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British Journal ofHaernatology, 1995, 91, 878-884

Combined cytogenetic, FISH and molecular analysis


in acute promyelocytic leukaemia at diagnosis
and in complete remission

MARCO MANCINI, MAURO NANNI,MICHELE CBDRONE,


DANIELA GIUSEPPEAVVISATI,
DIVERIO, ROBERTARICCIONI,
MARIAROSARIA DE CUIA, SUSANNA FENUAND GIULIANAALIMENA
Haematology, Department of Human Bioputhology,
University of Rome ‘LaSapienza’, Rome, Italy

Received 23 May 1995; acceptedfor publication 27 July 1995

Summary. This study reports the results of a simultaneous disease, all were also recognized by interphase FISH, and
application of cytogenetic fluorescence in situ hybridization eight were positive by metaphase FISH. Of the three samples
(FISH) and molecular analysis (RT-PCR) in 28 APL cases (23 negative at RT-PCR, all were also negative at the interphase
M 3 and five M3v; 26 studied at diagnosis and two at FISH. The results of this study indicate that: (a)the t(15;17)
relapse). FISH on metaphases identified the t(15;17) in all is present in all cases positive for the PML/RAR Q
cases who were positive for the PML/RAR a transcript by rearrangement, thus in virtually all true APLs; (b) standard
RT-PCR. Conventional cytogenetics revealed the t( 15;17) in cytogenetics, capable of unravelling the t( 15; 17) in only
only 68% of cases. However, it enabled the detection of 68% of cases, enables recognition of additional chromosome
additional chromosome changes in five cases, three of whom changes of potential clinical and prognostic significance; (c)
were M3v. 11 patients were also investigated during FISH on interphase nuclei is a reliable tool for the monitoring
complete remission (CR) by both FISH and RT-PCR, in of residual disease, with a sensitivity greater than that of
order to evaluate residual disease; the duration of CR at the FISH on metaphase cells and superimposable to that of RT-
time of analysis ranged between 1 and 1 6 months, with PCR.
three patients being studied twice. Comparison of RT-PCR
and FISH results showed a very good correlation. In fact, of Keywords: metaphase and interphase FISH, APL,
the 10 samples which were RT-PCR positive for residual cytogenetics, RT-PCR, residual disease.

Acute promyelocytic leukaemia (APL) is characterized by ment has, in fact, been indicated as a predictor of impending
well-documented biological and clinical features. These relapse, whereas its complete disappearance frequently
include: (1)the rearrangement between the promyelocytic correlates with long-term survival and, possibly, cure (Lo
(PML) and retinoic acid receptor o (RAR a )genes, located on COCOet al, 1992b; Huang et al, 1993; Miller et al, 1993).
chromosomes 1 5 and 17, respectively; this leads to the PML/ Southern blot and polymerase chain reaction (PCR)
RAR a fusion gene which encodes for the PML/RAR Q analyses, which enable the identification of a PML/RAR a
protein (Grignani et al, 1994), and (2) the high sensitivity of rearrangement in 100% of cases, enable a reliable molecular
the neoplastic population to differentiation therapy with all- diagnosis of APL and offer a potential tool for disease
trans retinoic acid (RA) (Huang et al, 1988; Degos et al, monitoring(Biondietal,1991.1992; Chenet al, 1991; Tong
1990; Warrel et al, 1991; Miller et al, 1992; Grignani et al, et al, 1992; Diverio et al, 1993). However, Southern blotting
1994). Furthermore, the presence of a specific genetic is a time-consuming and poorly sensitive technique, whereas
marker enables monitoring of the leukaemic clone during the use of PCR has so far been restricted to experienced
treatment and this has been shown to bear prognostic laboratories. Cytogenetic studies recognize the t( 15;17) in
implications. A persistent signal following induction treat- only 70-80% of APL cases (Berger et al, 1983; Larson et al,
1984;De Braekeleer & Lin, 1986). The discrepancy with the
Correspondence: Dr Marco Mancini, Haematology, Department of molecular results may be due to karyotypic analyses carried
Human Biopathology, University of Rome ’La Sapienza’. Via out on cells which do not belong to the neoplastic clone,
Benevento 6,00161 Rome, Italy. or to sub-microscopic rearrangements not detectable by

