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RAPID COMMUNICATION
A PML/Retinoic Acid Receptor CY Fusion Transcript Is Constantly Detected by
RNA-Based Polymerase Chain Reaction in Acute Promyelocytic Leukemia
Sylvie Castaigne, Nicole Balitrand, Hughes de The, Anne Dejean, Laurent Degos, and Christine Chomienne
The t(15; 17) translocation is specifically observed in patients
with promyelocytic leukemia (AML3). The chromosomal rearrangement juxtaposes the retinoic acid receptor a (RARa)
and PML genes, resulting in PMLI RARa fusion transcripts.
Our previous studies have shown that a polymerase chain
reaction (PCR) amplification product could be obtained from
the cDNA of the NB4 promyelocytic cell line from which the
chimaeric PML/RARa was cloned. We report here that in all
14 AMLB patients tested, reverse transcriptase-PCR (RTPCR) allows the detection of three specific fusion products. In
eight patients, one amplificationproduct was detected corresponding to the previously described abnormal fusion. Five
patients displayed a different amplified fragment correspond-
ing to a different fusion point. One other patient always
showed a third different-sized product. The different types of
fusion transcripts amplified were correlatedto the size of the
abnormal RARa transcripts detected in these patients by
Northern analysis, but did not prove determinant for either
the phenotypic features or the retinoic acid responsiveness
in AML3 cells in this group of patients. The consistent
identificationby RT-PCR of the fusion of the PML and RARa
genes in AMLB patientssuggest that this method will provide
a useful tool for the diagnosis and detection of minimal
residual disease in these patients.
o 1992 by The American Society of Hematology.
A
chain reaction (PCR) was subsequently performed on the
cDNA of the NB4 cells, yielding a single specific amplication product of 456 bp that was not detected in the control
HL-60 samples.
To investigate whether such a PMLIRARa fusion transcript could be found in AML3 patients, we used reverse
transcriptase-PCR (RT-PCR) on the RNA of 14 AML3
patients. We report that PMLIRARa fusion transcripts
were detected in all of the AML3 patients studied. The
sizes of amplification products obtained with the primer
pairs chosen confirm that the breakpoint in the RARa gene
is within the first intron in these 14 patients and that the
breakpoint of the PML gene occurs in at least two different
loci.
CUTE PROMYELOCYTIC leukemia (AML3) is a
distinct subtype of acute nonlymphocytic leukemia
with unique clinical, cytologic, and cytogenetic features.' In
1976, Golomb et aI2 first reported the occurrence of an
abnormal chromosome 17 in two patients with AML3. This
anomaly was shown to be part of a balanced translocation
t(15q+;17q-) in which genetic material from the long arm
(4) of chromosome 17 is transferred to the long arm of
chromosome 15. The breakpoints were assigned to 15q22
and 17q21.1 or 17q11.2.12.3 t(15;17) is detected in as many
as 90% of AML34 and has never been documented in any
other subtype of leukemia. In 1988, Mattei et a15 showed
that the retinoic acid receptor a gene ( R A R c x )mapped
~ ~ ~ to
chromosome 17q21 and suggested that it could be implicated in the pathogenesis of AML3. We and others have
since shown that the RARa gene is rearranged and its
expression altered in AML3
In one AML3derived cell line, NB4,1° we have shown that, through the
t( 15;17) translocation, the RARa gene is truncated and
fused to a new gene myl, renamed PML, shown by in situ
hybridization to be located on chromosome 15." This
genetic alteration resulted in a fusion 4-kb messenger RNA
(mRNA) transcript in the NB4 cell line that cohybridized
with probes from the PML and RARa genes. Polymerase
From the Laboratoire de Biologie Cellulaire Hkmatopoiitique,
Universiti Paris WI;Service de Mkdecine Nuclkaire; Service Clinique
des Maladies du Sang, Hapita1 Saint Louis; and Inserm 163, Institut
Pasteur, Paris, France.
