Leukemia (2003) 17, 1124–1129
& 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00
www.nature.com/leu
Patterns of BCR/ABL gene rearrangements by interphase fluorescence in situ
hybridization (FISH) in BCR/ABL+ leukemias: incidence and underlying genetic
abnormalities
D Primo1,3, MD Tabernero4, A Rasillo1,3, JM Sayagués1,3, AB Espinosa1,3, MC Chillón5, R Garcia-Sanz5, N Gutierrez5, M Giralt6,
A Hagemeijer7, JF San Miguel3,5 and A Orfao1,2,3
1
Servicio General de Citometrı́a, Spain; 2Departamento de Medicina, Spain; 3Centro de Investigación del Cáncer, University of
Salamanca, Spain; 4Unidad de Investigación, and 5Servicio de Hematologı́a, Hospital Universitario of Salamanca, Salamanca,
Spain; 6Servicio de Hematologı́a, Hospital Miguel Servet, Zaragoza, Spain; and 7Center for Human Genetics, University of
Leuven, Leuven, Belgium
Interphase fluorescence in situ hybridization (iFISH) is increasingly used for the identification of BCR/ABL gene rearrangements in chronic myeloid leukemia (CML) and acute
lymphoblastic leukemia (ALL). In the present study, we have
explored the incidence of both typical and atypical iFISH
patterns of BCR/ABL gene rearrangements in a series of 168
consecutive BCR/ABL+ patients – 135 CML, 31 precursor B-ALL
and two acute myeloblastic leukemia (AML) cases – and
established their underlying genetic alterations through further
molecular and chromosome analyses. Two different FISH
probes (Vysis Inc., Downers Grove, IL, USA) were used: the
LSI BCR/ABL dual color extra signal (ES) and the dual color
dual fusion BCR/ABL probe (D-FISH). Our results show that
most BCR/ABL+ patients (83%, including 88% of all CML, 61% of
ALL and one of two AML) displayed typical iFISH patterns of
either Major (M) BCR/ABL (87% of CML, 13% of ALL and one of
the two AML) or minor (m) BCR/ABL gene rearrangements (1%
of all CML and 48% of ALL cases) with the two probes. Further
molecular and cytogenetic studies confirmed the presence of
such typical rearrangements in all except one of these ALL
cases who had coexistence of an MBCR/ABL and an mBCR/
ABL gene rearrangement together with monosomy 9. In the
remaining 29 cases (17%), up to five different atypical iFISH
patterns were detected with the ES probe. Atypical iFISH
patterns were most frequently due to additional numerical
changes – most often supernumerary Philadelphia (Ph) chromosome (7%) but also gain or loss of chromosome 9 (1%) or 22
(1%). Deletion of 9q sequences proximal to the breakpoint were
also frequently observed with the ES probe (8%). Application of
the D-FISH probe showed that in most of these latter cases (5%)
deletion of 22q sequences distal to the breakpoint also
occurred. The remaining cases with atypical iFISH had cryptic
insertion of BCR in 9q34 (1%). Exact interpretation of each
iFISH pattern was supported by FISH on metaphases and
molecular determination of the BCR breakpoint. In summary,
our results indicate that despite the high incidence of typical
iFISH patterns of BCR/ABL gene rearrangements, atypical
patterns are also found in BCR/ABL+ acute leukemias; the
precise definition of the alteration present in individual cases is
dependent on metaphase studies and molecular definition of
the breakpoint.
