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ABSTRACT: The molecular basis for blast transformation of chronic myeloid leukemia (CML) remains
poorly understood. Cytogenetic alterations associated with CML blast crisis have previously been exten-
sively studied by conventional G-banding analysis. However the complexity of some chromosome
abnormalities or poor chromosome morphology or both has exceeded the resolution of G-banding anal-
ysis in a significant proportion of CML cases, and complex chromosome rearrangements have remained
unidentified. In this study, comparative genomic hybridization (CGH) was used to elucidate genome
imbalances in chronic phase or blast crisis samples or both from 12 CML patients. CGH and G-banding
results were compared, and discrepancies were further clarified by using multipaint chromosome anal-
ysis and locus-specific DNA probes. No imbalances were detected in the 4 early disease phase samples
studied. Eleven blast crisis samples were analyzed by G-banding and CGH, and the commonest genomic
abnormality detected was overrepresentation of the long arm of chromosome 8, which was detected in 5
patients. This overrepresentation was attributable to trisomy 8 in 4 patients, whereas amplification of
the entire long arm of chromosome 8 was detected in 1 patient. The formation of isochromosomes of the
long arm of chromosome 8 was observed as a mechanism for gene amplification in this patient. Addi-
tional material originating from chromosome 8 was also observed intercalated into three marker chro-
mosomes in peripheral blood metaphase spreads from this patient. These markers may further define
areas on chromosome 8 that harbor oncogenes implicated in transformation of chronic myeloid
leukemia. Elsevier Science Inc., 1999. All rights reserved.
blast crisis patients revealed that only 1 case, with blast sis of DNA from CML patients provides the potential for
cells of a lymphoid phenotype, carried an N-RAS muta- detecting occult genetic changes. The technique includes
tion [7]. cohybridizing differentially labeled tumor and reference
An increased expression of the c-MYC gene, which genomic DNA onto normal metaphase chromosomes. The
maps to chromosome locus 8q24, has been suggested to be fluorescence intensity ratio (FR) on each chromosome is
associated with disease progression in a proportion (20%) then measured to identify areas that are over- or underrep-
of CML cases [8]. The author speculates that an increase in resented in the tumor DNA sample. CGH has been widely
gene expression could serve as an alternative to trisomy 8. used in the past 67 years to study chromosomal imbal-
Finally, a small number (approximately 1%) of CML pa- ances in solid tumors [1622] and, to a lesser extent, he-
tients in blast crisis [9] harbor a t(3;21)(q26;q22), which re- matological malignancies [2327]. In the present study,
sults in fusion of the AML-1 and EVI-1 genes [10]. How- CGH has been used for the first time to study chromo-
ever, increased EVI-1 expression is seen in approximately somal abnormalities in CML patients. We have analyzed
70% of patients with CML myeloid blast crisis, only some 12 patients with CML, 11 of which had transformed into
of which have been shown to have abnormalities of chro- blast crisis.
mosome 3 detected by G-banding [11]. Whether increased
EVI-1 expression plays a causal role in CML blast crisis is
MATERIALS AND METHODS
currently unclear.
There is no evidence of a specific genetic abnormality Patient Samples
occurring consistently in CML blast crisis other than the Twelve patients with CML were studied. Eleven of 12
t(9;22) that may be responsible for disease progression. To were in blast crisis, whereas 1 patient remained in chronic
isolate regions of the genome that may harbor such genetic phase. For 6 of the 11 patients in blast crisis, a sample was
abnormalities, Mori et al. [12] performed allelotype analy- also available from an earlier phase of the patients dis-
sis for 30 CML patients (21 myeloid and 9 lymphoid cases) ease. Either peripheral blood or bone marrow samples that
by using 82 microsatellite markers. Loss of heterozygosity contained a high percentage of blast cells (.60%) were
$20% was detected on 1p (29%), 18p (20%), and 20q studied. Each sample was divided between short-term cell
(27%) in myeloid blast crisis and on 1p (50%), 4p (25%), culture for G-banding analysis and genomic DNA extrac-
7p (43%), 9p (29%), 18q (25%), 19p (43%), and 20q (33%) tion. In one case (patient 1), a peripheral blood sample
in lymphoid blast crisis. These data may identify new loci was available for DNA extraction, whereas the G-banding
where tumor suppressor genes responsible for CML dis- analysis was conducted on a bone marrow sample. For
ease progression reside. one patient (patient 5), G-banding analysis was not per-
Transformation from chronic stage to blast crisis of formed (Table 1).
