaphases present†
inv(X);(p11.4q24)
t(11,19)(q23;p13.3)
Previous
Disease
No
No
No
No
No
No
No
No
Hodgkin
disease,
stage IA
No
Prostate
cancer
Selective IgA
deficiency
No
No
No
No
No
No
aMPX, antibody immunohistochemical stains; AWOD, alive without disease; AWD, alive with disease; CNS, central nervous system; DOD, died of disease; MPX,
myeloperoxidase; ND, not done; PAS, periodic acid–Schiff; SBB, Sudan black B; +, positive; –, negative.
* Age is given in years unless otherwise stated. For all patients, bone marrow examination revealed hypercellularity. Conversions to traditional units of measure are as follows:
× 103/µL).
WBC count, divide by 0.001 (/µL); hemoglobin, divide by 10 (g/dL); platelet count, divide by 1.0 (×
† Relapse at 6 months, t(10;11)(p15;q23).
11 cases (4.5%) that fulfilled the FAB criteria for AML-M0;
90 cases of pediatric AML were reviewed for a similar
period at Riley Hospital for Children (Indianapolis) and
Children’s Memorial Hospital (Chicago, IL). Seven pediatric
cases (8%) also fulfilled the FAB criteria for AML-M0.
May-Grünwald-Giemsa–stained bone marrow smears
were reviewed for all cases to confirm the original diagnosis.
A minimum of 30% marrow blasts was required for inclusion in the study. Cytochemical stains of bone marrow aspirates were reviewed and included myeloperoxidase (cMPX),
Sudan black B (SBB), alpha-naphthyl acetate esterase,
alpha-naphthyl butyrate esterase, and periodic acid–Schiff
stains (Sigma, St Louis, MO).
Bone marrow trephine biopsy specimens were fixed in
B-5 for up to 6 hours, decalcified using 10% nitric acid for 2
hours, and embedded in paraffin. Four-mm-thick sections
were dried on polylysine-coated glass slides at 37°C
overnight, deparaffinized by serial passages in xylene and
graded alcohol series, and rehydrated in distilled water. H&E
and Gomori reticulin silver stains were performed routinely
on all samples. Immunohistochemical staining of the bone
marrow biopsy specimens for myeloperoxidase was
performed as previously reported.3 Briefly, deparaffinized
slides were covered with goat serum for 20 minutes, blotted,
194
Am J Clin Pathol 2000;113:193-200
and incubated overnight at 4°C with negative control serum
and the primary antibody, an antihuman myeloperoxidase
polyclonal antibody that reacts with neutrophil precursor
cells and granulocytes 4 (Dako, Carpinteria, CA). The
primary antibody was stained with biotin-conjugated goat
antimouse antibody (30 minutes; Kirkegaard and Perry
Laboratories, Gaithersburg, MD) and then with a peroxidaseconjugated streptavidin (30 minutes; Kirkegaard and Perry).
The enzyme was developed with 3,3´-diaminobenzidine
(Sigma). Immunohistochemical results were expressed as a
percentage of positively stained cells of the total number of
blast cells evaluated on a bone marrow biopsy section with
an artifact-free area of at least 10 mm 2 , as previously
described.3
Immunophenotypic analyses were performed on fresh
bone marrow aspirates with Profile, Elite (Coulter, Miami, FL)
or FACS-Scan (Becton Dickinson, San Jose, CA) flow
cytometers. Seven milliliters of lysing reagent (NH4Cl, pH =
7.3) was added to 500 µL of bone marrow. All samples were
then stained with the following panel of dual-color monoclonal antibodies directly conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), and peridinin chlorophyll
alpha protein: CD45-FITC/CD14-PE, CD7-FITC/HLADRPE, CD33-FITC/CD34-PE, CD15-FITC/CD13-PE, CD3© American Society of Clinical Pathologists
Hematopathology / ORIGINAL ARTICLE
❚Table 1❚
(Continued)
Extramedullary
Leukemia
Remission
Length of
Follow-Up
(mo)
Outcome
Adult
No
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
1
2
2
14
42
4
4
8
6
Unknown
DOD
DOD
DOD
AWD
AWOD
AWOD
AWD
AWOD
No
No
Yes
Yes
11
7
AWD
AWOD
No
7
DOD
Yes
Yes
Yes
Yes
Yes
Yes
10
6
14
9
13
9
AWOD
AWD
DOD
AWOD
AWOD
AWD
Pediatric
No
No
Yes (CNS, skin)
Yes (chloroma of iris)
No
No
No
FITC/CD16+56-PE, CD2-FITC/CD13-PE, CD11bFITC/CD11c-PE, CD10-FITC/CD19-PE, CD41-PE, CD61FITC, glycophorin-PE (Coulter Immunology, Hialeah, FL),
CD19-PE/kappa-FITC, CD19-PE/lambda-FITC (Kallestad,
Austin, TX), and terminal deoxynucleotidyl transferase (TdTFITC, Caltag Laboratories, Burlingame, CA). Monoclonal
antibodies were dispensed according to the manufacturer’s
recommendations or determined with titration for 5 × 105
cells. Leukemic blasts were evaluated for antigen expression
using 2 different gating strategies, including forward-angle
light scatter vs side scatter in 13 of 18 cases and multiparameter analysis (CD45 vs side scatter) in the remaining cases.
