1. No category

From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
1993 82: 712-715
Persistence of the 8;21 translocation in patients with acute myeloid
leukemia type M2 in long-term remission
G Nucifora, RA Larson and JD Rowley
Updated information and services can be found at:
http://www.bloodjournal.org/content/82/3/712.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
RAPID COMMUNICATION
Persistence of the 8;21 Translocation in Patients With Acute Myeloid
Leukemia Type M2 in Long-Term Remission
By Giuseppina Nucifora, Richard A. Larson, and Janet D. Rowley
The translocation between chromosomes 8 and 21,
t(8;21) (q22;q22), is the most frequent abnormality in
acute myeloid leukemia (AML) with French-American-British type M2 (FAB-MP) morphology. The breakpoints in this
translocation have been characterized at the molecular
level, and the genes involved areAML 1on chromosome 21
and €TO on chromosome 8. The rearrangement of the two
chromosomes results in a fusion gene and in the production of a consistent fusion transcript on the der(8) chromosome. We have used oligonucleotide primers derived from
both sides of the fusion cDNA junction and reverse transcription-polymerase chain reaction (RT-PCR) to analyze
six AML-M2 patients with a t(8;21) during various stages
of their disease. Two patients studied at diagnosis and one
studied at first relapse are alive off therapy and in
continuous complete remission for 8 3 t o 94 months. We
have detected the AMLIETO fusion transcript in recent peripheral blood samples from each of them. Three other patients also had a fusion transcript detected after l to 4
months in remission. Two of these patients subsequently
relapsed and died whereas the third patient is alive and in
continuous complete remission 70 months later. Thus, our
preliminary data suggest that cells with the translocation
are still circulating in t(8;21) patients in long-term remission. This finding raises serious questions regarding the
interpretation of positive results obtained only with this
technique that may not be suitable to decide appropriate
further treatment for patients in clinical remission.
0 1993 by The American Society o f Hematology.
0
DNA rearrangements can be detected by Southern blot analysis in 80% to 100% of the patient^.^.^ More importantly,
appropriate primers designed from the chimeric cDNA sequence from both sides of the junction can detect an identical size chimeric transcript by reverse transcriptase-PCR
(RT-PCR) in all of the patients tested.6 We used RT-PCR to
analyze peripheral blood (PB) samples recently obtained
from patients who had been diagnosed with AML-M2 and
the t(8;21) and treated as long ago as 8 years. Our analysis
shows that the chimeric transcript is still detectable in these
samples, indicating that cells with the 8;2 1 chromosomal
translocation are still present in patients with AML-M2 in
long-term remission.
NE OF THE central tenets of the management of patients with acute leukemia is that the cytogenetically
abnormal leukemic clone, present at diagnosis, disappears
during remission. Its reappearance is often used to confirm
a relapse. This view has now been called into question by
our ability to detect a very small number of abnormal cells,
in the range of 1 in IO4 or lo5,with the use ofthe polymerase
chain reaction (PCR). This technique is especially powerful
when used to detect the product of fusion genes that result
from chromosome translocations.
The 8;2 1 translocation with breaks at 8q22 and 21q22.3 is
a recumng translocation observed in approximately 20% of
patients with acute myeloid leukemia type M2 (AMLM2).’s2This translocation results in the fusion oftwo genes,
AMLl on chromosome 21, and E T 0 on chromosome 8,
with the formation of a chimeric gene AMLl/ETO on the
~
cloning of
derivative 8 [der(8)] c h r o m ~ s o m e .Molecular
the 8;2 1 translocation has provided genomic probes from
either side of the breakpoint junction; in several studies,
~
~~~
From the Section of Hematology/Oncology, Department of Medicine, and the Cancer Research Center, The University of Chicago,
Chicago, IL.
Submitted April 12, 1993; accepted April 29, 1993.
Supported by Department of Energy Grant No. DE-FGO286ER60408 (J.D.R.), National Institutes of Health Grant Nos. CA
42557 (J.D.R.) and CA40046 (R.A.L. and J.D.R.), andspastic Paralysis Research Foundation, Illinois-Eastern Iowa District Kiwanis International (J.D.R.). G.N. was supported by National Institutes of Health training Grant No. ST32CAO9273-14.
Address reprint requests to Janet Rowley, MD. Section of Hematology/Oncology, Department of Medicine, 5841 S Maryland Ave.
MC2115, Chicago, IL 60637.