CML is characterized in a majority of cases by secondary
chromosomal changes. Two main categories of alterations Cell Line Culture
are defined in CML blast crisis. Approximately 70% of
A BV173 cell line was derived from the peripheral blood
CML blast crisis cases are associated with at least one of
of a 45-year-old man with CML [28]. The cell line was
the following aberrations (major route); trisomy 8, i(17q),
grown in RPMI 1640 medium with 10% fetal calf serum at
trisomy 19, and an extra Philadelphia chromosome. These
378C in 5% CO2.
chromosomal changes can occur alone or in specific com-
binations. Major route changes can accumulate to common
combinations of chromosome abnormalitiesfor example, G-Banding Chromosome Analysis
18, i(17)(q), 1Ph. However some combinations are only High-resolution chromosome analysis was performed on
very rarely seenfor example, i(17q), 119 [9]. Whereas bone marrow and peripheral blood cultures in RPMI 1640
major route changes are associated with gain of genetic medium, with 20% fetal calf serum by using an ethidium
material, minor route changes are associated with losses of bromide synchronization technique and conventional Gi-
genetic material. CML blast crisis (minor route changes) emsa staining. Cells were treated prior to harvest with
include monosomy 7, monosomy 17, monosomy 21, loss colchicine for 10 minutes. Chromosome preparations were
of the Y chromosome, and t(3;21)(q26;q22) and are present observed under a Zeiss Axiophot microscope, images were
in 15% of cases. The various chromosomal changes are captured by Fujitsu camera, and karyotype analysis was
also often differentially associated with myeloid and lym- aided by a dedicated program-Quips XL (Vysis, Inc., Sur-
phoid blast crisis. Thus i(17q), 18, 119, and 121 are re- rey, UK).
ported to be involved mostly in myeloid disease [13] and
hypodiploidy, 27, 2Y chromosome, and abnormalities of Genomic DNA Extraction
chromosome 14 are associated with lymphoid blast crisis Patients with a high percentage of blast cells (.60%) in
[14], whereas double Ph has been found in lymphoid and their bone marrow or peripheral blood were chosen for
myeloid cases at a similar frequency [15]. this study (Table 1). Nucleated cells from the bone marrow
However, although cytogenetic alterations can be char- or peripheral blood samples were separated by using 1077
acterized in most patients in blast crisis, in approximately or 1119 Ficoll gradients. A mononuclear cell (MNC) frac-
one-third, no additional chromosome abnormalities other tion was recovered from the interface. Each sample was
than the t(9;22)(q34;q11) chromosome rearrangement are cytospun and stained, and the cell morphology was subse-
present. Comparative genomic hybridization (CGH) analy- quently analyzed to assess the percentage of blast cells in
Comparative Analysis in CML Blast Crisis 9
the resolution of the method. Translocations that were ysis failed to detect any chromosome imbalances. To clarify
present in the blast phase samples from patients 3 and 4 the observed chromosome alterations, multicolor chromo-
were confirmed by CGH to be balanced rearrangements. some painting (Fig. 3a) and FISH analysis with the use of a
G-banding information was not available for the sample chromosome 11-specific paint simultaneously with a
from patient 5 used in CGH analysis. No genetic imbal- BCR/ABL probe (Fig. 3b) was performed. A masked t(9;22)
ances were found in this sample by CGH. A bone marrow (q34;q11) involving chromosome 11 with a typical fusion
sample from patient 6 was shown by G-banding to be BCR/ABL FISH signal on the derived (22) chromosome
monosomic for chromosome 7 in only 10% of Ph positive was established (Fig. 3b). Patient 10 was described as hav-
cells. CGH failed to detect any imbalances presented in ing an inversion of chromosome 2 and a deletion of the
this low-level cell clone. The bone marrow sample from distal half of the long arm of chromosome 7 in all metaphases
patient 8 was found by G-banding to have a balanced (see Table 2). CGH confirmed the loss of 7q material; how-
translocation between chromosomes 11 and 22. Although ever, no imbalance was detected on chromosome 2, sug-
no typical Ph chromosome was present, one of the chro- gesting that the inversion was not associated with a ge-
mosome 9 homologs appeared shorter, with a banding pat- netic gain or loss. G-banded bone marrow metaphase cells
tern suggestive of a deletion at the 9q34 region. CGH anal- from patient 11 contained multiple chromosome abnor-