Leukemia (2003) 17, 1124–1129. doi:10.1038/sj.leu.2402963
Keywords: BCR/ABL; FISH; Ph chromosome; CML; ALL; AML
rearrangements in around 5% of all chronic myeloid leukemia
(CML) patients1–4 and a relatively high proportion of acute
lymphoblastic leukemia (ALL) cases.4,5 In addition, about 5% of
CML cases carry ‘masked’ translocations that can only be
detected by molecular techniques such as fluorescence in situ
hybridization (FISH) or RT-PCR.6
The first generation of BCR/ABL single fusion FISH probes
detected the fusion gene with high specificity but with a low
sensitivity (false positive rate X5%).7 A new generation of FISH
probes has been developed with probes extending up to 450 Kb
proximately to the ABL gene on 9q34 and in some probes also
with distant coverage on 22q.8 The rational was to define the
t(9;22) by two FISH events: a fusion signal and an extra signal
(ES) corresponding either to the remaining probe on the 9q+ or to
a second fusion on the 9q+, according to the breakpoint
localization in the BCR gene. With these new probes – dual
color extra signal (ES) and dual color dual fusion (D-FISH) – the
cutoff rate for false positives has dropped significantly to o1%.9
With the ES probes, the pattern of interphase FISH (iFISH) signals
observed is distinct for the major (M) and minor (m) breakpoint
in the BCR gene (MBCR or mBCR), whereas the D-FISH probe
design does not distinguish between these two events. Additional variations to the typical iFISH patterns can be caused by
numerical changes such as gain of the Philadelphia (Ph)
chromosome, and to deletions of sequences proximal to the
breakpoint on chromosome 9 or distal to the breakpoint on
chromosome 22.8–14
The aim of this study was to determine the incidence of
atypical iFISH patterns of BCR/ABL gene rearrangements and to
establish the specific underlying molecular abnormalities
carried by the neoplastic cells, in a large series of 168
consecutive BCR/ABL+ leukemias, which included CML, precursor B-ALL and AML cases.
Material and methods
Patients
Introduction
For many years, conventional karyotyping has been used as the
sole diagnostic tool for t(9;22). However, it has several
limitations that may lead to failure for detecting BCR/ABL gene
Correspondence: Professor A Orfao, Servicio General de Citometrı́a,
Laboratorio de Hematologı́a, Hospital Universitario Salamanca, Paseo
de San Vicente 58-182, Salamanca 37007, Spain;
Fax: +34 923 29 46 24
Received 5 August 2002; accepted 17 February 2003
A total of 168 consecutive patients – 135 CML, 31 precursor BALL and two AML – who were studied at the onset of the disease
and who showed BCR/ABL gene rearrangements by iFISH, were
included in the study. Diagnosis of CML and ‘de novo’ acute
leukemias was performed according to the FAB15 and the
WHO16 classifications, respectively; the EGIL criteria17 were
used for the immunological classification of acute leukemias.
In all cases, heparin-anticoagulated fresh bone marrow (BM)
aspirate samples were obtained at diagnosis and used for the
cytogenetic studies described below.
Patterns of BCR/ABL gene rearrangements by interphase fluorescence
D Primo et al
Conventional karyotyping
BM aspirate samples were cultured for 24 and 48 h following
standard procedures.18 Chromosomes were stained by Giemsa
and the karyotypes described according to the ISCN recommendations.
FISH studies
FISH analysis for the t(9;22) translocation was initially
performed on both interphase nuclei and metaphase chromosomes using the LSI-BCR/ABL dual color ES probe (Vysis Inc.,
Downers Grove, IL, USA) (Figure 1). In addition, the LSI-BCR/
ABL D-FISH DNA probe (Vysis Inc.) (Figure 1) was also used in a
subgroup of 49 patients – 37 CML, 11 precursor B-ALL and one
AML ANL. Whenever appropriate, the CEP 9 probe (Vysis Inc.)
and a biotinylated probe that simultaneously identifies the
centromeric repetitive DNA sequences of chromosomes 14 and
22 (Cambio, Cambridge, UK) were also used.
FISH analyses were performed on BM cells prepared
according to conventional cytogenetic techniques and placed
on slides. Hybridization with fluorochrome-labelled FISH
probes was performed according to the recommendation of
the manufacturers with slight modifications.19 For the biotinylated probe, appropriate immunologic detection of hybridization was performed using avidin–FITC.7
Fluorescence signals were evaluated using a BX60 fluorescence microscope (Olympus, Hamburg, Germany) and a 100
oil objective. For all slides measured, the number of unhybridized cells in the areas assessed was o1% and only those spots
with a similar size, intensity and shape were counted (minimum
of 200 cells/sample). The iFISH patterns obtained in normal cells
– two green, two red, no fusion signals – are shown in Figure 2
for both the ES (pattern N) and the D-FISH (pattern n) probes.
Based on the analysis of nuclei from 20 BCR/ABL BM samples,
a case was considered to be BCR/ABL+ once the percentage of
BCR/ABL+ nuclei was X0.9% (mean number plus two standard
deviations of nuclei showing a typical MBCR/ABL iFISH
pattern).