Peridinin chlorophyll alpha protein–conjugated CD45 was
used with dual-color antibodies for multiparameter analysis.5
A marker was considered positive when 20% or more of
the gated blast population stained with an antibody relative
to an appropriate isotype control.6 The intensity of antigen
brightness also was assessed in all cases by review of the
histogram data.
Cytogenetic analysis was performed on direct and 24hour unstimulated bone marrow cultures using a trypsinGiemsa banding procedure. Results were reported using the
International System for Human Cytogenetic Nomenclature.7 A minimum of 20 analyzed cells was required for a
© American Society of Clinical Pathologists
diagnosis of normal chromosomes. Clinical outcome and
follow-up data were obtained through chart reviews and
physician interviews.
Comparisons between immunophenotypic profiles were
analyzed by using the Fisher exact test. Comparisons
between continuous variables, such as WBC counts and
platelets, used the Mann-Whitney U test since the data were
not distributed normally.8
Results
Of 333 cases reviewed, 11 adult and 7 pediatric acute
myeloid leukemias fulfilled the FAB criteria for AML-M0.
Clinicopathologic findings for both groups are summarized
in ❚Table 1❚. The adults were 9 men and 2 women ranging
from 31 to 84 years of age (median, 60 years). Two adults
had secondary AML after previous therapy for malignant
neoplasms. One patient was treated successfully for stage IA
Hodgkin disease with chemotherapy and radiation therapy
10 years before the current diagnosis; the second was treated
surgically for prostatic carcinoma without chemotherapy 1
year before the diagnosis of leukemia. The remaining 9
adults had de novo AML. There were 4 boys and 3 girls in
the pediatric group; 4 of 7 were infants younger than 8
months of age. All children had de novo AML.
WBC counts at diagnosis for adults varied from 1,000 to
38,800/µL (1.0-38.8 × 10 9 /L; median, 9,400/µL [9.4 ×
109/L]), in contrast with higher WBC counts observed in the
pediatric group (median, 19,600/µL [19.6 × 109/L]). Blasts
were observed in the peripheral blood specimens of all
patients at the time of diagnosis. All patients demonstrated
mild to moderate anemia; thrombocytopenia was variable
and more prominent in adults (median, 52 × 103/µL [52 ×
109/L]) than in children (median, 204 × 103 /µL [204 ×
109/L]; P = .020). Leukemic blasts in pediatric and adult
cases of AML-M0 usually were medium to large with scant
agranular cytoplasm. Nuclei were round to oval with fine
chromatin and prominent nucleoli ❚Image 1❚.