12 S. M. Gribble et al.
Figure 2 Diagrammatic representation of chromosomal gains and losses detected by CGH in CML
blast crisis patient bone marrow/peripheral blood genomic DNA. Results are presented for the chro-
mosomes found to be abnormal in the patients. Affected chromosomes are represented by numbered
ideograms. Lines on the left of the chromosome ideogram indicate a loss and lines on the right indi-
cate a gain of genomic DNA. The number at the end of each data line identifies the patient carrying
the imbalance. High-level amplification (shown by a thick line) of chromosome 8q was present in
patient 9.
malities, but a detailed G-banding analysis was not possi- pared (Table 3). G-banding analysis of the bone marrow
ble, because of poor banding morphology. CGH analysis of sample of patient 1 showed multiple chromosome abnor-
this sample revealed an overrepresentation of genetic ma- malities in addition to the t(9;22)(q34;q11) (Table 2). Be-
terial derived from the long arm of chromosome 1. cause a bone marrow sample was not available, CGH anal-
ysis was performed by using peripheral blood collected at
Discrepancies Between G-Banding and CGH Results the same time, and an overrepresentation of the entire
Three patients (patients 1, 9, and 12) were shown to have chromosome 6 was found as the only genetic imbalance
discrepant results when CGH and banding data were com- (Fig. 2).
Figure 3 (a) Multipaint chromosome analysis of bone marrow metaphase chromosomes obtained from patient 8
at the accelerated phase of disease, with paints specific for chromosomes 8 (red), 9 (purple), 11 (yellow), and 22
(green). There is no visible t(9;22). Acrocentric chromosomes show cross-hybridization signals at centromeric sites
(green). Arrows indicate the t(11;22), (b) bone marrow metaphase chromosomes from patient 8 hybridized with
chromosome 11 paint (yellow), and the BCR/ABL DNA probes, to show the fusion gene located on the der(22)
t(11;22).
Comparative Analysis in CML Blast Crisis 13
In patient 9, two discrepancies were seen between G-band- identified this marker as der(6)t(8;6;8)(8qterq13::6q21(?)
ing data and CGH FR profiles. Whereas G-banding showed c6p23(?)::8q22qter) (Fig. 4e). In total, seven c-MYC DNA
the loss of one of the homologs of chromosome 6 in a hybridization signals were seen in the metaphase cells
highly rearranged karyotype, CGH showed a lack of imbal- from this patient, all of which were located to marker
ance of chromosome 6. FISH with a chromosome 6 paint chromosomes (Fig. 4b). In addition, signals from the chro-
revealed, in addition to a normal 6 homolog, three further mosome 8 paint were found in the structure of another
marker structures containing genetic material from chro- two markersa small acrocentric and a larger metacentric
mosome 6. Further analysis with the use of a cocktail of chromosome. However, neither of these markers hybrid-
chromosome paints established the identity of the marker ized to the c-MYC DNA probe. Additional painting analy-
structures as der(6)t(8;6;8), der(9)t(6;9), der(6)t(X;6) (Table 3). sis demonstrated that both markers were due to nonbal-
The precise banding involvement was not possible to as- anced rearrangements (Fig. 4g and f), but it was not possible
certain. The presence of occult fragments of chromosome 6 to precisely ascertain the regions of chromosome 8 in-
elsewhere in the genome explains the normal CGH profile. volved in each marker.
A second discrepancy was also identified between the A bone marrow sample from patient 12 was shown to
G-banded karyotype and the CGH profiles of a peripheral have blast cells with a lymphoid immunophenotype and
blood DNA sample from patient 9. G-banding described no other abnormalities apart from the t(9;22)(q34;q11).
the possible presence of two isochromosomes of the long However, CGH analysis of this sample revealed overrepre-
arm of chromosome 8. CGH analysis confirmed the pres- sentation of the entire chromosome 8 consistent with tri-
ence of extra material originating from 8q (Fig. 2). How- somy (Table 3 and Fig. 2). No sample was available with
ever, the FR profile indicated a nonuniform amplification which to investigate this discrepancy further.