The publication costs of this article were defiayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-49 71/93/8203-0044$3.00/0
712
MATERIALS
AND METHODS
Patients’ and normal control samples. PB samples were recently obtained with informed consent from three patients in longterm remission (patient nos. 4, 5, and 6, Table I). The red blood
cells (RBCs) were separated by centrifugation, and the leukocytes
were removed and used for RT-PCR analysis. Although bone
marrow (BM) samples were not evaluated, there was no clinical
evidence of recurrent disease in these patients. In addition, we had
frozen leukocytes from BM samples taken at diagnosis and in early
remission for these patients, for another patient in long-term remission (no. 3, Table l), and for two other t(8;21) AML patients who
later relapsed (patient nos. 1 and 2, Table 1). The karyotypes ofthe
leukemia cells at diagnosis or at relapse of all of the patients analyzed have been reported previously.6The cell line Kasumi-l established from a patient with AML-M2 and the t(8;Zl) was used as a
positive contr01.~The TK-6 (American Type Culture Collection,
Rockville, MD) and the M L 1 cell lines and PB samples from two
individuals who did not have AML were used as negative controls.
These cells do not contain the t(8;21). The cell lines were grown in
RPMI 1640 with 10%fetal bovine serum FBS and were maintained
under standard conditions.
RNA preparation. PolyA RNA was prepared from 0.8 to 3 X
IO7 PB leukocytes or BM cells or cell lines with the Fast Track
mRNA isolation kit (Invitrogen, San Diego, CA) according to the
manufacturer’s instructions. All of the polyA RNA isolated (0.1 to
1 pg) was used for the synthesis of the first-strand cDNA necessary
6/0od, Vol82, No 3 (August 1). 1993: pp 7 12-715
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
713
PERSISTENCE OF t(8;21) IN LONG-TERM REMISSION
Table 1. Clinical Characteristics of Patients With AML-M2 and the t(8;21), and Results of the Cytogenetics,
Southern Blots, and PCR Analyses
Diagnosis
% of t(8;21) cells'
Southern blot/PCR
Source
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
07/05/85
42
n.d.1n.d.
BM
04/03/85
100
i1n.d.t
BM
1 i/o5/a 1
94
n.d.1n.d.
BM
05119/83
94
n.d./n.d.
BM
10/22/85
100
t / n .d.t
BM
12/04/a4
100
+/n .d.t
BM
Months in CR1
% of t(8;2 1) cells"
Southern blot/PCR
Source
4
12
1
9
0
0
0
0
n.d./+
BM
-1n.d.t
BM
n.d./+
BM
n.d./t
BM
84
n.d.
n.d./t
PB
94
n.d.
n.d./t
PB
Alive, NED
88+
Alive, NED
99+
Months in CR1
% of t(8;21) cells.
Southern blot/PCR
Source
1211 1/85
5
-1n.d.t
0M
05/29/87
91
+/n.d.t
BM
First relapse
% of t(8;2 1) cells.
Southern blot/PCR
Source
1 1/04/86
50
+/n.d.t
BM
Months in CR2
% of t(8;2 1) cells'
Southern blot/PCR
Source
1
1
1
0
-/+t
0
0
-/+
BM
BM
n.d./+
BM
Months in CR2
% of t(8;21) cells'
Southern blot/PCR
Source
09/30/85
38
+/+t
BM
81
n.d.
n.d./+
PB
Second relapse
% of t(8;21) cells'
Southern blot/PCR
Source
07/28/88
25
+/tt
BM
Status
Follow-up (mos in CR)
Died
Died
Alive, NED
70t
Alive, NED
85+
Abbreviations: NED, no evidence of disease; n.d., not determined; CR1, first complete remission; CR2, second complete remission.
Cells analyzed by cytogenetics.
t Data from reference 6.
for PCR amplification. The synthesis was performed with the
cDNA First Strand Synthesis kit (Pharmacia LKB Biotechnology,
Piscataway, NJ) in a total reaction volume of 15 pL.
PCR amplification and Southern blot analysis. For each PCR
amplification, 4 pL of the cDNA reaction volume was used.