8 78 01995 Blackwell Science Ltd


Combined Cgtogenetic, FlSH and Molecular Analysis in APL 879
conventional cytogenetics (Lo COCO et al, 1992a). This with a Zeiss microscope Axiophot equipped with a triple-
technique is, nonetheless, useful for the detection of band pass filter.
chromosome changes in addition to the t(15:17), or of Hybridization signals were evaluated independently by
variant translocations such as t(11;17) (Licht et ul. 1995), or three microscopists in 1000 nuclei.
of not transcribing RAR cr rearrangements. Molecular biology. (A) RNA extraction. Mononuclear cells
Fluorescence in situ hybridization (FISH) has greatly were separated by Ficoll-Hypaque centrifugation and total
increased the potential of standard cytogenetics towards RNA extracted according to the method of Chomczynski &
identifying chromosomal or gene rearrangements both in Sacchi (1987). The quality of RNA was assessed in an
metaphase or interphase cells. The feasibility and usefulness ethidium bromide-stained 1.5% agarose gel containing 2.2
of this technique in APL has been previously recognized mol/l formaldehyde.
(Speicher et al, 1993). (B)RT-PCR amplification. In vitro reverse transcription of 1
With the aim of better assessing the sensitivity and role of pg of APL total RNA to cDNA was performed using a
FISH in detecting the t( 15:17) and/or the PML/RAR a commercial kit (GeneAmp RNA kit, Perkin Elmer-Cetus). The
rearrangement as compared to both classic cytogenetics and RT-PCR conditions and the oligonucleotides used to amplify
PCR, we have applied these three techniques simultaneously the PML,/RAR a cDNA have been reported elsewhere (Diverio
to 28 cases, classified as APL according to morphological et al, 1993). Amplification of the normal RAR a cDNA was
criteria and molecular grounds. Samples were collected at performed to further verify RNA integrity and the efficiency of
diagnosis and/or at relapse, and in a proportion of cases also the reverse transcription reaction. To assess the sensitivity of
after the achievement of complete remission (CR), in order to the RT-PCR assay, total RNA isolated from one APL patient
evaluate minimal residual disease (IvlRD). was serially diluted by mixing with the myeloid cell line GF-D8
RNA, as previously described (Diverio et al, 1993).
MATERIALS AND METHODS
RESULTS
Patients. Bone marrow (BM) specimens were collected
from 28 patients (16 females, 12 males; median age 34 Clinical data
years, range 2-77) with morpho-cytochemical diagnosis of The main clinical and haematological data of the 28 cases
FAB M3 (23 patients) or M3v (five patients), observed are summarized in Table I. 23 patients were diagnosed
between June 1991 and July 1994 at the Haematology morphologically as having the classic M3 form and five were
Department of the University ‘La Sapienza’ of Rome. Of the classified as M3v. CR was achieved in 24/28 APL patients
28 patients, 26 were studied at diagnosis, and two at relapse. after induction therapy.
11 patients were also studied in CR after induction therapy: Cytogenetics
three of them (nos. 6, 8 and 10) were analysed twice at The cytogeneticresults are reported in Table 11. Evaluable data
different time intervals. were obtained in 20126 patients studied at diagnosis and in 21
Cytochemistry and immunology. Morphological and cyto- 2 patients observed at relapse. A mean number of 1 5
chemical analyses were performed by Wright-Giemsa, metaphases (range 12-30) could be analyzed for each
myeloperoxidase, chloroacetate esterase, alpha naphtyl- sample. Of the 26 patients studied at diagnosis, 18 (69%))
acetate and butyrate esterase in all patients. revealed the standard t(15;17)(q22;q21). In 14 cases (13 M3
The immunophenotypic characterization was performed and one M3v). this was present as an isolated abnormality,
on Ficoll-Hypaque-isolated mononuclear cells using a panel whereas in four, two of which with M3v (13 and 25) and one
of monoclonal antibodies, as previously reported (De Rossi et (24) with a percentage of microgranular blasts > 25% it was
al, 1990). associated to additional chromosome changes: these consisted
Cytogenetics. GTG-banded chromosomes of BM cells were of a t(6;17)(q23;q21),a del(l7)(p12)and a + 8 (two cases),
studied according to standard methods and classified respectively. Two cases had an apparently normal karyotype,
according to the ISCN (199 1). whereas six (five M3 and one M3v) were not evaluable
In situ hybridization. (A) A library of chromosome 17 because of the poor quality of the metaphases.
labelled with biotin (Oncor, Gaithesburg, Md.) was used as Of the two cases analysed at relapse, one with a M3v( 7)
probe for chromosomal in situ suppression (CISS) hybridiza- showed an add( 7)(pl5) chromosome associated to the
tion on metaphase cells. Hybridization and detection of t(15;17) and the other displayed an apparently normal
chromosome 1 7 sequences were carried out according to the karyotype.
protocol of Wiegant et al(l991). Samples were visualized on
a photomicroscope (Zeiss Axiophot) equipped with a double- In situ hybridization on metaphase cells
band pass filter. An average of 29 metaphases (range 15-98) could be
(B) A mixture of digoxigenin-labelled cosmid DNA probes analysed for each case at first examination (Table II). In all
specific for the 17q21 chromosome breakpoint and a biotin- 24 cases examined at diagnosis and the two cases analysed
labelled cosmid DNA probe specific for the 15q22 chromo- at relapse, CISS-hybridization technique clearly demon-
some breakpoint (Oncor, Gaithesburg, Md.) were used on strated the t(15:17), and also in poor quality metaphases
interphase cells. Dual-colour hybridization was performed as not analysable by standard cytogenetics (Table 11). In all 26
previously described by Arnoldus et a l ( l 9 9 0 ) and according cases the t( 15:17) coexisted with normal cells at a rate
to the manufacturer’s instructions. Samples were evaluated ranging between 30% and 93% of examined cells.