Submitted December 2, 1991; accepted April 9, 1992.
Supported by grants from lilssociation de la Recherche contre le
Cancer, La Ligue Nationale contre le Cancer, and a Contrat de
Recherche Clinique.
Address reprint requests to Christine Chomienne, MD, Laboratoire
de Biologie Cellulaire Hkmatopoi'ktique, Centre Hayem, Hapita1 Saint
Louis, 1 av Claude Vellafaux, Paris 75010, France.
The publication costs of this article were defrayed 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 1992 by The American Society of Hematology.
0006-49711921 7912-0038$3.00/0
3110
MATERIALS AND METHODS
Cells. Bone marrow cells were collected from 14 patients with
confirmed diagnosis of AML3 (French-American-British [FAB]
classification)I2 after informed consent and
on Ficoll
gradients. NB4 is a stable promyelocytic leukemic cell linelo
derived from a primary culture of blast M3 cells. HL60 is a myeloid
cell line derived from an M2 acute nonlymphoblastic 1e~kemia.I~
RNA isolation. RNA was isolated by the guanidium isothiocyanate-cesium chloride centrifugation method described by Chirgwin et al.I4 One microgram of RNA was used for the RT-PCR
reaction.
Amplification methods. The amplification method used was
that described by Wang et al.15 One microgram of total cellular
RNA was reverse transcribed to cDNA by an incubation of 10
minutes at room temperature, 15 minutes at 42"C, and 5 minutes at
99°C in a total volume of 20 p.L that contained 1X PCR buffer (10
mmol/L Tris-HC1, 50 mmol/L KCl), 5 mmol/L MgC12, 1 mmol/L
each dNTP (Pharmacia, Piscataway, NJ), 50 U Moloney murine
leukemia virus (M-MLV) reverse transcriptase (Perkin ElmerCetus, Nonvalk, CT),and 2.5 pmol/L of random hexamers. The
reaction volume was increased to 50 pL with additional PCR
buffer, 2.5 U of Taq Polymerase (Perkin-Elmer Cetus), and 30
pmol of each specific PML and RARa primer. Samples were
overlaid with 80 pL of light mineral oil (Sigma, St Louis, MO) and
amplification was performed in a thermocycler. Reaction times
consisted of an initial denaturation of 94°C for 3 minutes, annealing at 58°C for 1.5 minutes, and elongation at 72°C for 2 minutes,
followed by 35 additional cycles (1 minute at 94T, 1 minute at
58"C, and 1 minute at 72°C). One-fifteenth (15 ILL)of the PCR
fractionated
Blood, Vol79, No 12 (June 15). 1992: pp 3110-3115
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RT-PCR IN ACUTE PROMYELOCYTIC LEUKEMiA
3111
product was size-fractionated by electrophoresis in a 1.5% agarose
gel stained with ethidium bromide. The gels were transferred onto
a Nylon membrane by the method of Southern and were subsequently hybridized to 5’ end-labeled oligonucleotides with 32P
using polynucleotide kinase. After washing in 2X SSC, 0.1%
sodium dodecyl sulfate (SDS), autoradiography was performed
with Kodak XAR-5 film (Rochester, NY)for 12 hours. Oligonucleotides were purchased from Genset (Paris, France) and the
sequences are described in Fig 1. Sensibility and specificity of the
amplification method was tested by diluting 1 pgof total RNAofeither
NB4 cells or patient number 13 bone marrow cells with increasing
quantities of HL-60 RNA. One-tenth of the amplification product
was analyzed by transfer and hybridization with a 5‘ end-labeled
oligonucleotide specific for the fusion I and fusion I1 transcripts.
Contamination was avoided by strict technical methods. In all
experiments, two negative controls were carried through all the
steps with the other samples. Confirmation of the results was
assessed by performing the RT-PCR at least twice for each sample,
and simultaneously running known negative samples for the primer
pairs tested. Negative samples were defined as such after repeated
assays and the demonstration of integrity of isolated RNA with
actin gene amplification.
