Leukemia (1998) 12, 1718–1723
 1998 Stockton Press All rights reserved 0887-6924/98 $12.00
http://www.stockton-press.co.uk/leu
Fluorescence in situ hybridization for the detection and monitoring of the Ph-positive
clone in chronic myelogenous leukemia: comparison with metaphase banding
analysis
A Cuneo1, R Bigoni1, B Emmanuel2, E Smit3, GM Rigolin1, MG Roberti1, A Bardi1, N Piva1 G Scapoli1, G Castoldi1,
H Van Den Berghe2 and A Hagemeijer2
1
Department of Biomedical Sciences – Hematology Section, University of Ferrara, Italy; 2Centre for Human Genetics, KUL Leuven, Belgium;
and 3Department of Cell Biology and Genetics Erasmus University, Rotterdam, The Netherlands
In order to analyze the efficiency of interphase FISH for the
detection and monitoring of Ph+ cells in chronic myelogenous
leukemia (CML) under interferon (IFN) treatment, the following
experiments were performed: (1) 98 specimens derived from 32
patients were analyzed in parallel by dual-color FISH and by
conventional chromosome analysis (CCA). A 300/200 kb
BCR/ABL probe was used in all tests and a smaller 35.5/39 kb
probe was tested in parallel in 22 BM samples; (2) 30 BM
samples were prepared by direct harvest and by 24-h culture
and were analyzed in parallel; (3) PB and BM samples obtained
simultaneously from 11 patients were analyzed. The cut-off
point for the recognition of BCR/ABL fusion was set at 2.4%,
calculated as the mean percent of false positivity in 11 controls
plus 3 s.d. A very close correlation was observed (r = 0.994, r2
= 0.988, P ⬍ 0.0001) between the percentages of Ph+ cells as
assessed by CCA and by interphase FISH in 98 samples (26 at
diagnosis). There was a moderate overestimation of the frequency of Ph+ cells by FISH with respect to CCA, that was more
evident at low-to-medium values of Ph positivity. Seven specimens without Ph+ metaphases (17–50 cells analyzed) were
shown to carry 2.5–8% interphase cells with BCR/ABL fusion.
Similar percentages of BCR/ABL+ nuclei were recorded in 22
samples hybridized using the 300/200 kb and the 35.5/39 kb
probe-sets (variation range: 0–5%, mean 2.3%). A very good
correlation between the frequency of Ph+ interphase cells was
observed when analyzing in parallel BM preparations after
direct harvest and after 24-h culture. Underestimation of the
percentage of BCR/ABL+ cells was noted to occur in 2/11 PB
samples, compared to BM samples, the remaining nine cases
showing superimposable results at either sites. We arrived at
the following conclusions: (1) dual-color FISH enables an
accurate detection and monitoring of the size of the Ph-positive
clone in CML at diagnosis and after IFN-therapy; (2) FISH is
more accurate than CCA, especially at low levels of Ph-positive
cells; (3) testing of directly harvested BM samples is feasible
and accurate, giving the opportunity to perform centralized
FISH analysis in the context of multicentre trials; (4) the percentage of BCR/ABL+ PB cells usually, though not invariably,
reflects the frequency of mutated cells in the BM.
Keywords: CML; FISH; t(9;22) detection
monitoring of the hematologic response to modern treatment,
including interferon (IFN) and/or bone marrow transplantation.1,2 Because the precise quantification of Ph-positive
cells is clinically important,3 growing attention has been
devoted over the last 5 years to fluorescence in situ hybridization (FISH), potentially permitting the study of large numbers
of cells, including non-cycling cells, in a relatively short
time.4–6 While the reliability of this molecular cytogenetic
method in recognizing Ph-positive leukemias has been
unequivocally documented,7 definite demonstration of its
efficiency in estimating the size of the abnormal clone was
not provided. Indeed, the relatively high frequency of falsenegatives, deriving from incomplete hybridization, and of
false-positives, deriving from the erroneous counting of artifactual spots in interphase cells, represented an area of concern, prompting scientists to test several probe combinations
in dual-color or triple-color hybridization experiments.8–10
While clearly improving the sensitivity and the specificity of
this method, these efforts yielded variable results concerning
the correspondence of the frequencies of Ph-positive cells as
assessed by FISH and by CCA.11,12
In order to compare the results of CCA and FISH in the
estimation of the size of the Ph-positive clone in CML under
IFN treatment, 98 specimens derived from 32 patients were
studied by karyotype analysis and by dual-color interphase
FISH, using two sets of BCR/ABL probes, differing in the size
of the human DNA insert. A comparison was made between
FISH data obtained on non-cultured samples and on 24-h culture samples, as well as between data obtained from peripheral blood (PB) and bone marrow (BM).