0 1995 Blackwell Science Ltd, British Journal of Haematology 91: 878-884


880 Marc0 Mancini et a1
Table I. Biological and clinical data of 28 patients with a morphological diagnosis of APL.

BM-blasts

Immunology
Disease PB-WBC
Case Agelsex Morphology status DIC x109/1 % DR CD34 CD33 CD13 CD9 0 2 Therapy Outcome
-
1 2/F M3 Dia Yes - 85 0 0 65 77 70 ND Idarubicin + cytarabine CR
2 14/F M3 Dia Yes 2.7 90 2 2 78 77 85 72 Idarubicin + cytarabine CR
3 26/F M3 Re1 NO - - - _ - - - - ATRA CR
4 30/M M3v Dia Yes 238 98 7 25 84 79 96 ND Died before treatment Dead
5 32/F M3 Dia Yes 1.6 60 - - - - - - Idarubicin CR
6 28/M M3 Dia Yes 15.7 90 2 2 79 87 ND 2 Idarubicin+ cytarabine CR
7 2O/M M3v Re1 Yes 19.7 78 - - - - - - ATRA CR
8 29/M M3 Dia Yes 3.2 80 0 0 81 26 ND 3 ATRA + idarubicin CR
9 38/F M3 Dia Yes 0.4 92 1 1 78 35 ND 2 ATRA + idarubicin CR
10 53/F M3 Dia Yes 1.3 81 1 0 80 90 85 1 ATRA + idarubicin CR
11 63/M M3 Dia Yes 1.7 93 3 0 85 75 86 0 ATRA + idarubicin CR
12 19/F M3 Dia Yes 2.0 80 0 0 69 49 80 2 ATRA + idarubicin CR
13 56/M M3v Dia Yes 2.5 93 8 16 72 60 90 2 ATRA + idarubicin CR
14 64/F M3 Dia Yes 4.0 79 3 1 61 77 93 2 ATRA + idarubicin Dead
15 77/F M3 Dia Yes 1.2 83 0 0 91 66 93 ND ATRA Dead
16 51/F M3 Dia Yes 1.5 80 0 0 90 72 87 2 ATRA + idarubicin CR
17 50/M M3v Dia Yes 18.2 90 1 0 90 4s 88 1 ATRA + idarubicin Dead
18 lS/F M3 Dia Yes 1.4 85 18 1 90 89 90 ND Idarubicin CR
19 28/M M3 Dia Yes 0.6 83 0 0 96 12 95 ND Idarubicin + cytarabine CR
20 51/F M3 Dia Yes 0.8 79 2 0 53 46 ND ND Idarubicin CR
21 14/M M3 Dia Yes 2.3 9s 4 9 80 60 67 ND ATRA + idarubicin CR
22 65/M M3 Dia NO 1.7 74 3 4 93 57 91 1 ATRA + idarubicin CR
23 36/F M3 Dia Yes 2.3 9s 1s 19 88 83 90 21 +
ATRA idarubicin CR
24 64/P M3 Dia Yes 18-9 94 15 30 72 61 91 28 ATRA + idarubicin CR
25 9/F M3v Dia Yes 3.1 80 2 1 76 69 ND 3 ATRA + idarubicin CR
26 37/M M3 Dia Yes 1.1 83 8 4 58 20 ND 1 ATRA + idarubicin CR
27 40/M M3 Dia Yes 7.9 75 4 11 60 57 5 8 ATRA + idarubicin CR
28 31/F M3 Dia Yes 5.5 76 5 0 34 78 ND 5 ATRA + idarubicin CR

Dia. diagnosis: Rel, relapse: ND, not done: ATRA. all-trans retinoic acid: CR, complete remission.