Routine cytochemical stains for myeloperoxidase and
SBB demonstrated less than 3% positivity in leukemic
blasts in all cases. Alpha-naphthyl butyrate esterase and
alpha-naphthyl acetate esterase stains also were negative; 2
pediatric and 2 adult cases demonstrated focal globular
periodic acid–Schiff positivity (Table 1). Immunohistochemical aMPX stains were performed on fixed bone
marrow particle sections or biopsy specimens in 9 adults
and 5 children (Table 1). The aMPX staining was more
sensitive than cMPX in both patient groups. Seven of 9
adult cases demonstrated positive aMPX staining in
leukemic blasts varying from 2% to 15%; aMPX staining in
2 of 5 pediatric cases was 2% and 10%, respectively. In 5
Am J Clin Pathol 2000;113:193-200
195
Kotylo et al / PEDIATRIC AND ADULT AML-M0
❚Table 2❚
Immunophenotypic Findings for Patients With
Acute Myeloid Leukemia (M0)*
Case No.
❚Image 1❚ (Case 11) Bone marrow aspirate of adult with
acute myeloid leukemia (M-0). Leukemic blasts with
prominent nucleoli and scant cytoplasm with minimal
granulation (Wright-Giemsa, ×1,000).
adults and 2 children in whom cMPX stains were entirely
negative, aMPX staining was observed.
Immunophenotypic profiles are summarized in ❚Table 2❚.
Statistically significant differences were observed in CD34
(P = .003) and TdT (P = .011) expression in pediatric vs
adult AML-M0. Adult AML-M0 was characterized by an
immature myeloid profile and frequently expressed CD34
and TdT in combination with the myeloid antigens CD33,
CD13, or both. Blasts in adult cases demonstrated bright
expression of CD34 (11/11), CD13 (10/11), CD33 (8/11),
and HLA-DR (10/11); CD33 and CD13 were expressed in
7 cases. Nuclear TdT was positive in 6 of 10 cases and was
weak in contrast with prominent expression of CD34 and
myeloid antigens. Four of 11 adult AML-M0 samples also
were positive for CD7. The CD7 activity observed in 3 of
these cases was less prominent than myeloid antigen
expression; the fourth case displayed equal intensity of
CD7 and myeloid antigens on leukemic blasts. The latter
case (case 6), a CD7+ adult case of AML-M0, which was
cMPX negative but aMPX positive, was evaluated with
multiparameter analysis to exclude lymphocytes from the
analysis. CD15 (5/9) and CD11b or CD11c (6) were
expressed in adult cases of AML-M0 and were of variable
intensity.
In contrast with the adult group, leukemic blasts of pediatric cases of AML-M0 typically displayed a more mature
myeloid phenotypic profile ❚ Figure 1❚. TdT studies were
negative in all 6 pediatric cases of AML-M0 evaluated for
196
Am J Clin Pathol 2000;113:193-200
Adult
1
2
3
4
5
6
7
8
9
10
11
Pediatric
1
2
3
4
5
6
7
Gate†
CD45
HLA-DR
TdT
CD34
CD13
1
1
1
1
1
2
1
2
1
1
2
98
80
74
93
95
99
98
73
94
93
73
86
81
81
91
12
30
54
87
82
37
96
6
63
47
7
87
4
52
2
ND
15
56
84
88
71
43
69
98
89
92
92
92
95
68
65
62
38
32
81
13
81
82
24
65
1
1
1
2
2
1
1
95
82
98
98
72
96
97
62
82
97
88
90
6
91
4
1
ND
2
8
2
2
81
1
1
86
2
2
1
50
6
1
25
48
45
37
Cyto µ, cytoplasmic mu heavy chain; ND, not done; SIg, surface immunoglobulin;
TdT, terminal deoxynucleotidyl transferase.
* Data are given as percentage of positivity.
† 1 indicates forward angle light scatter vs side scatter; 2, CD45 vs side scatter.
this marker; CD34 activity was present in only 2 cases.
Bright CD33 positivity was observed in all pediatric AMLM0 samples and was associated with negative or relatively
dim CD13 in 5 of 7 cases. Weak CD7 coexpression was
observed in 4 pediatric cases of AML-M0, 1 of which also
was aMPX positive. CD15 was positive in 5 of 6 cases;
CD11b and CD11c were positive in 2 of 3 cases tested for
these antigens.
All pediatric and adult cases of AML-M0 tested were
negative for CD3, CD14, CD19, CD20, CD10, CD41/CD61
and surface immunoglobulin expression. One pediatric and 1
adult case of AML-M0 displayed CD16/56 activity.
