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
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).
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
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
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