The results of this study are presented and discussed, with
reference to the definition of the possible role and conditions
for the employment of FISH in the detection and monitoring
of Ph-positive cells in CML.
Introduction
Materials and methods
Chronic myelogenous leukemia (CML) is a myeloproliferative
disorder that results in the clonal expansion of a mutated stem
cell carrying the translocation t(9;22)(q34;q11), associated
with the BCR/ABL fusion gene. The demonstration of this molecular cytogenetic defect by conventional chromosome analysis (CCA), by Southern blotting and/or by the sensitive reverse
transcriptase polymerase chain reaction (RT-PCR) technique,
is a major diagnostic criterion. In addition, the estimation of
the relative size of the abnormal clone is important in the
Correspondence: A Cuneo, Istituto di Ematologia – Università di Ferrara, Via Savonarola, 9 44100 Ferrara, Italy; Fax: 532 212142
Received 26 February 1998; accepted 1 July 1998
Patients
Thirty-two adult patients with Ph-positive CML, treated by IFN
at a dosage of 5 MU/m2/day, were included in the present
study. Patients’ ages ranged between 16 and 65 years (median
56.5), with a male to female ratio of 0.77. The presence and
frequency of Ph+ cells was ascertained for clinical purposes
in these patients in 98 occasions. Twenty-six patients were
studied by CCA at diagnosis, whereas no information on the
disease status at the time of first cytogenetic investigation was
available in two patients. Four patients were referred to our
Institutions for the first time after 12–60 months of IFN therapy. Of these 32 patients, 26 were studied sequentially (20
starting from diagnosis); a total of 66 analyses were performed
FISH vs CCA for monitoring Ph+ CML
A Cuneo et al
at 6–12 month intervals (1–4 tests in each patient, median 2)
on specimens collected over a 6–84 month follow-up. Among
the patients studied during IFN therapy, six were classified as
complete cytogenetic responders based on 20–50 analyzed
metaphases (one with absence of BCR/ABL fusion at RT-PCR
analysis, five with persistent BCR/ABL+ cells by RT-PCR, data
not shown), nine as major responders (ie 1–33% Ph+ cells by
CCA) and 11 as minor responders or non-responders (ie 33–
99% Ph+ cells at CCA).
(24 h). Direct harvests and short-term cultures of BM mononuclear cells were prepared by colcemid block, hypotonic
shock and fixation; (3) 11 PB and BM samples, which were
obtained simultaneously from follow-up cases and tested in
parallel. Freshly aspirated PB and BM cells were centrifuged
in heparinized sterile tubes at 200 g for 10 min and the buffy
coat was aspirated. Then, a drop of each cell suspension was
used to ascertain the percentage of lymphocytes and of nonlymphoid elements (May–Grünwald Giemsa staining). The
remaining cells were directly submitted to hypotonic treatment and fixation.
Cytogenetic analysis
CCA was performed on bone marrow (BM) mononuclear
cells, obtained by centrifugation over a 1.077 mg/ml density
gradient and cultured for 24 h in RPMI additioned with 30%
fetal calf serum (FCS). The preparation of cytogenetic samples
according to standard methods,13 included mitotic block by
colcemid, hypotonic shock in 0.075 m KCl solution and fixation in a 3:1 methanol/acetic-acid solution. Whenever possible, a minimum of 20 metaphases, and usually more, were
analyzed to calculate the percentage of Ph-positive cells.
FISH studies
Controls:
Cytogenetic preparations from 11 normal BM
samples (ie lymphomas without BM involvement, or autoimmune thrombocytopenias), were used as negative controls
to set the cut-off point for the recognition of Ph+ cases.
Dilution samples:
In order to ascertain the reproducibility
of FISH as a means of assessing the percentage of Ph-positive
cells, 10 specimens were obtained from five patients at diagnosis and five patients at follow-up. 1:1 dilutions of these Ph+
cases were prepared using cells obtained from cytogenetically
normal controls. BM mononuclear cells were collected from
patients and controls after centrifugation over density gradient.