In 5/8 cases in CR who were studied only once (Table 111: leukaemia) was 3.3% (range 1*8-3.9%)with a standard
nos. 1 , 2 , 5, 7 , 9 , 16,20 and 22) CISS hybridization analysis deviation of 0*4% These apparent fusion signals were
revealed the t(15;17) in 1-12% of the metaphases (an interpreted as due to coincidental overlapping of the PML
average of 100 metaphases was analysed for each case, and RAR a domains. A PML/RAR a fusion in the test
range 19-283), whereas three cases were negative (Table specimens was therefore diagnosed if > 4.5% (mean f 3 SD
III; nos. 1,7 and 22). The other three cases (Table III; nos. 6, of the controls) (Fig 1).
8 and lo), who were studied twice during CR.were positive (B) APL patients. 11 cases were analysed in CR following
at the first determination and negative at the second. induction treatment. Cases 6, 8 and 10 were studied twice.
In 11/14 specimens fusion signals were detected in a rate
In situ hybridization on interphase cells greater than the cut-off level of 4.5% (range 5*7-10-7%)
The 15q22 labelled breakpoint resulted in a green signal and established in the controls, thus indicating persistence of
the 17q2 1in a red signal. Normal cells showed four separate residual disease. Three cases (nos. 7 , 8 [2nd control] and 22)
signals (two green and two red), whereas all translocations showed a percentage of fusion signals in the normal range
involving the fusion of PML and RAR Q regions resulted in a (Fig 1,Table III).
close red-green signal, plus two separated red and green
signals. Only overlapping or touching signals were classified Molecular biology
as positive for PML/RAR a rearrangement. The 28 cases were studied by RT-PCR for the presence of
(A) Controls. The mean percentage of interphase cells with PML/RAR a fusion gene transcripts (26 at diagnosis and two
an apparent PML/RAR a fusion signal, evaluated in 11 at first relapse). A positive RT-PCR was observed in all 28:
selected specimens (normal, BM donors, and patients in CR 15 cases had a breakpoint in bcr 1 or 2, and 13 in bcr 3
after a BM transplantation for chronic myelogenous (Table 11).

0 1995 Blackwell Science Ltd. British Journal of Haemutobgy 91: 878-884


Combined Cytogenetic, FISH and Molecular Analysis in APL 88 1
Table 11. Cytogenetic, FISH and RT-PCR data of 28 patients.

Disease CISS RT-PCR


Patient status Morphology Karyotype t(15;17) (type of breakpoint)

1 Dia M3 NE ND pos (bcr 1)


2 Dia M3 46,XX ND pos (bcr 1-2)
3 Re1 M3 46,XX pos pos (b 3)
4 Dia M3v 46,XX,t(l5:17) POS pos (bcr 3)
5 Dia M3 NE POS pos (bcr 1-2)
6 Dia M3 46,XY,t(l5:17) POS pos (bcr 1)
7 Re1 M3v 46,XX,add(7),t(15;17) POS pos (bcr 3)
8 Dia M3 46,XY,t(15;17) POS pos (bcr 1)
9 Dia M3 46,XX POS ~ o (bcr
s 2)
10 Dia M3 46,XX.t(15;17) POS pos (bcr 1)
11 Dia M3 46,XY,t(15:17) Pos Pos ( b a 1)
12 Dia M3 46,XX,t(l5;17) POS pos (bcr 1)
13 Dia M3v 46,XY,t(6;17),t(15:17) Pos pos (bcr 3)
14 Dia M3 46,XY,t(l5:17) POS pos (bcr 3)
15 Dia M3 NE POS pos (bcr 1)
16 Dia M3 NE POS pos (bcr 1)
17 Dia M3v NE Pos pos (bcr 1)
18 Dia M3 46,XX.t(15;17) Pos ~ o (sb a 3)
19 Dia M3 46,XY,t(l5:17) POS pos (bcr 1)
20 Dia M3 46,XX,t(15;17) POS pos (bcr 1)
21 Dia M3 46,XY,t(15;17) POS pos (bcr 3)
22 Dia M3 46,XY,t(15:17) POS pos (bcr 3)
23 Dia M3 46,XX,t(15;17) POS pos (bcr 3)
24 Dia M3 46,XX,del(l7p),t(15;17) pos pos (h 3)
25 Dia M3v 47,XX,+8~(15;17) pos P (bcr 3)
26 Dia M3 47,XY,+8,t(l5;17) pos pos (bcr 3)
27 Dia M3 NE POS pos (bcr 3)
28 Dia M3 46 ,XX,t(15;17) POS pos (bcr 1)

Dia. diagnosis: Rel, relapse: NE. not evaluable: ND, not done.

Table 111. Results of FISH and RT-PCR in the BM cells of patients studied at CR.