Cytogenetic findings are summarized in Table 1. Three
adult and 1 pediatric case demonstrated trisomy 13 as the
sole abnormality. Trisomy 8 was present in 2 adults. An
11q23 breakpoint was present in 2 children at diagnosis. A
third infant (case 5), whose cytogenetic testing was unsuccessful at diagnosis, had an 11q23 breakpoint at relapse 6
months later. Only 2 of all cases of AML-M0 had multiple
cytogenetic abnormalities.
Clinical outcomes are summarized in Table 1. None of
the adult cases demonstrated extramedullary leukemic infiltrates during the course of disease. One adult refused treatment and was lost to follow-up 1 month after diagnosis. The
remaining 10 patients were treated with different multiagent
chemotherapy protocols. Two adults did not achieve remission and died of disease 2 months after diagnosis; a third
achieved remission but experienced relapse 6 months later
© American Society of Clinical Pathologists
Hematopathology / ORIGINAL ARTICLE
❚Table 2❚
(Continued)
CD2
CD3
CD14
CD15
13
5
10
50
15
68
96
6
18
10
53
18
6
10
12
17
ND
35
1
15
9
2
12
3
8
5
12
ND
6
1
4
5
3
7
6
1
4
2
1
2
1
6
4
2
7
54
32
64
16
17
40
25
ND
ND
3
7
14
15
47
4
77
86
37
9
ND
ND
14
34
22
80
15
41
29
50
ND
ND
ND
ND
1
2
2
ND
ND
3
1
ND
60
5
2
1
5
1
1
2
2
1
3
1
1
ND
ND
ND
ND
ND
1
1
1
ND
ND
2
3
5
8
1
1
ND
1
1
ND
3
ND
ND
ND
ND
1
ND
1
1
1
1
ND
1
2
2
5
4
3
1
7
1
4
2
1
4/2
9/1
6/ND
7/ND
3/ND
11/ND
1/2
1/1
ND/1
ND
19/ND
25
14
60
3
19
37
42
27
1
ND
ND
1
13
38
5
1
1
ND
1
6
4
5
4
1
1
2
4
1
ND
5
65
83
38
73
77
ND
ND
ND
ND
57
8
50
ND
ND
ND
ND
79
3
74
2
77
1
ND
1
2
1
1
1
1
1
1
2
1
15
1
1
3
6
2
2
3
1
1
ND
1
5
2
1
1
ND
1
1
1
1
5
1
11
5
2
5
15
3/5
1/1
1/1
7/ND
1/1
2/3
1/1
A 64
50
A
Cyto µ
CD20
SIg
CD19
CD41/61
D 50
C 50
Count
B
90 LS
CD11b CD11c CD16/56 CD10
Count
Adult
6
72
23
93
10
98
83
45
53
60
7
Pediatric
77
85
93
84
78
94
58
CD7
Count
CD33
B
0
.1
1,000
CD45
B
0
.1
1,000
CD45
.1
CD34
1,000
CD33
1,000
0
0
0
.1
H 24
G 50
Count
A
CD13
1,000
Count
F 50
90 LS
.1
CD34
E 64
0
0
1,000
Count
0
.1
.1
CD13
1,000
.1
1,000
CD33
❚Figure 1❚ Multiparameter histogram flow cytometric analysis. A-D, Adult acute myeloid leukemia (AML M-0). E-H, Pediatric AML
M-0. Note leukemic blasts (gate A) lack CD34 in the pediatric case, in contrast to CD34+ in the adult case. LS, light scatter.
and died of disease 14 months after diagnosis. Seven other
patients achieved remission and are alive with (n = 3) or
without (n = 4) recurrent disease at follow-up times ranging
from 4 to 42 months.
Limited follow-up for pediatric cases of AML-M0
ranged from 6 to 14 months. Two patients had
extramedullary disease at diagnosis. All were treated with
multiagent chemotherapy protocols; 1 received a cord blood
transplant (case 6). One patient did not achieve an initial
complete remission and died of disease 7 months after diagnosis. The remaining 6 pediatric patients achieved an initial
© American Society of Clinical Pathologists
complete remission, 5 of whom are alive with or without
recurrent disease.
Both patient groups included males and females; no
specific gender-associated clinicopathologic features were
observed in the present study.