Two 24-h cultures were set up in parallel; then, equal numbers of cells from the Ph-positive sample and from the normal
sample were mixed and submitted to hypotonic treatment and
fixation. Parallel hybridization experiments were performed
on slides prepared from non-diluted and diluted samples.
CML specimens:
FISH studies were performed on the
same 98 BM specimens that had been previously analyzed by
CCA as described above. Forty analyses were performed in
patients, either at presentation or at follow-up, having ⬎66%
Ph+ metaphases; 26 specimens were tested having 34–66%
Ph+ metaphases, 23 were tested with 1–33% Ph+ cells and
nine without Ph+ cells in 17–50 analyzed metaphases. The
slides for FISH analysis were prepared from methanol/acetic
acid-fixed samples, which had been stored at −20°C for ⬍1–
24 months.
Variable
conditions:
Additional FISH experiments
included: (1) 22 BM samples (11 at diagnosis, 11 at followup), which were hybridized using two probe sets (see below),
in order to check whether the interpretation of the results of
interphase FISH could be influenced by the probe size; (2) 30
BM samples obtained at follow-up, which were hybridized in
parallel after direct harvesting and after short-term culture
Probes, hybridization and signal screening
The first probe
set, which was used in all 98 experiments, consisted of a BCR
probe of approximately 300 kb in size, which was directly
labeled with SpectrumGreen fluorophore and of an ABL probe
of 200 kb in size, directly labeled with SpectrumOrange fluorophore (Vysis, Downers Grove, IL, USA). The second probe
set included the cos51 having a 35.5 kb BCR insert and the
cos ABL19 with an insert of 39 kb (Dept of Cell Biology and
Genetics, Erasmus University, Rotterdam, The Netherlands).
Hybridization conditions were as follows: After a 1-h incubation with RNAse (100 ␮g/ml 2 × SSC) at 37°C, the slides
were washed in 2 × SSC three times and then incubated at
37°C with 10 mg pepsine/100 ml 0.01 N HCl, for 10 min. The
slides were subsequently fixed at room temperature with 1%
formaldehyde in PBS with 50 mM MgCl for 10 min, dehydrated in ethanol series and air dried. The slides were
denatured in 70% formamide in 2 × SSC at 70°C for 120 s,
quenched on ice, dehydrated on ethanol series and air dried
before use. A commercially available probe/hybridization solution, consisting of 7 ␮l LSI hybridization buffer, 1 ␮l bcr/abl
probe, and 2 ␮l purified water, for a total of 10 ␮l each slide
(Vysis) was heated for 5 min in a 73°C water bath and applied
to prewarmed slides to proceed to overnight hybridization in
a humidified box at 37°C. The second set of probes was labelled with a nick-translation kit according to the manufacter’s
instruction (Boehringer Mannheim, Mannheim, Germany) and
the DNA resuspended in 50% formamide, 10% dextran sulfate in 2 × SSC to a final concentration of 40 ng/␮l and coprecipitated with 40 ␮g of cot-1 for 100 ng of cosmid probes. The
hybridization solution was denatured at 70°C for 4 min, and
subsequently incubated at 37°C for 40 min to allow the preannealing of repetitive sequences. Overnight hybridization was
allowed to proceed as above.
Post-hybridization procedures for both the probe-sets
included 3 × 5-min washes in 55% formamide in 2 × SSC at
39°C, 3 × 5-min washes in 2 × SSC at the same temperature,
a single 5-min wash in 4 × SSC/0.05% Tween 20 at room temperature, each with intermittent agitation. The detection of the
second probe set was performed with alternating layers of
fluoresceinated avidin, biotinylated goat antiavidin 5 ␩g/␮l
(Vector Laboratories, Burlingame, CA, USA), sheep antidigoxigenine-rhodamine, and donkey antisheep Texas red, to
obtain a double layer for each fluorochrome. Each incubation
was followed by 3 × 5-min washes in 0.1 M Tris-HCl/0.15 M
NaCl/0.05% Tween 20. A Dapi/vectashield antifade was
applied on to the slides to counterstain double-stranded DNA.
One hundred to 300 nuclei were evaluated in each sample
on a fluorescence microscope equipped with a three-band
pass filter; images for illustration purpose were obtained using
a cooled charge-coupled device (CCD) camera (Vysis
Advanced Imaging System).