1125
Molecular analysis of BCR/ABL transcripts
RNA was extracted from washed BM mononuclear cells by the
guanidium thiocyanate method.20 RT-PCR analysis of the fusion
gene transcripts was carried out according to the guidelines of
the Biomed-1 Concerted Action.21 This procedure includes a
reverse transcription of the RNA and a double round PCR of the
cDNA, with the identification of the amplicons through agarose
gel electropheresis and ethidium bromide staining. The procedure also includes the check of the RNA integrity through
amplification of the normal ABL RNA and a control PCR (shifted
PCR) for confirmation or exclusion of false-positive results.
Results
The great majority of the patients analyzed (139/168, 83%)
including most CML (119/135, 88%) and ALL (19/31, 61%) and
one of two AML cases, displayed typical iFISH patterns of BCR/
ABL gene rearrangements involving either the MBCR (pattern A
from Figure 2; n ¼ 122) or the mBCR breakpoints (pattern B from
Figure 2; n ¼ 17), with the ES probe. Pattern A consisted of one
fusion-der (22)-, one green-nonrearranged 22- and two red-der
(9) plus the nonrearranged chromosome 9- signals; in pattern B
the red signal corresponding to der(9) is replaced by a second
fusion. Most cases with a typical MBCR/ABL pattern (pattern A)
corresponded to CML cases (117 out of 122 MBCR/ABL+ cases;
96%) and only a few were patients with ALL (n ¼ 4) or AML
(n ¼ 1). In turn, all except two cases with a typical mBCR/ABL
iFISH pattern (pattern B) corresponded to ALL patients (n ¼ 15);
the other two were CML. Additional iFISH analyses with the DFISH probe were performed in 20 and six cases with typical
TO THE CENTROMERE
TO THE TELOMERE
9q34 region
ABL gene – 225 Kb
ASS gene – 65 Kb
Exon 1b
5’
Exon 1a
Exon 2
Exon 11
3’
CHROMOSOME 9
LSI-ES probe
D-FISH probe
650 Kb
22q11.2 region
TO THE CENTROMERE
m
B
C
R
IgLV segments
5’
CHROMOSOME 22
LSI-ES probe
BCR gene
TO THE TELOMERE
M
B
C
R
3’
300 Kb
D-FISH probe
600 Kb
300 Kb
600 Kb
Figure 1
Schematic representation of the two different BCR/ABL gene probes used in the present study: LSI BCR/ABL extra signal dual color
(ES) probe and D-FISH BCR/ABL dual color dual fusion (D-FISH) probe.
Leukemia
Patterns of BCR/ABL gene rearrangements by interphase fluorescence
D Primo et al
1126
D-FISH probe
ES- probe
+
Typical BCR/ABL interphase FISH patterns
A
ab
B
Atypical BCR/ABL+ interphase FISH patterns
C
cd
D
e
E
f1
F
f2
G
g
Interphase FISH patterns of Normal BCR/ABL nuclei
N
n
Figure 2
Representative interphase FISH (iFISH) patterns found with the LSI BCR/ABL extra signal dual color (ES) probe (panels in the left
column) and the D-FISH BCR/ABL dual color dual fusion (D-FISH) probe (panels in the right column). Representative photographs and schemes of
nuclei carrying typical MBCR/ABL (panels A and ab) and mBCR/ABL (panels B and ab) gene rearrangements are shown in the upper part of the
figure. In the middle part, atypical BCR/ABL iFISH patterns are shown: supernumerary Philadelphia (Ph) chromosome with either MBCR/ABL gene
rearrangements (panels C and cd) or mBCR/ABL gene rearrangements (panels D and cd) alone or supernumerary Ph with mBCR/ABL gene
rearrangements associated with gain of the nonrearranged chromosome 22 (panels E and e); atypical BCR/ABL fused gene rearrangements on
chromosome 9 or 9q deletion of the rearranged chromosome 9 (panels F and f1); coexistence of der(9q) and der(22q) deletions (panels F and f2)
and; mBCR/ABL gene rearrangements associated to 9q deletion of the non-rearranged chromosome 9 (panels G and g). The lower part of the figure
displays representative iFISH patterns found in normal nuclei for the two probes used: panels N (ES probe) and n (D-FISH probe).
Leukemia
Patterns of BCR/ABL gene rearrangements by interphase fluorescence
D Primo et al
1127
patterns A and B, respectively. All these cases showed an
identical and typical iFISH pattern with this probe (pattern ab
from Figure 2), this probe not allowing distinction between
MBCR and mBCR breakpoints.