Discussion
Flow cytometric immunophenotyping and/or electron
microscopic demonstration of the presence of myeloperoxidase
Am J Clin Pathol 2000;113:193-200
197
Kotylo et al / PEDIATRIC AND ADULT AML-M0
often are necessary to confirm the true myeloid nature of
AML-M0 and distinguish it from a primitive leukemia of
lymphoid origin. Unfortunately, electron microscopic or
other ultrastructural studies frequently are labor intensive,
cumbersome, and not readily available at many institutions.
Immunophenotyping and cytochemical or immunocytochemical stains are used more frequently for determination
of cell lineage.9,10 The present study is the first to include
evaluation and comparison of the clinicopathologic features
of pediatric and adult cases of AML-M0 in males and
females. It also is the first report, to the best of our knowledge, to compare standard cytochemical with polyclonal
antibody immunocytochemical myeloperoxidase stains in a
series of well-characterized cases of AML-M0.
The first report of a series of AML-M01 consisted of 8
adults and 2 children. All cases were positive for at least 1 of
the myeloid antigens, CD13 or CD33. Five cases coexpressed CD7, while nuclear TdT was positive in 1 of 4 tested
for this antigen. Ultrastructural myeloperoxidase studies
were helpful for confirming myeloid lineage in selected
cases. Pediatric phenotypic profiles were not distinguished
separately and described in that report.1
Sempere et al11 studied antigen expression by using
fluorescent microscopy in 11 adult cases of AML-M0 (9
men, 2 women). All cases expressed CD13 or CD33; CD34
was noted in 3 of 4 cases tested and TdT in 1 of 7. Coexpression of the lymphoid markers, including CD4 and CD7, also
was observed in several cases. These authors concluded that
these T-cell antigens were not lineage restricted and could be
observed on early myeloid cells.
Another study12 characterized 25 cases of adult AMLM0 in 13 women and 12 men. These authors also compared
standard cMPX stains with monoclonal aMPX tissue stains.
They reported all aspirates as cMPX and SBB negative (<3%
positivity), but the corresponding methanol-fixed cytospin
aspirate materials were aMPX positive in all cases. Five cases
also were reported as “faintly” butyrate positive at diagnosis.
Flow cytometric immunophenotyping of AML-M0 samples
demonstrated a “stem cell” pattern, with TdT and CD34 positivity frequently observed in combination with CD13 or
CD33. aMPX positivity was useful for confirming myeloid
lineage in 4 cases that were entirely negative for myeloid antigens. Eighty percent of the cases of AML-M0 in that study12
coexpressed at least 1 of the lymphoid-lineage associated
antigens, including TdT, CD7, CD4, CD2, CD5, CD10, and
CD19. Cytogenetic studies revealed complex karyotypes in
53% of patients with AML-M0, with frequent abnormalities
of chromosomes 5, 7, 8, and 13. A poor outcome was
reported in these cases of AML-M0 compared with other
FAB myeloid subtypes.
A recent report13 of AML-M0 consisted of 17 adults,
including 10 men and 7 women. These investigators noted a
198
Am J Clin Pathol 2000;113:193-200
similar primitive phenotypic profile and observed TdT and
CD34 expression in all cases tested. Positivity for CD13,
CD33, or both also was observed in all 17 samples, frequently
in association with lymphoid markers, such as CD7, CD19,
CD20, and CD22. These lymphoid-associated markers were
relatively dim or weak in contrast with bright myeloid antigen
activity. Abnormal clonal karyotypes were found in 6 of 14
cases; abnormalities of chromosomes 7 and 13 were the most
common finding. The aMPX stains of 12 bone marrow aspirates or biopsy specimens demonstrated a lower incidence of
peroxidase activity than in previously described reports, with
significant staining observed in only 2 of 12 samples tested.
An isolated study14 including 3 pediatric cases of AMLM0 described weak or negative TdT and CD34 antigen
activity. However, most AML-M0 studies15-18 consist of
limited numbers of adults with few or no pediatric patients.
The morphologic features of AML-M0 were similar in
both of our patient groups. Bone marrow specimens typically
were hypercellular and replaced with primitive agranular
blasts. Significant cytologic dysplasia was not observed in
peripheral blood or bone marrow samples in the present series.