Norrhem blotring. Northern blotting and hybridization with
RARa CDNA probes was performed as already described!
RESULTS AND DISCUSSION
Total RNA from leukemic cells of 14 patients with AMW
(Table 1) were subjected to RT-PCR using PML and
RARa oligonucleotides (Fig 1). In all patients, an amplification product was detected. In eight patients (nos. 1, 2,
and 4 through 9), the size of the amplified products (456 bp)
obtained with 0 7 (PML) and 0 8 (RARa) or 0 7 (PML)
and 0 6 (RARa) oligonucleotides was identical to that
observed in the NB4 cell line. The junction of coding
sequences was the same in each case as the products
hybridized with a fusion site oligonucleotide sequence
(010) based on the previously described fusion cDNA
sequence (fusion type I)I6 (Fig 2). In one patient (patient
no. 3), the two different primer pairs amplified a 50-bp
shorter amplification product.
Patients no. 10 through 14 were repeatedly shown negative with the 0708 and 0 7 0 6 primer pairs. In these five
patients, a different size amplification product was obtained
with a PML oligonucleotide (M7) 536-bp upstream from
the fusion point type I (Fig 3). The M701 primer pair
amplified a product of 230-bp that hybridized with internal
oligonucleotides that were located within the PML and
RARa sequences (data not shown) as with an oligonucleotide based on the PMLIRARa fusion sequence (referred
to subsequently as type 11) recently described by Kakizuka
et all7 (Fig 3).
Thus, in 12 of these 14 randomly selected patients with
AMW, an amplification product of either type I or type I1
fusion transcript was obtained. Two patients showed different patterns: patient no. 3 RNA was amplified to a
smaller amplification product of 400 bp (Fig l), but which
nevertheless hybridized specifically with the fusion type I
site-specific oligonucleotide; patient no. 9 RNA displayed
both aqplification products, but only hybridized specifically
TYPE I FUSION
5’
M7
07
+
.
)
i
I
I
Fusion point
Alternative spliced
exon
RARa
01 :
5‘ CCMGClTClTGCAGCCCTCACGA 3
06:
SGGClTGTAGATGCGGGGTAG 5
08:
5’ GCTGGGCACTATCTClTCAG 3
PML
Fig 1. Schematic diagram of the ollgonucieotlde
primers located on PML/RAhfusion cDNA resulting
from the t(15;17) described in the NB4 cell line.3.15
PCR amplificationand detection oligonucleotides are
indicated.
07 :
5CAGAGAGAGTGMGGCCCAG3
M7:
5’ ATGGClTCGACGAGlTCMGG 3‘
Fusion sequences
5’ GAGGCAGCCAlTGA 3
010 :
32 :
5’ TCMTGGCTITCCC 3’
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3112
CASTAIGNE ET AL
Table 1. Clinical and Biologic Data of the 14 AML3 Patients
Patient
No.
1
SexlAge
Type of
M3*
Status
t(15; 17)
Fl23
M3
2 relapse
+
2
3
MI75
Fl73
M3v
M3v
De novo
De novo
ND
4
5
6
7
8
MI52
M/49
MI31
Fl29
Fl76
M3
M3
M3
M3
M3v
1 relapse
De novo
De novo
De novo
De novo
+
+
ND
9
10
Fl54
MI82
M3
M3v
1 relapse
De novo
+
+
11
12
13
14
MI52
MI47
MI47
F125
M3
M3v
M3
M3
De novo
1 relapse
1 relapse
De novo
+
+
+
+
+
+
+
Northern
Abnormal
Amplification
Transcripts
(kb)
Product
(bp)
1.2
4
4
1.4
2
2.5
3.5
4
ND
ND
ND
1.1
1.9
4
ND
2.9
3.6
3.8
3.6
3.6
ND
Response to RA
In Vivo
PR
450
450
400
450
450
450
450
450
450
230
230
230
230
230
In Vitro ( O h )
97
Early death
NT
100
ND
CR
CR
Early death
CR
Early death
98
83
100
60
85
CR
Early death
65
85
CR
NT
CR
CR
100
70
95
a7
In vivo response to RA: patients were treated with all-trans RA 45 mglm2/d. In vitro responseto RA: patients leukemic cells were incubated with or
without all-trans RA 1O-6mol/Lfor 5 days. Valves are the percentage of differentiated cells (treated-control).