As may be seen in Table 1, further metaphase and molecular
studies showed that all except one case with typical iFISH
patterns had conventional t(9;22) involving either the MBCR
(pattern A in Figure 2) or the mBCR breakpoints (pattern B in
Figure 2); in the remaining case metaphase studies showed that
pattern B was because of a double Ph associated to loss of
der(9); coexistence of both p190 and p210 transcripts were
found in this ALL patient.
In the remaining 29 cases (17%) – 16 CML, 12 ALL and one
AML – up to five different atypical iFISH patterns were detected
with the ES probe (patterns C to G in Figure 2). In order to
identify the exact molecular/genetic abnormalities involving the
BCR and ABL genes in each of these atypical iFISH patterns,
further PCR studies, conventional cytogenetics and/or FISH
analyses of both metaphase and interphase chromosomes 9 and
22, were performed.
As illustrated in Figure 2 and shown in Table 1, pattern C (two
fusion, one green and two red signals) was found in five cases
(3%) (three CML and two ALL) all of whom had p210 transcripts
compatible with the occurrence of an MBCR breakpoint;
metaphase studies performed in these cases confirmed the
presence of an extra fusion signal, as compared to a typical
iFISH MBCR/ABL pattern (pattern A), because of the presence of
a supernumerary Ph (Table 1). In turn, all cases showing nuclei
with three fusion signals associated with one red and either one
(pattern D; n ¼ 8, 5%) or two (pattern E; n ¼ 1, 1%) green signals
were ALL and had p190 transcripts/mBCR breakpoint; analysis
of metaphase chromosomes confirmed in all except one of these
ALL cases that the additional third fusion signal, as compared to
typical mBCR/ABL iFISH pattern B, was because of a supernumerary Ph; in the other case, who had atypical pattern D,
instead of corresponding to an additional Ph, the third fusion
Table 1
Discussion
In the present study, we show that the use of a new generation of
BCR/ABL FISH probes in interphase nuclei of a large series of
BCR/ABL+ leukemias is associated with the observation of a
Distribution of typical and atypical Interphase FISH patterns with the ES probe in BCR/ABL+ leukemias studied at diagnosis (n=168)
iFISH pattern with
BCR/ABL ES probea
Chromosome localization of signalsb
F
R
G
1G (22)
1G (22)
1G (22)
A: 1F 2R 1G
B: 2F 1R 1G
1F (Ph)
2F (Ph; 9q+)
2F (Ph, Ph)
2R (9,9q+)
1R (9)
1R (9q)
Cc: 2F 2R 1G
Dd: 3F 1R 1G
2F
3F
3F
3F
1F
1F
2R
1R
1R
1R
1R
1R
E: 3F 1R 2G
Fe: 1F 1R 1G
G: 2F 1G
was because of an additional der(9) In the ALL case who
displayed pattern E with a double Ph, the second green signal
(BCR probe) observed was because of an additional gain of a
nonrearranged chromosome 22.
Among all atypical iFISH patterns, pattern F (one fusion, one
red and one green signals) was the most frequently observed: 14
cases (8%), corresponding to 13 CML and one AML. Despite
having an identical iFISH pattern with the ES probe, this group
was shown to be genetically rather heterogeneous. Accordingly,
metaphase studies showed that in some of these CML cases with
p210 transcripts (11/13 cases) extensive deletions of 9q
sequences proximal to the breakpoint occurred explaining the
loss of one red signal – as compared to typical MBCR/ABL iFISH
pattern A; in contrast, in the other two CML patients the
presence of a single red signal was because of the existence of
cryptic insertion of BCR in 9q34. In the remaining patient with
atypical iFISH pattern F corresponding to an AML, p190
transcripts were detected and loss of a fusion signal, as
compared to typical mBCR/ABL iFISH pattern B, was explained
by the existence of an extensive deletion of der(9q). The
application of the D-FISH probe in these cases further showed
the existence of deletions on 22q sequences distal to the
breakpoint – pattern f2 from Figure 2 – in nine of the 11 CML
cases who had del(9q) but not in the other two cases.
Pattern G (two fusions, one green and no red signal with the
ES probe) was found in one ALL case who had p190 transcripts.
Metaphase studies confirmed the absence of an ABL probederived red signal because of del(9q) of the nonrearranged
chromosome 9.