FISH RT-PCR
Clinical status at
Case FISH and PCR date CISS t(15;17) PML/RAR a PML/RAR 01

1 CRx 1 month
2 CR x 2 months
5 CRx 1 5 months
6 CRx 1month
CR x 16 months
7 CR x 1 month
8 CR x 1 month
CR x 1 5 months
9 CRxl month POS POS POS
10 CRx 1.5 months POS POS POS
CR x 2 months neg POS Pos
16 CRx 1month POS POS P oS
20 CR x 2 months POS POS POS
22 CRx 12 months neg neg neg

CR, complete remission: ND, not done.

0 1995 Blackwell Science Ltd. British Journal of HuernatoZogy 91: 878-884


882 Marc0 Mancini et a1
12 -
%Of
NUCLEI

10 -

8-

6-

4-
Fig 1. Percentage of nuclei with PML/RAR LY
fusion signals in 11 controls and 11 API,
2-
patients in CR. The cut-off level was obtained
from the statistical evaluation of the controls:
the mean f3 SD of false-positivenuclei is

0- PML/RAR 01 fusion. Number in oarentheses is


1 11 1 11 the duration of CR (in months) it FISH date.
Controls APL patienta *Second control.

Eleven patients were investigated also following the chromosome morphology are the main limitations of the
achievement of CR (Table 111): two of them (nos. 8 and 10) cytogenetic investigation.
were studied twice. Nine cases studied within 1 or 2 months Recently the ability of FISH to identify the t( 15;17) in
after CR showed positive RT-PCR. Three cases (nos. 7 , 8 [2nd cases of APL with an apparently normal karyotype has been
control] and 22) investigated at 1, 1 5 and 1 2 months from reported, thus suggesting that this technique represents an
CR, respectively, displayed negative RT-PCR. Case 5 had important tool for the recognition of the aberration (Speicher
persistent positive RT-PCR 15 months after CR (Table III). et al, 1993). We have applied painting of chromosome 1 7 in
26/28 cases, 24 at diagnosis and two at first relapse, with
the aim of: (a)confirming the power of FISH in detecting the
DISCUSSION
t(15:17), and (b) evaluating the real percentage of t(15:17)-
The risk of a potentially fatal coagulopathy, as well as the positive cells in a large number of PCR-positive patients.
unique clinical response to RA in APL, requires an accurate Using this approach, clear-cut results were obtained even in
and prompt diagnosis of this disease (Gralnik & Sultan, cases with chromosomes of poor morphology; moreover, a
1975; Rodeghiero et al, 1990; Tallman & Kwaan, 1992). higher number of metaphases (a median of 29 per case) was
Though the identification of the hypergranular APL form analysable over a short period of time, compared to standard
is usually easy, a misclassification of some AML cases with cytogenetics. The t(l5;17) was detected in all cases positive
signs of maturation as APL may occur: also in cases of M3v for the PML/RAR N fusion transcript by PCR, including those
the distinction from acute myelomonocytic leukaemia may not evaluable or negative by conventional G-banding
represent a diagnostic problem (Castoldi et al, 1994). method (Table 11). These results further support the
However, in both the classic and variant forms of APL the suggestion that virtually all true APLs carry the t(15;17)
PML/RAR a! hybrid gene is a diagnostic hallmark. It is and indicate that CISS hybridization is an easy method to
therefore mandatory that this is searched for when a unravel the anomaly, with a sensitivity which appears
diagnosis of APL is suspected (Grignani et al. 1994). The superimposable to that of molecular analyses.
use of molecular biology (Southern, PCR) enables the Cytogenetics is, however, of fundamental importance in
identification of RAR n rearrangements or PMT,/RAR a detecting additional karyotypic changes which may occur in
transcripts in 100% of cases, whereas cytogenetics recog- 10-30% of cases. Berger et aZ(1991), in a series of 73 APLs
nizes the t(15;17) in only 70430% of APL cases (De examined at diagnosis, observed additional chromosomal
Braekeleer & Lin, 1986; Warrel et d,1991; Lo COCOet al, changes in 2 0 cases with no significant differences between
1992a; Grignani et al, 1994). the M3 and M3v forms. In our series of 28 patients, 22 of
In the present study cytogenetic analysis of 28 cases with whom were cytogenetically evaluable, we found chromo-
a diagnosis of APL confirmed at molecular level identified the some anomalies in addition to the t(15;17) in five cases
t( 15;17)in 1 9 of them (68%)(Table 11).This finding is in line (Table 11: nos. 7, 13, 24, 25 and 26). three of which were
with previously reported cytogenetic results for APL (Berger M3v and one M3 with a high number of microgranular
et ul, 1983; De Braekeleer & Lin, 1986). A low number of blasts. The anomalies consisted of a t(6;17)(q23;q21). an
evaluable metaphases (a mean of 15 per case) and a poor add( 7)(pl 5 ) , a del (17)(p12) (one case each) and trisomy 8