*M3 (FAB cytologic classification).
Abbreviations: ND, not done: NT, not treated by RA: CR, complete remission; PR, partial remission.
with the fusion type I-specific oligonucleotide (Figs 2 and
3). Unfortunately, absence of available material did not
allow us to sequence these different products. Similar
results have already been reported by other groups (P.G.
Pelicci and Z. Chen, personal communications, 1991).
In one patient (patient no. 6), the bone marrow chromosome analysis, made after a 24-hour culture, showed only 2
abnormal mitosis with the t(15;17) among 31 normal metaphases; RT-PCR allowed us to identify the abnormal fusion
product (Fig 2). The level of sensitivity of detection of these
different amplification products was tested by diluting the
sample RNA into HL-60 RNA before RT-PCR amplifica-
tion. The 0 7 0 8 amplification product was detected at 0.01
pg and at 0.001 pg for M701 (Fig 4). The fact that 0 7 is
within the exon that is alternatively spliced in PML explains
the weaker sensitivity of the 0 7 0 8 primer pair.
The M701 primer pair allowed us to detect the different
PML/RARa amplification products in all 14 patients. In
patients no. 10 through 14, the 230-bp type I1 fusion
transcript was observed; and in patients no. 1 through 9, this
primer pair allowed us to detect two fusion-specific amplication products of 709 and 565 bp that, according to the
described PML sequence, correspond (1) to the previously
amplified 456-bp product of type I fusion transcript ob-
Fig 2. RT-PCR analysis of AML3 patient samples.
The oligonucleotides chosen were 0 7 (PML) 0 8
(RARu) for patients no. 1 through 8, and 07 (PML) 0 6
(RARu) for patient no. 9. (A) Ethidium bromidestained gel. Patient numbers correspond t o those of
Table 1. Lane H is the control (H20sample). Molecular
size marker (Hae 111 digest of 0x174 DNA) is shown in
the last lane. (E) The RT-PCR products were transferred and hybridized with the type I site fusion
oligonucleotide 010..
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3113
RT-PCR IN ACUTE PROMYELOCYTIC LEUKEMIA
Fig 3. RT-PCR analysis of AML3 patient samples.
The oligonucleotides chosen were M7 (PML) and 01
(RARa). (A) Ethidium bromide-stained gel. Patients
numbers correspond t o those described in Table 1.
(E) The RT-PCR products were transferred and hybridized with the type II fusion site oligonucleotide S2'.
tained with the primer pair 0708, and (2) to the normal
alternative splicing of the 144-bp exon of PML.
In the two types of fusion PML/RARa recently published,I6,l7 the fusion point in RARa is the same and
corresponds to the junction between the intron of the
RARa gene that separates the A and B domain of RARa
and its downstream exon. These data and our results are
consistent with the previous observation that the t( 15;17)
breakpoint occurs within this intron of the RARa gene.1R-*'
The fusion points within the PML cDNA appear, however,
M7°1
O7O8
1 Y9
1Y 8
0.1 y9
0.1 Y 9
0.01 yg
0.01 yg
0.001 yg
0.001y9
0.1ng
0.01 ng
H20
0.1ng
A
VI
m
0.01 ng
0)
m r F c
m s r
m= r- . 0o -. qg e
F
0
6
0
0
4420
0
B
Fig 4. Sensitivity of the RT-PCR-amplified PML/RARa fusion transcripts with (A) 0708 or (E) M701 primer pairs. The quantity of sample
RNA diluted in HL-60 RNA is indicated. One-tenth of the amplification
reaction was analyzed by slot blot (A and E) or Southern transfer IC).