(Ph,
(Ph;
(Ph;
(Ph;
(Ph)
(9)
Ph)
Ph, 9q+)
9q+, 9q+)
Ph, 9q+)
1F (Ph)
2F (22, 9)
(9,9q+)
(9)
(9)
(9)
(9)
(9)
1R (9)
0R
1G
1G
1G
2G
1G
1G
Number of cases (%)
CML
(n=135)
ALL
(n=31)
3 (2%)
0
0
0
11 (8%)
2 (1%)
1G (22)
1G (22)
0
0
Interpretation
p210
p190
p190 and
p210
p210
p190
p190
p190
p210
p210
t(9;22), MBCR
t(9;22), mBCR
t(9;22), +Ph, monosomy 9,
MBCR and mBCR
t(9;22), +Ph, MBCR
t(9;22), +Ph, mBCR
t(9;22), +9q+, mBCR
t(9;22), +Ph, +22, mBCR
t(9;22), MBCR, del(9q+)
Cryptic insertion of BCR in
9q34, MBCR
t(9;22), mBCR, extensive del(9q+)
t(9;22), mBCR, extensive del(9q)
AML
(n=2)
117 (87%) 4 (13%) 1
2 (1%) 14 (45%) 0
0
1 0
(22)
(22)
(22)
(22)
(22)
(22)
RT-PCR
transcripts
2 (6%)
7 (23%)
1
1
0
0
0
0
0
0
0
0
0 1f
1 0
p190
p190
a
Patterns as observed with the LSI BCR/ABL ES (ES) probe (Figure 1); corresponding patterns with the dual fusion BCR/ABL (D-FISH) probe
(Figure 1) are illustrated in Figure 2 in parallel. F=fusion signal, R=red signal, G=green signal.
b
Data based on metaphase FISH results and use of enumeration probes for chromosomes 9 and 22; Ph=der(22) t(9;22)(q34;q22), 9q+= der(9)
t(9;22).
c
In two CML and one ALL cases, mosaicism with nuclei showing pattern A and in a subpopulation pattern C.
d
7/8 ALL with pattern D showed nuclei with pattern B in a significant proportion of cells.
e
Nine cases showed del(9q) in association with del(22q) with the D-FISH probe; in the other two cases only del(9q) was found with the D-FISH
probe.
f
Mosaicism with pattern B in 78% and pattern F in 15% of the nuclei.
Leukemia
Patterns of BCR/ABL gene rearrangements by interphase fluorescence
D Primo et al
1128
variable number of different iFISH patterns. The most frequently
detected patterns with the ES probe corresponded to typical
BCR/ABL gene rearrangements involving the MBCR (one
fusion, one green (BCR probe) and two red (ABL probe) signals)
and the mBCR (two fusion, one green and one red signals)
breakpoints; as expected, distinction between these two breakpoint regions could not be achieved with the D-FISH probe.
Although both patterns A and B were found both in CML and
ALL cases, the former was more frequently observed in CML
while the latter was usually associated with ALL. Interestingly, in
all ALL showing a typical MBCR/ABL pattern, iFISH studies
showed the presence of MBCR/ABL gene rearrangements not
only in blast cells but also in residual neutrophils (data
not shown). These results could suggest that these patients
might actually correspond to blast crisis of CML arising
from a multipotent stem cell, who present as an apparently
‘de novo’ ALL.
Since additional karyotypic changes (eg supernumerary Ph,
gain or loss of chromosomes 9 and 22, as well as deletions of 9q
and 22q) can occur in BCR/ABL+ CML, ALL and AML cases,8–14
other atypical iFISH patterns could be expected as observed in
around one-sixth of all cases studied; conversely, cases with
additional cytogenetic changes could present as a typical
pattern and potentially be misdiagnosed with iFISH. Exact
interpretation of the atypical iFISH patterns obtained in
individual cases was dependent on the analysis of metaphases
complemented with molecular definition of the breakpoint.
Accordingly, metaphase analyses associated to molecular
studies showed that in cases with atypical iFISH patterns
presence of an additional Ph and deletions of 9q sequences
proximal to the breakpoint, were the two most frequent
underlying genetic abnormalities. However, none of these two
abnormalities was associated with one single atypical iFISH
pattern.