By using the 300/200 kb probe, the t(9;22)-BCR/ABL fusion
1719
FISH vs CCA for monitoring Ph+ CML
A Cuneo et al
1720
was recognized directly at the microscope in the presence of
unequivocal overlapping of red/green signals (yellow fusion
signal); whereas the random positioning of two red and two
green spots was interpreted as indicative of the absence of
BCR/ABL fusion (Figure 1). In the presence of touching
red/green signals, the image was visualized on the screen and
only those cells with unequivocal overlapping were scored as
Ph-positive. Signal screening was performed by one of us (RB)
who had no knowledge of the results of CCA. Cells with
touching and/or overlapping green/red signals were scored as
Ph-positive in those samples hybridized with the smaller
35.5/39 kb cosmid probes.
Results
Table 1
Outcome of FISH studies in 10 samples
% fusion signals (200 cells observed)
Undiluted
(FISH)
92
90
79
65
64
42
28
24
22
10
1:1 dilution
(FISH)
45
42
39
30
30
20
15
11
9
4
Controls, dilution samples and probe-sets
The cut-off point for recognizing a case as Ph-positive
(300/200 kb BCR/ABL probe) was set at the mean percent
value of cells exhibiting a fusion signal that was observed in
normal controls, plus 3 s.d. (0.748% + 3 × 0.564% = 2.44%).
A 2.3% cut-off was used with the smaller 35.5/39 kb probe
(mean false positivity rate 0.727%).
A very good overlap was observed between the expected
values and the observed values obtained by FISH analysis of
dilution samples, as shown in Table 1.
The estimated frequencies of Ph+ cells using the 300/200 kb
probes were not significantly different as compared with those
observed using the smaller 35.5/39 kb probes, with a 2.3%
mean variation (range 0–5%) in 22 specimens (Figure 2).
Frequency of Ph-positive cells: CCA vs interphase
FISH
The mean percentage of Ph-positive metaphases in 26 patients
analyzed at diagnosis was 81.1%, as compared with a 83.7%
mean percentage of interphase cells with fusion signals. In the
remaining 72 samples (including 70 samples obtained after
IFN treatment plus two samples without information on the
disease status), 38.3% and 41.4% of Ph-positive cells (mean
values) were found by CCA and by interphase FISH, respectively. Globally, the observed differences in the frequency of
Ph-positive cells in these 98 tests ranged between 0% and 9%,
median value 4%: a close correlation was observed between
the percentage values of Ph-positive cells as assessed by CCA
and by interphase FISH (see Figure 3, r = 0.994; r2 = 0.988;
P ⬍ 0.0001).
Observed differences in the frequency of Ph+ cells in three
cytogenetic groups (low, medium and high percentage of Ph+
metaphases), corresponding to different categories of cytogenetic response in clinical trials, are shown in Table 2.
FISH detected 2.5–8% interphase cells with BCR/ABL fusion
in seven out of nine specimens taken from six patients without
Ph+ metaphases (17–50 cells evaluated) after IFN therapy (see
Table 3).
FISH in non-cultured samples and 24-h cultures
A total of 30 samples was analyzed by FISH after direct harvesting and after 24-h culture, yielding a very good correspondence between the observed values (r = 0.997, r2 = 0.995; P
Figure 1
Large overlapping signals (yellow) in a BCR/ABL-positive CML at diagnosis, studied with the 300/200 kb probes (left panel). An
example of hybridization pattern with the smaller 35.5/39 kb BCR/ABL probe is shown in the right panel. The intensity of the signals is weaker
and those cells with signals in close proximity to each other (arrows) were scored as BCR/ABL positive.
FISH vs CCA for monitoring Ph+ CML
A Cuneo et al
in PB samples were 6–36% of all white blood cells, whereas
they were less than 6% in BM samples.
Discussion
Figure 2
Correlation between the frequency of Ph+ interphase
nuclei as determined by 35.5/39 kb BCR/ABL probes (Y-axis) and by
300/200 kb probes (X-axis) in 22 cases.
Figure 3
Linear regression line (X-axis: % cells positive by FISH;
Y-axis: % cells positive by CCA), showing the correlation between the
frequency of t(9;22) as assessed by conventional cytogenetics and by
interphase FISH using a 300/200 kb BCR/ABL probe in 98 tests.
