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Blood, 15 September 2002, Vol. 100, No. 6, pp. 2267-2267
CORRESPONDENCE
To the editor:
Transcription of AML1/ETO in bone marrow and cord
blood of individuals without acute myelogenous leukemia
The translocation t(8;21), AML1/ETO, represents a
frequent aberration in de novo acute myelogenous leukemia (AML) and is
detectable in up to 40% of AML FAB M2.1 In constitutively
transgenic mice, AML1/ETO abrogates fetal hematopoiesis, but in
inducible transgenic mice AML1/ETO is not leukemogenic per
se.2-4 AML1/ETO is detectable in stem cells of patients in
complete continuous remission (CCR) and only an increasing transcript
number indicates a forthcoming clinical relapse.5,6 We
investigated whether AML1/ETO transcripts are also present in bone
marrow (BM) aspirates of 18 adults (22 to 76 years old) without
neoplasia, 4 adults (25 to 76 years old) with hematopoietic neoplasia
(non-Hodgkin lymphoma [NHL], myelodysplasia syndrome [MDS]), and
156 cord blood (CB) samples from healthy newborns. The samples were
investigated by 3 independent laboratories in Goettingen, Vienna,
and Hannover. Of 22 adult bone marrow samples, 6 (27%) were AML1/ETO-positive
(Figure 1), of which 2 derived from
patients with NHL. Of all samples, 2 exhibited neoplastic cells (NHL,
MDS), of which one was AML1/ETO-positive.
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| Figure 1.
Transcription of AML1/ETO in adult bone marrow from
individuals without AML.
In the upper panel the 185-bp amplification product of the nested
reverse trancriptase (RT)-PCR is shown in the positive control
cell lineage Kasumi-1 (K) and 6 bone marrow samples of
individuals without AML followed by the primary and nested negative
controls (P, N, respectively). In the lower panel, the corresponding
internal positive controls using an abl RT-PCR are shown. We used 2%
to 3% of the total cDNA of each sample as PCR template. Goettingen
primary PCR, AML1-A 5'-ACC TCAGGTTTGTCGGTCG-3' (bp 1976 to 1974), ETO-B
5'-GAACTGGTTCTTGGAGCTCCT-3' (bp 2211 to 2231). Goettingen nested PCR,
AML1-C 5'-AAA AGCTTCACTCTGACCATCA-3' (bp 2008 to 2029), ETO-D
5'-GGCATTGTTGGAGGAGTCAG-3' (bp 2173 to 2192). Vienna primary PCR,
AML1-1 5'-AGCCATGAAGAACCAGG-3' (bp 1941 to 1960), ETO-1
5'-AGGCTGTAGGAGAATGG-3' (bp 2265 to 2281). Vienna nested PCR, AML1-2
5'-TACCACAGAGCCATCAAA-3' (bp 2062 to 2079), ETO-2
5'-GTTGTCGGTGTAAATGAA-3' (bp 2229 to 2246). Hannover real-time PCR was
performed as described.7 All nucleotide sequences refer to
Miyoshi et al.8 An abl RT-PCR served as internal positive
control and specificity of amplification was confirmed by cycle
sequencing as described.4 The sensitivity of the AML1/ETO
RT-PCRs was 10 6 (cell-in-cell dilution of Kasumi-1 in
HL60). RNA preparation, reverse transcription, and PCR were performed
in separate laboratories and in repetitive negative controls
contaminations were not observed.
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Of 156 CB samples, 63 (40%) were AML1/ETO-positive. We subjected 6 positive CB samples to real-time polymerase chain reaction (PCR) to
determine the AML1/ETO copy number. Typical normalized ratios
(AML1/ETO/housekeeping gene copies) of patients in CCR are
5 × 105 to 1 × 103, whereas patients
with newly diagnosed AML range between 0.1 and 2.14.7 The
ratios of the 6 CB samples ranged between 1.9 × 105
and 7.8 × 103, of which 3 out of 6 were comparable to
patients with CCR. Assuming similar transcriptional levels, the number
of AML1/ETO-positive cells in half of all positive healthy newborns may
resemble that of patients with AML in CCR. We postulate that positive cells are either generated by permanent
mutagenesis or are derived from aberrant hematopoietic stem cells.
Since the gene fusion AML1/ETO is prone to be induced by radiation in
vitro,9 an ongoing generation in all age groups by
external mutagens may explain our observations. In this model it seems
unlikely that AML1/ETO-positive cells have a survival advantage;
otherwise, a much higher incidence in the elderly should be expected,
though our observations may be influenced by the better cDNA quality of
the cord blood samples. On the other hand, the t(8;21) may be generated in early hematopoiesis.
Positive cells will then permanently derive from a positive stem cell
pool but only few positive individuals may attract secondary genetic
alterations and progress to AML. The latter mechanism is supported by
the recent report of Wiemels et al on the detection of AML1/ETO in
neonatal blood spots of children who developed a corresponding AML with
more than 10 years latency.10
Jorg Basecke, Lukas Cepek, Christine Mannhalter, Jurgen Krauter, Stefanie Hildenhagen, Guenter Brittinger, Lorenz Trumper, and Frank Griesinger
Correspondence: Jorg Basecke, Divison of Hematology and
Oncology, University of Goettingen, Robert-Koch-Stra e 40, D-37075
Goettingen, Germany; e-mail: jbaesec{at}gwdg.de.
Acknowledgments
J.B. is supported by a grant from the Deutsche José
Carreras Leukämie-Stiftung. We are grateful to Professor B. Wormann (Klinikum Braunschweig, Germany) for stimulating discussion and scientific support and to Midia Jlussi for technical assistance.
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