Accordingly, gain of a Ph was usually characterized by the
presence of an additional fusion signal in both MBCR/ABL+ and
mBCR/ABL+ cases, with the two probes. However, among cases
in the latter group, gain of an extra fusion signal was not specific
of supernumerary Ph, since it was also observed in association
with gain of der(9). In turn, not all ALL cases showing a
supernumerary Ph had this atypical iFISH pattern: in one case,
supernumerary Ph was associated with an additional gain of the
nonrearranged chromosome 22 that translated into a different
iFISH pattern; in another patient, gain of a Ph was associated
with del(9q) and; in a third ALL, a double Ph coexisting with
both p210 and p190 mRNA transcripts in association with
monosomy 9 was observed leading to a typical iFISH pattern.
The presence of a double Ph in CML as well as in ALL has been
previously reported as a secondary genetic change.22 In line
with this, in almost all cases with a supernumerary Ph, nuclei
carrying a double Ph chromosome coexisted with a variable
proportion of nuclei displaying typical iFISH patterns. In line
with the previous observations, the presence of a double Ph with
mBCR breakpoints was exclusively found among ALL patients
while gain of a second Ph in MBCR/ABL+ cases was observed
both among CML and ALL cases. Interestingly, our results
suggest that among MBCR/ABL+ cases studied at diagnosis, the
presence of a double Ph would be more frequently observed in
ALL than in CML.
Coexistence of p190 and p210 transcripts have been reported
in ALL23 and could be due either to the presence of a double Ph
chromosome, each carrying a different breakpoint, or, more
commonly, to the presence of a typical MBCR/ABL translocation
in which the p190 is generated by alternative splicing of MBCR/
ABL mRNA.24
Leukemia
Der(9q) deletions were found only among MBCR/ABL+ CML
and in one AML; with the ES probe they constantly translated
into loss of one red signal from der(9q). A similar incidence of
del(9q) to that found in our study has been previously described
in CML.8,13,14 Such deletions typically expand up to 5.5 Mb14
on chromosome 9q sequences proximal to the breakpoint site of
the translocation, therefore, hampering visualization of the
residual ABL probe on der(9). Interestingly, all MBCR/ABL+
nuclei from all these CML cases showed del(9q) confirming that
both abnormalities – t(9;22) and del(9q) – might represent a
single one-step event.14 Most CML cases with del(9q) also had
deletions of 22q sequences distal to the breakpoint. As
expected, this latter abnormality could only be detected with
the D-FISH probe and it also involved all BCR/ABL+
nuclei. Although both mBCR/ABL and MBCR/ABL gene
rearrangements have been reported in AML,25 to the best of
our knowledge this is the first report in which coexistence of an
extensive del(9q) and mBCR/ABL gene rearrangements are
described in AML.
Despite the association found between loss of a red signal in
MBCR/ABL+ nuclei and extensive del(9q), this atypical iFISH
pattern was not specific of this cytogenetic abnormality.
Accordingly, an identical pattern was found in two CML cases
who were Ph-negative and had no del(9q). Informative
metaphase FISH analyses showed in both cases a cryptic
insertion of BCR in 9q34. The exact mechanisms responsible
for the aberrant localization of the BCR/ABL fused gene on
chromosome 9 instead of chromosome 22 are not entirely clear.
Previous reports on Ph CML cases in which the BCR/ABL
fusion gene is located on chromosome 9q34 suggest that either
an interstitial insertion of the BCR gene from chromosome 22
into the ABL gene on chromosome 9 or two successive
translocations including a standard t(9;22) followed by a
translocation between der (9q) and der(22q) could occur.26 In
the two cases reported here, insertion of 50 BCR sequences
could not be confirmed through chromosome painting (data not
shown) probably because of the small size of the fragment
inserted in the ABL gene. It should be noted that no interphase
nuclei with a typical MBCR/ABL iFISH pattern were observed in
any of these cases, which could help to explain the underlying
mechanism for this genetic abnormality.
In summary, our results indicate that although iFISH can
correctly diagnose many BCR/ABL+ patients, exact interpretation of individual cases requires the application of the probes on
metaphases complemented with molecular definition of the
breakpoint.
Acknowledgements
MD Tabernero is supported by a grant from the MCYT, programa
Ramón y Cajal. JM Sayagues is supported by a grant (02/9103)
from Ministerio de Sanidad y Consumo, Madrid, Spain.
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