Q 1995 Blackwell Science Ltd, British Journal of Huemutology 91: 878-884


Combined Cytogenetic, FISH and Molecular Analysis in APL 883
(two cases). The APL variant form, although bearing the REFERENCES
PML/RAR a protein and responding to RA treatment,
exhibits clinical, cytological and, possibly, molecular dis- Arnoldus, E.P.J.. Wiegant, J., Noordmeer. LA.. Wessels. J.W.,
Beverstock. G.C.. Grosveld, J.C., van der Ploeg, M. & Raap. A.K.
tinctive features, suggesting that it may represent an
(1990) Detection of Philadelphia chromosome in interphase
evolutive form of APL. When compared to classic APL, the
nuclei. Cytogenetics and Cell Genetics, 54.108-111.
M3v shows the presence of hyperleucocytosis at onset, a Berger, R., Bernheim, A,, Daniel, M.T., Valensi, F. & Flandrin. 1.
more severe coagulopathy, the expression of the CD2 (1983) Cytological types of mitosis and chromosomal abnormal-
antigen, and a higher incidence of BCR3 breakpoint in the ities in acute leukemia. Leukemia Research, 7, 221-236.
PML gene. This type of breakpoint has been recently related Berger, R.. Le Coniat, M., Derre, J.. Vecchione, D. & Jonveaux. P.
to poor prognosis (Huang et al, 1993; Grignani e t a ] , 1994; (1991) Cytogenetic studies in acute promyelocytic leukemia: a
Vahdat et al, 1994). The presence of karyotypic anomalies in survey of secondary chromosomal abnormalities. Genes, Chromo-
addition to the t( 15;17) in the M3v may therefore be related somes and Cancer, 3, 332-337.
to an increased chromosome instability due to tumour Biondi, A.. Rambaldi, A., Alcalay. M.. Pandolfi, P.P., Lo Coco. F..
Diverio. D., Rossi, V., Mencarelli. A., Longo, L., Zangrilli, D..
progression, in a fashion similar to that observed when
Masera. G.. Barbui, T., Mandelli, F.. Grignani, F. & Pelicci. P.G.
chronic myelogenous leukaemia enters acute phase (Heim & (1991) RAR-rw gene rearrangements as a genetic marker for
Mitelman, 1987). The evaluation of the prognostic sig- diagnosis and monitoring in acute promyelocytic leukemia. Blood.
nificance of additional karyotypic changes, as well as their 77, 1418-1422.
nonrandom association with M3v, requires further inves- Biondi, A.. Rambaldi, A., Pandolfi, P.P., Rossi, V., Giudici. G..
tigations. Alcalay. M., Lo Coco, F., Diverio. D., Pogliani, E.M., Lanxi, E.M.,
Monitoring of residual disease through the presence of the Mandelli, F., Masera, G., Barbui, T. & Pelicci, P.G. (1992)
genetic marker is another very important issue in APL the Molecular monitoring of the My1 retinoic/acid receptor-cu fusion
incidence of CR is, in fact, very high, and survival after gene in acute promyelocytic leukemia by polymerase chain
therapy is probably long-term in this disease (Grignani et al, reaction. Blood. 80, 492-497.
Castoldi, G.L., Liso. V.. Specchia, G . & Tomasi. P. (1994) Acute
1994). In a recent study Zhao et aZ(1995) applied the CISS to
promyelocytic leukemia: morphological aspects. Leukemia. 8.
detect residual leukaemic cells in 10 APL patients in CR; 1441-1446.
although less sensitive than PCR, owing to its confinement Chen. Z., Chen, S.J., Tong, J.H., Dong, S., Huang, W., Chen. Y..
to dividing cells only, FISH appeared to be more efficient than Xiang. W.M., Zhang, L., Song, X., Qian, X.S., Wang, Z.Y., Chen. Z.,
conventional cytogenetics. In our study, 11APL patients in Larsen. C.J. & Berger, R. (1991) The retinoic acid o receptor gene