Primers I and I1 and the conditions for the PCR reactionshave been
described.6The amplification produces a DNA fragment of 339
bp.6 The probe used for the Southern blot analysis of the electrophoresed PCR reactions was a 0.77-kb fragment obtained by PCR
amplification of an AMLI/ETO cDNA as previously described.6
RESULTS
Clinical analysis. All patients had received intensive remission induction and consolidation chemotherapy,
usually with cytarabine and an anthracycline andfor with
high-dosecytarabine regimens. No maintenancechemotherapy was administered. One of the patients (no. 5) received a
syngeneic BM transplant. Four of the patients (nos. 1
through 4) had a relapse. Patient 2 was studied by PCR after
4 months in first clinical and cytogenetic remission (CRl),
and the patient relapsed 3 months after the marrow sample
was taken. Patients 1 and 3 were studied by PCR only in
second remission and patient 4 only at first relapse and second remission. In none of the remission marrow samples
were metaphase cells with the t(8;2 1) observed. Four of the
patients (nos. 3 through 6 ) currently remain in complete
remission more than 5 to 8 years after last chemotherapy.
Analysis of the PCR amplification reactions. We had
previously analyzed leukemia samples from all of the patients at diagnosis or relapse and detected either a rearrangement in the AMLl gene by Southern blot analysis or a fusion transcript by RT-PCR.6 Using RT-PCR, we analyzed
all ofthe early and long-term remission samples. We consistently observed a faint band of 339 bp in ethidium bromidestained gels representing the chimeric AMLI/ETO mRNA
junction only in samples from the six patients and from the
Kasumi-1 cell line but not from the TK-6 and M G l cell
lines or the control individuals (not shown). Figure 1A
shows the autoradiogram of the Southern blot for the six
patients during early remission. Figure 1B shows the results
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
NUCIFORA, LARSON, AND ROWLEY
714
B
A
1
2
3
4
5
6
7
8
1
2
3
4
5
6
7
Fig 1. (A) Autoradiogram of the PCR-amplified products from the cell line Kasumi-1, carrying the t(8;21) (lane 1). the t(8;21) patients
early in hematologic and cytogenetic remission (patients 1 to 6, lanes 2 to 7). and the myeloid cell line ML-1, (lane 8). The karyotypes of the
patients at diagnosis have been described:' corresponding numbers of patients 1 to 6 in reference 6 are: patients 5,9, 12, 14, 16, and 20.
(B)Autoradiogram of the PCR-amplified products from the cell line Kasumi-1, carrying the t(8;21) (lane 1). the PB of the patients in
long-term remissions (patients 4. 5 . and 6: lanes 2, 3, and 4). the cell line TK-6 (lane 5 ) . and two control individuals (lanes 6 and 7). The
probe used was a 0.77-kb chimeric fragment obtained from a t(8;21) chimeric c D N A . ~
from recent PB samples for 3 of the 4 long-term patients
(nos. 4, 5, and 6); we were not able to obtain a recent blood
sample from patient 3.
DISCUSSION
We have used RT-PCR with primers mapping on both
sides of the A M L l / E T O chimeric cDNA junction to detect
the fusion transcript in PB samples obtained recently from
three patients who were diagnosed and successfully treated
for AML-M2 with the t(8;21) more than 6 to 8 years ago
(patients 4 through 6, Table I). Our results show that these
patients still have circulating leukocytes with the t(8;2 1)
producing the chimeric AMLI/ETO transcript. We studied
samples from two of these patients at initial diagnosis and
during remission (nos. 5 and 6) and one of them during first
relapse and subsequent remission (no. 4). All ofthe patients
at diagnosis or relapse had DNA rearrangements on standard Southern blot analysis, and they amplified the same
size chimeric AMLI/ETO mRNA junction on RT-PCR
during early or long-term remission. So far we have only
studied three patients who had sequential samples from
diagnosis or relapse through long-term remission, and thus
we cannot extrapolate our results to all patients. We have
studied one additional patient after 1 month and 4.5 years
in remission whose cells had the t(8;2 1) at diagnosis. Several
analyses on this patient's early remission BM and more recent blood sample failed to detect an amplified chimeric
message on RT-PCR. We did not have any sample of this
patient at diagnosis to analyze for the presence of the chi-
meric transcript, but it is possible that some long-term survivors may actually be cured.
The t(8;21) is a recumng chromosomal abnormality that
is closely associated with a characteristic morphologic subtype of AML and a favorable response to chemotherapy.'-''
The great majority of these patients (in some series 100%)
will achieve complete hematologic remission, and the translocation is then no longer detectable in BM metaphase cells
by cytogenetic methods. After additional remission-consolidation chemotherapy, these remissions are generally durable, and relapses after more than 2 years in remission are
uncommon. Indeed, AML-M2 with t(8;21) is generally felt
to be among the most "favorable" subtypes of AML because of its predictably high rate of cure (about 60%).Characteristically, the arrest in maturation ofgranulocytic differentiation is not complete in this subtype of leukemia, and at
diagnosis it is likely that many circulating mature granulocytes are in fact the progeny of the neoplastic clone. It is
remarkable that even after 8 years in clinical remission (and
in one case after BM transplantation), cells that appear to be
derived from the leukemic lineage continue to circulate in
the blood.