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
CASTAIGNE ET AL
3114
to be at least of two sorts. Although the genomic organization of PML gene is not yet completely described, our
results may imply that t( 15;17) breakpoints must also lie
within specific introns of the PML gene. This finding would
be in accordance with the previously described chromosomal translocations, resulting in a fusion protein, that all
lie within one specific intron [the E2A gene in the t(1;19),22
the can gene in the t(6;9),23and the bcr gene in t(9;22) acute
leukemias].24
Interestingly, we noted a strong correlation between the
type of amplification product obtained and the size of the
abnormal RARa transcripts we had previously detected by
Northern analyses and reported in these patients? It
appears that the type I fusion amplification product corresponds to the 4-kb and the type I1 to the 3.6-kb transcript
(Table 1 and Fig 5). No specific clinical or biologic feature
of these AML patients could be correlated with the two
different breakpoints detected in the PML gene, namely,
M3 versus M3 variant subtype and response to all-trans RA
in vitro and in vivo (Table 1) or induction of hyperleucocytosis by ATRA. Both fusion proteins exhibit altered RARa
transactivating properties, suggesting that both may equally
contribute to the leukemogenesis of the d i ~ e a s e . ' ~ JThe
~.~'
data further stress that the region implicated in the breakpoints of the PML gene is probably not determinant for
phenotypic features and RA responsiveness in AMW cells.
This study shows that RT-PCR analysis in AMW patients allows for the molecular detection of the PML/
RARa fusion transcripts, now one of the biologic hallmarks
of this disease. We and others have recently shown that
all-trans RA therapy is an efficient alternative to chemotherapy in AML patient~.Z-~~
Accurate diagnosis is essential as
all-trans RA is ineffective both in vitro and in vivo in other
AML subtypes.28The absence of peripheral blasts and poor
cellular bone marrow aspirates render cytogenetic and in
vitro differentiaton studies difficult in some patients. Finding the t(15;17) can be masked by the normal mitoses
p t 4
13
10
7 k
-
-4
36
-32
RARa
-23
Fig 5. Analysis of RARa gene expression in RNA from four AML3
patients and the HL-60 cell line. Arrows indicate the abnormal 4-kb
and 3.6-kb RARa transcripts.
originating from erythroblasts and this effect can last even
after 24 hours of culture,29as it happened in patient no. 6.
The detection of the fusion transcript on a small quantity of
cells by RT-PCR will provide a useful complementary
diagnostic tool for AML3, namely, in the M3 variant
subtype, which is sometimes difficult to assess. It will,
furthermore, allow studies of minimal residual disease that
have not yet been evaluated either after conventional
chemotherapy or after differentiation therapy with all-trans
RA. The demonstration that the RNA-based PCR method
can accurately identify specific PML/RARa sequences in
all patients with AMI3 is an essential prerequisite for these
investigations and therapeutic guidelines.
ACKNOWLEDGMENT
We are indebted to the physicians who have transmitted the
patients' samples to the laboratory, to M. Collyn (Unit6 Inserm
124, Lille), A. Bernheim (Institut Gustav Roussy, Villejuif), R.
Berger (Unit6 lnserm 301), and M.T. Daniel (Laboratoire
d'Himatologie, Hdpital Saint Louis, Paris, France).
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1992 79: 3110-3115
A PML/retinoic acid receptor alpha fusion transcript is constantly
detected by RNA-based polymerase chain reaction in acute
promyelocytic leukemia
S Castaigne, N Balitrand, H de The, A Dejean, L Degos and C Chomienne
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