CR were investigated with FISH both on interphase and on is frequently disrupted in its 5’ part in Chinese patients with acute
metaphase nuclei, by using probes for the PML/RAR a: gene promyelocytic leukemia. Leukemia, 5, 288-292.
or specific for the entire chromosome 17. The results were Chomczynsky, P. & Sacchi, N. (1987) Single-step method of RNA
compared with those based on RT-PCR analysis. Of these 11 isolation by acid guanidinium thiocyauate-phenol-chloroform
patients, two were re-analysed both by FISH and PCR, and extraction. Annals of Biochemistry, 162.156-160.
one only by FISH (Table III). FISH applied to interphase cells De Braekeleer. M. & Lin,C.C. (1986) The occurrence of the 15:17
translocation in acute promyelocytic leukemia. Cancer Gmetics
was positive in all cases which were positive for the PML/
and Cytogenetics, 19, 311-319.
RAR a transcript by RT-PCR. The cases negative at the Degos, L., Chomienne, C.. Daniel, M.T.. Berger, R., Dombret. H..
molecular analysis showed a percentage of rearranged Fenaux. P. & Castaigne, S. (1990) Treatment of first relapse in
nuclei lower than the cut-off level. CISS hybridization, acute promyelocytic leukaemia with all-trans retinoic acid. Luncct,
applied to metaphases, revealed the presence of the t( 15;17) 336, 1440-1441.
in only eight of the 10 RT-PCR-positive samples, though it De Rossi, G., Avvisati, G., Coluzzi. S., Fernu. S., Lo Coco, P.. Lopex.
enabled the evaluation of an extremely large number of M., Nanni, F., Masqualetti. D. & Mandelli, F. (1990) Immunolo-
metaphases per patient (median 100, range 19-283). gical definition of acute promyelocytic leukaemia (FAR M3): a
Based on the results of this study, it emerges that FISH on study of 39 cases. European Journal of Haematology, 45. 168-
171.
interphase nuclei using probes exploring the PML/RAR Q
Diverio, D., Pandolfi, P.P.. Biondi, A.. Avvisati. G., Petti. M.C.,
junction represents an effective tool for detecting MRD in Mandelli. F., Pelicci, P.G. &Lo Coco. F. (1993) Absence of RT-PCR
APL patients. Its high sensitivity appears greater than that of detectable residual disease in patients with acute promyelocytic
CISS hybridization and superimposable to that of RT-PCR. It leukemia in long-term remission. Blood, 82, 3556-3559.
represents an easy procedure, which enables an accurate Gralnik, H.R. & Sultan, C. (1975) Acute promyelocytic leukaemia:
quantitative assessment of the PML/RAR cy rearranged cell haemorrhagic manifestation and morphologic criteria. British
fraction. The combined use of the FISH and PCR techniques Journal of Haematolgy, 29, 373-376.
in prospective studies may be of relevance towards a better Grignani, F., Fagioli. M., Alcalay. M.. Longo, L., Pandolfi, P.P., Ilonti.
definition of the reproducibility of these two methods in the E.. Biondi, A., Lo Coco. F., Grignani, F. & Pelicci, P.G. ( I YY4)
evaluation of MRD and of its significance with respect to the Acute promyelocytic leukemia: from genetics to treatment. Blood,
outcome of the disease. 83.10-25.
Heim, S. & Mitelman. F. (1987) Cancer Cytogenetics. Alan R. Liss.
New York.
ACKNOWLEDGMENTS Huang. W., Sun, G.L., Li,X.S., Cao, Q.. Lu, Y., Jang, GS.. Zhang. P.Q..
Chai. J.R., Wang, Z.Y., Waxman, S. & Chen, S.J. (1993) Acute
We thank Professor Robert Foa for critically reading the promyelocytic leukemia: clinical relevance of two major PML-
manuscript. M. Cedrone is an AIRC fellow. RAR A isoforms and detection of minimal residual disease by