along the long arm of chromosome 8 (Fig. 4h). FISH with a
chromosome 8 paint confirmed the presence of two copies
DISCUSSION
of the isochromosome 8q (Fig. 4c) and revealed the pres-
ence of an acrocentric marker that was interpreted as In this paper, we report the first use of CGH to search for
del(8)(p11::p23.1) (Fig. 4d). In addition, another marker genetic changes in samples from patients with CML blast
structure, a metacentric chromosome, which was not iden- crisis. Our data underline the frequency of chromosome 8
tified by G-banding, was found to carry a signal from the abnormalities and demonstrate the utility of CGH for iden-
chromosome 8 paint. Hybridization signals from the chro- tification of marker chromosomes.
mosome 8 paint were seen on both arms in this marker. To
ascertain whether these ectopic hybridization signals orig- Chromosome 8 Abnormalities in CML
inated from the long arm of chromosome 8, the c-MYC DNA The most frequently abnormal chromosome in the CML
probe was applied together with a chromosome 8-specific samples studied by a combination of molecular cytoge-
paint (Fig. 4e). Further multicolor chromosome painting netic techniques was chromosome 8; 5 of 12 patients had
Figure 4 (a) CGH of genomic DNA isolated from the peripheral blood of patient 9. Note that the p arm of chro-
mosome 8 is predominantly red to indicate underrepresentation of this region in the test DNA. Note that the q arm
of chromosome 8 is bright green to indicate overrepresentation of this region in the test DNA. The X chromosome
is visibly greener in appearance owing to competition between female test DNA and male reference DNA. (b)
Metaphase chromosomes and interphase nuclei in peripheral blood from patient 9 hybridized with c-MYC-specific
DNA probe (red). Note a total of seven c-MYC hybridization signals. (cg) G-banded analysis/chromosome paint
analysis of marker chromosomes in peripheral blood of patient 9. Chromosome 8 paint (green), chromosome 14
paint (red), X chromosome paint (yellow), and c-MYC-specific DNA probe (red). (h) CGH fluorescence ratio profile
of chromosome 8 for patient 9. The threshold values 0.85 (left-hand side) and 1.15 (right-hand side) are indicated
by dotted lines.
an abnormal chromosome 8, presenting as either a trisomy a cell clone that is BCR/ABL negative and not in the dis-
8, an i(8)q, or amplification of a region of chromosome 8. ease cell clone. There are two explanations for this obser-
Trisomy 8 is present in approximately 35% of CML blast vation: first, the trisomy 8 is an early disease clone; and,
crisis patients, either as the sole abnormality or in con- second, the trisomy 8 is not disease related. The clinical
junction with additional changes [9]. Several studies have significance of a BCR/ABL negative trisomy 8 clone re-
monitored the occurrence of trisomy 8 in myeloid and mains unclarified. The results presented in this paper re-
lymphoid CML blast crisis, with conflicting results. Ali- vealed two i(8)q chromosomes as secondary chromosomal
mena et al. [33] suggested that the frequency of occurrence changes in a CML patient with lymphoid blast crisis. This
of trisomy 8 is similar in lymphoid blast crisis and mye- change has been documented in only one previous CML
loid blast crisis. Conversely, Nanjangud et al. [34] found report [37]. The most likely pathogenic effect of i(8q) is to
that only 1 of 22 lymphoid blast crisis patients and 13 of multiply potential candidate genes located on the long
28 myeloid blast crisis patients carried a third copy of arm of chromosome 8.
chromosome 8. Further reports by Anastasi et al. [35] and Amplification of the long arm of chromosome 8 has
Ariyama et al. [36] described trisomy 8 as being present in been reported to occur in non-Hodgkin lymphoma by CGH
Comparative Analysis in CML Blast Crisis 15
[24] and in T-prolymphocytic leukemia by G banding [38] Chromosomal Deletions That Mark the Site of Tumor
but not frequently in CML. A recent review by Forozan et Suppressor Genes
al. [39] emphasized the presence of several common ge- Chromosome deletions affecting chromosomal regions 1p
netic imbalances revealed in a series of CGH studies of and 9p in patients 3 and 9, respectively, were detected by
solid tumors. One common imbalance affects chromosome both G banding and CGH. These deletions may indicate ar-
8 and is manifested by loss of the short arm accompanied eas of the genome containing tumor suppressor genes im-
by amplification of the long arm. This genetic imbalance is plicated in CML blast crisis. Visible deletion of the chro-
clearly demonstrated by a CGH FR profile for chromosome mosomal region (1)(p34p36) is not a common finding in
8 in patient 9 in this study (Fig. 4h). The same FR profile CML blast crisis. However, microsatellite PCR has been
for chromosome 8 has been established by CGH analysis used to demonstrate loss of heterozygosity on 1p in 37%
of prostatic cancer [19], small-cell lung tumors [40] and of cases of CML blast crisis [12]. These data suggest that 1p
hepatocellular carcinomas [41]. The gain of 8q and loss of contains a tumor suppressor gene or genes with a role in
8p phenomenon has been invariably observed together the progression of CML to blast crisis.