The role of molecular methods of detection of subclinical
leukemia is currently being explored. PCR methods that
can amplify a unique nucleotide sequence from I in IOs
cells can detect residual disease with a sensitivity that far
exceeds standard light microscopy or routine cytogenetics.
The t(8;21) AML is not the only malignancy in which minimal residual disease has been detected by PCR methods
during long-term remission."*'* As in these other cases, the
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
PERSISTENCE OF t(8;21) IN LONG-TERM REMISSION
biologic and clinical significance of the presence of cells
carrying specific abnormalities associated with cancer are
still unknown. The expression of the chimeric protein could
represent only one of the modifications necessary for the
development of cancer and leukemia, and the affected clone
would require additional mutations to express the transformed phenotype. Alternatively, it is possible that these
t(8;2 1) cells are in fact completely transformed, but that cell
proliferation is repressed by an unknown mechanism. However, as illustrated by our patients who have remained in
continuous remission for over 5 years, further cytotoxic chemotherapy or BM transplantation is not an appropriate response to a positive test result. Longer follow-up is required
to gain the appropriate perspective on our relatively
disquieting observations. However, they suggest that a new
paradigm regarding the implications of translocation-carrying cells may be required.
ACKNOWLEDGMENT
We thank Dr Michelle M. LeBeau for cytogenetic analysis and
Karen M. Daly for assistance in obtaining these patients’ samples.
REFERENCES
I . Mitelman F, Heim S. Quantitative acute leukemia cytogenetics. Genes Chromosom Cancer 5 5 7 , 1992
2. Fourth International Workshop on Chromosomes in Leukemia, 1982. Cancer Genet Cytogenet 11:249, 1984
3. Erickson P, Gao J, Chang K-S, Look T, Whisenant E, Raimondi S, Lasher R, Trujillo J, Rowley JD, Drabkin H: Identification of breakpoints in t(8;21) AML and isolation of a fusion transcript with similarity to Drosophila segmentation gene runt. Blood
80: 1825, 1992
715
4. Miyoshi H, Shimizu K, Kozu T, Maseki N, Kaneko Y, Ohki
M: The t(8;21) breakpoints on chromosome 21 in acute myeloid
leukemia clustered within a limited region of a novel gene, AMLI.
Proc Natl Acad Sci USA 88:10431, 1991
5 . Tighe J, Daga A, Calabi F: Translocation breakpoints are clustered on both chromosome 8 and chromosome 2 I in the t(8;2 1) of
acute myeloid leukemia. Blood 81592, 1993
6. Nucifora G, Birn D, Erickson P, Ciao J, Le Beau MM, Drabkin HA, Rowley JD: Detection of DNA rearrangements in the
AMLI and E T 0 loci and of an AMLI/ETO fusion mRNA in patients with t(8;21) AML. Blood 81:888, 1993
7. Asou H, Tashiro S, Hamamoto K, Otsuji A, Kita K, Kamada
N: Establishment of a human acute myeloid leukemia cell line (Kasumi-1) with 8;21 chromosome translocation. Blood 77:203 1,199 1
8. Bitter MA, LeBeau MM, Rowley JD, Larson RA, Golomb
HM, Vardiman JW: Association between morphology, karyotype,
and clinical features in myeloid leukemias. Hum Path01 18:211,
1987
9. Mitelman F, Heim S: Cancer Cytogenetics. New York, NY,
Liss, 1987
10. Swirsky DM, Li YS, Matthews JG, Flemans RJ, Rees JKH,
Hayhoe FGJ: 8;2 I translocation in acute granulocytic leukaemia:
Cytological, cytochemical and clinical features. Br J Haematol
56:199, 1984
11. Lange W, Snyder DS, Castro R, Rossi JJ, Blume KG: Detection by enzymatic amplification of bcr-ab1 mRNA in peripheral
blood and bone marrow cells of patients with chronic myelogenous
leukemia. Blood 73:1735, 1989
12. Pignon JM, Henni T, Amselem S, Vidaud M, Duquesnoy P,
Vernant JP, Kuentz M, Cordonnier C, Rochant H, Goossens M:
Frequent detection of minimal residual disease by use of the polymerase chain reaction in long-term survivors after bone marrow
transplantation for chronic myelogenous leukemia. Leukemia
4:83, 1990