The phenotypic profiles observed in adults were similar to
those noted in previous reports and consisted predominantly of
an “immature” myeloid pattern with frequent expression of
HLA-DR, CD34, and nuclear TdT in combination with
myeloid antigens CD33, CD13, or both. Weaker coexpression
of the T-cell antigens CD7 or CD2 was observed in 4 of 11
cases (36%). In contrast, the pediatric cases demonstrated a
more mature myeloid profile. All cases lacked TdT and only 2
expressed CD34; all were CD33+, while CD13 expression
was relatively less prominent or negative. Weaker coexpression of CD7 and or CD2 also was observed in 4 of 7 cases
(57%). No coexpression of other lymphoid antigens, including
CD3, CD10, CD19, CD20, or surface immunoglobulin, was
detected in any patient in our study.
Standard cytochemical stains were performed on all
cases and revealed negative or weak (<3%) myeloperoxidase
or SBB reactivity. The aMPX staining performed with a polyclonal rabbit antihuman antibody on paraffin-embedded bone
marrow particle preparation or biopsy specimen was more
sensitive than cMPX staining of bone marrow tissues. Seven
cases in our series (5 adults, 2 children) that were negative
with cMPX stains demonstrated positive staining using the
aMPX technique. These results are similar to those noted in
previous studies that have suggested immunologic stains may
be a more sensitive method for myeloperoxidase detection
than cMPX.4,19-22 The polyclonal aMPX used in the present
retrospective study may be used on fixed bone marrow material rather than methanol-fixed cytospin material. Flow cytometric studies in the present series were performed using a
whole-blood lysis technique and, in many instances, multiparameter analysis with selective gating on leukemic blasts.
© American Society of Clinical Pathologists
Hematopathology / ORIGINAL ARTICLE
Leukemic cells were stained with a consistent panel of monoclonal antibodies conjugated with similar fluorochromes
throughout the study. The combination of flow cytometric
analysis, cytochemical stains, and immunohistochemical
studies described herein was particularly useful for cases in
which only fixed bone marrow–embedded tissue was available for study or for confirming myeloid lineage in leukemias
that coexpressed various lymphoid antigens.
Cytogenetic abnormalities were detected in 8 of 11 adult
cases and 4 of 5 pediatric cases of AML-M0. One additional
pediatric case for which the cytogenetic analysis was unsuccessful at diagnosis had an abnormal karyotype at relapse. In
our series of patients with AML-M0, trisomy 13 was present in
3 older men and one 14-year-old boy; in all 4 cases, this was
the sole abnormality, which is consistent with findings of
previous reports.23,24 In contrast with previous studies that have
reported frequent complex abnormalities in AML-M0, only 2
of the present cases (1 adult and 1 pediatric) had complex
abnormalities.25 One adult and 3 pediatric cases demonstrated
breakpoints at 11q23. This breakpoint frequently is observed in
monoblastic leukemia, and its presence in these cases is consistent with previous observations that some cases of AML-M0
may, in fact, represent primitive monoblastic proliferations.
The adult group consisted primarily of men, while the
pediatric group consisted of both boys and girls. Statistically
significant clinicopathologic gender differences were not
observed in the present pilot study.
Limited clinical follow-up and a small sample preclude
analysis of overall survival in our patient groups. However,
the different phenotypic profiles observed in children and
adults in the present series suggest that AML-M0 may represent a different disease process in these patient populations.
The present study also revealed that polyclonal aMPX
staining of fixed bone marrow tissues may be useful for
confirming myeloid lineage, especially in cases with negative cMPX or lymphoid antigen coexpression. Additional
multicenter cooperative group studies26 are necessary to
determine the clinical significance of AML-M0 in children
and adults.
From the 1Indiana University School of Medicine, Indianapolis,
2Children’s Memorial Hospital, Chicago, IL, and 3College of
Physicians and Surgeons of Columbia University, New York, NY.
Address reprint requests to Dr Kotylo: Riley Hospital for
Children, Dept of Pathology and Laboratory Medicine, Room
0959, 702 N Barnhill Dr, Indianapolis, IN 46202-5200.
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