with other genomic changes, either balanced or unbal- Patient 9, who had a lymphoid blast crisis, has a cyto-
anced abnormalities. This finding is interesting for two genetic visible deletion of (9)(p13pter) detected by both G
reasons: first, a concomitant gain and loss implicates the banding and CGH. This finding is consistent with a recent
existence of paired genetic changes, which may be respon- study by Sill et al. [4], who reported, by loss of heterozy-
sible for tumor progression; and, second, the abnormality gosity studies, that 50% of lymphoid blast crisis patients
appears to be universal, occurring in many types of carci- are homozygously deleted for the p16 gene, which locates
nomas and in hematological malignancies. to chromosome band 9p21.
The Involvement of Marker Chromosome Structures Differential Retention in Bone Marrow and Peripheral
in CML Blood of Distinct Clonal Subpopulations
The present understanding of secondary chromosomal G-banding analysis of bone marrow metaphases from pa-
changes in CML patients is based on G-banding analysis. tient 1 revealed complex multiclonal chromosomal abnor-
The resolution of the G-banding method is highly depen- malities. In marked contrast, CGH analysis of peripheral
dent on good chromosome morphology. Because high- blood DNA from the same patient revealed an extra copy
quality chromosome preparations from leukemic cells are of chromosome 6 as the only detectable abnormality (Ta-
difficult to produce, cytogenetic analysis often fails to ble 2, Fig. 2). These observations further support the view
identify complex chromosomal rearrangements. The oc- that some cytogenetically abnormal cell clones may be
currence of unidentifiable marker chromosomes as sec- preferentially retained in the bone marrow [48, 49].
ondary chromosomal changes is not a rare event in CML This study has shown that CGH is better than G-band-
blast crisis. A search of the cases published in Mitelmans ing and other FISH techniques in providing genomic im-
catalog of chromosome abnormalities [42] enabled us to balance information in all cell types, not just dividing
calculate that about 18% of CML blast crisis patients, with cells. In addition, patient samples that fail to grow in cul-
a t(9;22)(q34;q11), appear to manifest with additional ture can be analyzed by CGH to look for common chromo-
marker chromosomes. The clinical or biological signifi- somal blast crisis changes such as trisomy 8. CGH can be
cance of marker chromosomes is unknown. Our results il- used to identify the components of marker chromosomes
lustrate how CGH and molecular cytogenetics can be present in CML blast crisis patient samples and, when
sources of insight into the pathogenesis of blast crisis. used in conjunction with conventional cytogenetic tech-
Discrepancies between G-banding and CGH findings re- niques, can clarify complex karyotypes.
vealed the involvement of chromosome 6 in the blast cri-
sis of patient 9 (Tables 2 and 3). G banding showed a loss
of chromosome 6, whereas CGH produced a normal chro- Research in the authors laboratories is supported by the Kay
Kendall Leukaemia Fund and the Leukaemia Research Fund. The
mosome 6 profile. Chromosome painting located chromo-
authors would like to acknowledge M. Phillips, for valuable tech-
some 6 material in the structure of two marker chromo- nical assistance, and Dr. N. Cross, Hammersmith Hospital, S.
somes that G banding failed to identify. Rearrangements OConnor, Leeds General Infirmary, Dr. B. Jennings and Dr. J.
involving chromosome 6 have previously been described Pearson, Norfolk and Norwich Hospital, for the contribution of
in the blast crisis of CML (9 cases in Mitelmans catalog CML patient samples.
[42]); however, these reports are limited. The documented
cases of translocations involving chromosome 6 are re-
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