⬍ 0.0001) (Figure 4). Observed differences ranged between 0
and 8%, median value 2%.
FISH in PB and BM samples
As shown in Table 4, moderately higher frequencies of Phpositive cells were detected by FISH in BM samples than in
PB samples in 11 patients, with observed variations ranging
between 7 and 26.3% (median value 12%). The lymphocytes
Despite being relatively quick and simple and showing an
excellent correlation with clinical outcome,15–17 CCA has the
following limits: (1) It is a relatively insensitive assay; under
the conditions in use in published clinical trials, where 20–
30 karyotypes were analyzed in the majority of cases3,16,17 this
technique cannot theoretically detect a disease ⬍3–5% of the
bone marrow cells. This problem may be complicated by the
presence of hypocellular or fibrotic marrow yielding a scanty
dilute aspirate. (2) It is not reliable in the quantification of
minor clones, because, under these circumstances, few
observed events cause a wide shift in the resulting percentage
of Ph-positive cells. (3) It is dependent on the ability of BM
cells to divide in culture. This aspect may be potentially
important in those patients treated by IFN, a molecule having
a potent inhibitory effect on the growth of Ph-positive cells.
(4) In the absence of circulating granulocyte precursors, BM
samples are required, making the frequent assessment of the
frequency of Ph-positive cells impractical.
Although FISH has several potential advantages over
CCA,18–20 it has not been standardized yet, with special reference to the estimation of the frequency of Ph-positive cells.21
Because a number of technical factors, including the type
of probes, the quality of the sample cells and the size of
interphase nuclei,8–10,21,22 were shown to significantly influence the interpretation of results, we tested two probe combinations and prepared the slides for FISH analysis by processing all samples by a standard cytogenetic method,
including swelling of the cells by hypotonic treatment.
To reduce the potentially high false-positive rate of
BCR/ABL fusion in interphase cells, deriving from random juxtaposition of the large 300/200 kb probe signals,10 we adopted
the restrictive criterion of counting as Ph positive only those
nuclei with at least partially overlapping signals (Figure 1) with
a resultant 2.4% lower cut-off rate for the recognition of
BCR/ABL-positive cases. Interestingly, no significant difference
was observed in the estimation of the numbers of Ph-positive
nuclei in 22 patients using these large probes and the smaller
35.5/39 kb BCR/ABL probe (see Figure 2), producing touching
signals or minimal red/green overlapping in Ph-positive
nuclei.
Globally, an excellent correspondence was observed
between CCA and FISH in the estimation of the size of the
Ph-positive clone, with moderate overestimation of the frequency of Ph-positive cells by FISH occurring both in
untreated patients (diagnostic samples) and in follow-up
samples. These differences were more significant at low-tomedium values of Ph positivity than at high values (see Table
2 and Figure 3). Noteworthy, similar data were reported by
Amiel et al5 in their comparative study of CCA and FISH in
the detection of residual disease in 11 CML patients.
These findings, together with the demonstration of a very
good overlap of expected/observed rate of Ph-positive nuclei
in dilution experiments, suggest that dual-color FISH may
represent an accurate method for the quantification of the
Ph-positive clone both at diagnosis and after IFN therapy
The employment of FISH may have implications in clinical
practice: it is noteworthy that a minority of BCR/ABL-positive
cells persisted after IFN therapy in seven out of nine specimens showing no Ph+ cell in 17–50 analyzed metaphases.
1721
FISH vs CCA for monitoring Ph+ CML
A Cuneo et al
1722
Table 2
Comparison of CCA and FISH in three cytogenetic groupsa
Group 1 (0–33% Ph+)
(32 samples)
% Ph+
Mean
Range
Group 3 (⬎66%*)
(40 samples)
Group 2 (34–66%)
(26 samples)
CCA
FISH
CCA
FISH
CCA
FISH
9.5
0–30
15.4
2–39
50
35–65
57
42–73
85.6
70–100
87.9
75–99
Difference of % Ph+ cells
Mean
Range
6.7
2–9
7.8
4–9
4
0–9
a
These three groups, with low (0–33%), medium (34–66%) and high (⬎66%) frequency of Ph-positive metaphase cells were separated
because they correspond to different categories of cytogenetic responses in clinical trials (ie complete/major, minor, minimal).