0 1995 Blackwell Science Ltd, British Journal of Huematology 91: 878-884


884 Marc0 Mancini et a1
retrotranscriptase/polymerase chain reaction to predict relapse. acute promyelocytic leukemia by reverse transcription polymer-
Blood, 82,1264-1269. ase chain reaction assay for the PML/RARA fusion mRNA. Blood.
Huang, M., Yu-Chen. Y.,Shu-Rong, C.. Chai, J., Lin. Z., Long, J. & 82,1689-1694.
Wang, Z. (1988) Use of all-trans retinoic acid in the treatment of Rodeghiero, P., Avvisati, G., Castman, G., Barbui. T. & Mandelli, F.
acute promyelocytic leukemia. Blood, 72, 567-572. (1990) Early deaths and anti-hemorragic treatments in acute
ISCN (1991) Guidelines for Cancer Cytogenetics, Supplement to an promyelocytic leukemia: a GIMEMA retrospective study in 268
International System for Human Cutogenetic Nomenclature (ed. by F. consecutive patients. Blood, 7 5 , 2112-2117.
Mitelman). Karger, Basel. Speicher, M.R., Janch, A.. Parr. A. & Becher, R. (1993) Delineation
Larson, R.A., Kondo. K., Vardiman, J.W., Butler, A.E., Golomb. H.M. of translocation t(15;17) in acute promyelocytic leukemia by
& Rowley, J.D. (1984) Evidence for a 15;17 translocation in every chromosomal in situ suppression hybridization. Lt.ukem.a
patient with acute promyelocytic leukemia. American lournal of Research, 17, 359-364.
Medicine, 76, 827-831. Tallman, M.S. & Kwaan, H.C. (1992) Reassessing the hemostatic
Licht, J.D., Chomienne C., Goy, A.. Chen. A., Scott, A.A., Head, D.R., disorder associated with acute promyelocytic leukemia. Blood, 79,
Michaux, J.L., Wu, Y., DeBlasio, A., Miller, W.H., Jr, Zelenetz, 543-553.
A.D., Willman. C.L.. Chen, Z.,Chen, S.J., Zelent. A,, MacIntyre. E., Tong, J.H., Dong, S.. Geng, J.P., Huang, W., Wang, Z.Y.,Sun, G.L.,
Veil, A,, Cortes, J., Kantarjian, H. & Waxman. S. (1995) Clinical Chen, S.J., Chen, Z., Larsen, CJ. & Berger, R. (1992) Molecular
and molecular characterization of a rare syndrome of acute rearrangements of the My1 gene in acute promyelocytic leukemia
promyelocytk leukemia associated with translocation (11:17). (APL M3) d e h e a breakpoint cluster region as well as some
Blood, 85,1083-1094. molecular variants. Oncogene, 7, 311-316.
Lo Coco, F.. Diverio, D., D’Adamo, F.. Awisati, G.. Alimena, G.. Vahdat, L., Maslak, P., Miller, W.H.. Eardley. A., Heller, G.,
Nanni, M., Alcalay, M., Pandolfi, P.P. & Pelicci, P.G. (1992a) PML Scheinberg, D.A. & Warrell, R.P., Jr (1994) Early mortality and
RAK-a rearrangement in acute promyelocytic leukaemias appar- retinoic acid syndrome in acute promyelocytic leukemia: impact
ently lacking the t( 15;17) translocation. European Journal of of leukocytosis, low-dose chemotherapy, PML/RAR N isoform.
Haematology, 48, 173-176. and CD13 expression in patients treated with all trans retinoic
Lo Coco, F., Diverio, D.. PandoK, P.P., Biondi, A.. Rossi, V., Avvisati, acid. Blood, 84, 3843-3847.
G.. Rambaldi. A., Arcese, W., Petti. M.C., Meloni. G., Mandelli. P., Warrel, R.P. Jr.,Frankel. S.R., Miller, W.H.. Jr, Scheinberg. D.A.. Itri.
Grignani. F.. Masera. G.. Barbui, T. & Pelicci, P.G. (1992b) L.M., Hittelman, W.N., Vyas, R., Andreeff. M., Tafuri. A.,
Molecular evaluation of residual disease as a predictor of relapse in Jakubowski. A., Gabrilove, J., Gordon. M.S. & Dmitrowsky. E.
acute promyelocytic leukaemia. Lancet. 340, 1437-1438. (199 1) Differentiationtherapy for acute promyelocytic leukaemia
Miller, W.H.. Jr, Kakizuka, A., Frankel. S., Warrel, R.P., Jr, De Blasio, with tretionin (all-trans-retinoic acid). New England Journal of
A.. Levine, K., Evans, R.M. & Dmitrowsky, E. (1992) Reverse Medicine, 324,138 5 - 13 9 3.
transcription polymerase chain reaction for the rearranged Wiegant, J., Ried, T.. Nederlof, P.M., van der Ploeg, M., Tanke, H.J. &
retinoic acid receptor a clariEes diagnosis and detects minimal Raap, A.K. (1991)In situ hybridization with fluoresceinated DNA.
residual disease in acute promyelocytic leukemia. Proceedings of Nucleic Acids Research, 19, 3237-3241.
the National Academy of Sciences of the United States of America. 89, Zhao. L., Chang. K.S., Estey, E.H.. Hayes, K., Deisseroth, A.B. & Liang.
2694-2698. J.C. (1995) Detection of residual leukemic cells in patients with
Miller. W.H., Jr. Levine. K., De Blasio, A.. Frankel. S.. Dmitrowsky, E. acute promyelocytic leukemia by the fluorescencein situ hybridm-
& Warrel, R.P.. Jr (1993) Detection of minimal residual disease in tion method: potential for predicting relapse. Blood, 8 5 , 4 9 5-499.

0 1995 Blackwell Science Ltd, British Journal ofHaematology 91: 878-884

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