Table 4
Table 3
Outcome of cytogenetic and FISH investigations in six
patients with ‘complete’ cytogenetic response
Patient
1
2
3
4
5
6
Duration of
IFN therapy
months
CCA
No. of Ph+
cells
FISH
% BCR/ABL
fusion %
17
30
18
22
26
36
60
36
12
0/40
0/34a
0/45
0/50
0/50
0/17a
0/20a
0/50
0/43
7
8
5
⬍1b
⬍1b
8
2.5
3
7
a
Few cells obtained at BM aspiration.
RT-PCR negative.
b
Figure 4
Correlation between the frequency of BCR/ABL fusion in
30 BM samples cultured for 24 h (Y-axis) and directly harvested (Xaxis).
Outcome of FISH analysis on PB and BM in 11 patients
Percentage of Ph-positive cells
Case
1
2
3
4
5
6
7
8
9
10
11
CCA
BM FISHa
PB FISH
%
lymphocytes
(PB)
65
25
70
75
55
60
90
80
85
40
15
68
32
68
75
64
65
86
79
86
50
25
60
25
60
66
50
38.7
78
53
74
25
5
6
7
10
6
NA
36
15
20
5
10
30
NA = not available.
a
BM lymphocytes were ⬍6% in all cases.
Because the stratification of CML patients under IFN treatment
according to the degree of karyotypic response (ie complete,
major, minor or minimal) proved to be prognostically significant,4 the precise determination of the frequency of mutated
cells may be important, especially in those patients with an
inadequate number of analyzable metaphases.
Frequent sampling may be required in the follow-up of
cytogenetically responder patients, especially after detection
of initial cytogenetic relapse and the possibility of directly
assessing the percentage of Ph-positive cells on PB samples
would be very practical in a clinical setting. For this reason,
we studied in parallel BM samples prepared by direct harvesting and by 24-h culture, documenting an excellent correspondence between observed values by either method and
subsequently compared the results obtained on direct harvests
of PB and BM samples. Moderate underestimation of the percentage of Ph-positive cells was observed in PB preparations
compared with BM samples and a positive correlation was
noted between the degree of underestimation and the percentage of PB lymphocytes. Similar findings were recently
reported by Muhlman et al,23 who calculated the proportion
of BCR/ABL+ nuclei relative to the percentage of myeloid
elements, in order to reduce counting errors deriving from the
presence of B or T lymphocytes. The involvement of lymphocytes in CML is a matter of controversy and this issue was
recently studied and reviewed by Haferlach and colleagues,24
who showed that 25–50% of lymphocytes carried BCR/ABL
fusion in CML chronic phase.
FISH vs CCA for monitoring Ph+ CML
A Cuneo et al
However, in our study a significantly lower percentage of
Ph-positive nuclei compared to BM samples, was detected in
two cases (Nos 10 and 11, Table 4) even when considering
the percentage of lymphocytes as a possible correction factor.
A similar intriguing phenomenon was noted in 2/6 cases by
Sinclair et al,10 who suggested that selective retention and/or
destruction of Ph-positive granulocytes may occur in the BM.
The precise mechanisms underlying this phenomenon remain
obscure and it is noteworthy that both our cases and those by
Sinclair et al were treated by IFN. At variance with these findings is the observation of comparable levels of BCR/ABL+ cells
in PB and BM granulocytes in other studies.9,25
In conclusion, we have shown that dual-color FISH enables
an accurate detection and monitoring of the size of the Phpositive clone in CML under IFN. While our findings are
reassuring with respect to the reliability of conventional cytogenetics in the follow-up of CML, interphase cytogenetics
offers the advantage of being more sensitive and accurate,
especially in cytogenetically responder patients, having a minority of Ph-positive cells. Sequential testing of stored fixed
cytogenetic preparations of BM samples without prior culture
provides excellent results, giving the opportunity of
performing centralized FISH analysis in the context of
multicentre trials.
Although the direct estimation of Ph-positive cells on PB is
as reliable as the traditional assessment on BM in the majority
of cases, unpredictable underestimation of the size of the Ph+
clone may occur on PB samples, suggesting that major
responses on PB should be confirmed on BM samples.
Acknowledgements
This work was supported by Biomed I, CA CT 1994–1703 to
to AH and AC and by MURST fondi 40% and 60% to GLC
and AC.
9
10
11
12
13
14
15
16
17
18
19
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