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Comparative Genomic Hybridization in Chronic B-Cell Leukemias Shows
a High Incidence of Chromosomal Gains and Losses
By Martin Bentz, Karin Huck, Stanislas du Manoir, Stefan Joos, Claudius A. Werner, Konstanze Fischer,
Hartmut Dohner, and Peter Lichter
In chronic B-cell leukemias, fluorescence in situ hybridization
revealedchromosomalgainsandlosses
not detectedby
has greatly improved the ability to detect certain chromobanding analysis.In 8 of these 13 cases, discrepancieswere
somal aberrations,as cellsin all phases ofthe cell cycle are
furtherinvestigatedusingothermethods,
and in allinanalyzed. To obtain a comprehensiveview of chromosomal
stances, the CGH findings were confirmed. A limitation of
gains and losses, we applied the recently developed techdetecting small deleted regions by CGH was found in one
nique of comparative genomic hybridization (CGH)
to 28 paexample of 18p.In conclusion, ourdata showthat the results
tients with chronic B-cell leukemias. CGH results
were comof banding analysesin chronic B-cell leukemias often do not
pared with those obtained by chromosome banding analysis reflect the chromosomal changes in the predominant cell
and interphase cytogenetics.In 19 of the 28 cases, chromoclone. Thismay be one explanation for
the as yet poor corresomal imbalances were detected, including amplified DNA
lation between cytogenetic findings and clinical course in
sequences in three instances. The most common aberrations this group of neoplasms.
included gains of chromosomal material on 8q and12as
0 1995 by The American Societyof Hematology.
well aslossesof6q.
l l q , 13q. and 17p. In 13cases,CGH
C
HRONIC B-CELL lymphocytic leukemia (B-CLL) is
the most prevalent leukemia inadults and accounts
formore than 30% of all leukemiacasesinEuropeand
North America. Although chromosomal banding analysis
has
shown a number of recurrent aberrations in B-CLL, the sole
prognosticallyrelevant chromosome abnormalitythat has
been identified in multivariate analyses is a complex karyotype associatedwith an inferior outcome.‘ In contrast, in
acute leukemias a number of specific chromosomal changes
with high
prognostic
impact
have
been
The difference between these two groups of leukemias may be due to
difficulties inidentifying aberrations of the leukemiccell
clone in B-CLL.Even when B cell-specific mitogensare
used, the leukemic cells may exhibit a low proliferative activity, and in about half of the cases, no chromosomal abnormalities This
are
is, at part,
least
in
due
to
mitotic cells arising from nonleukemic T lymphocyte^.^^^ A
number of fluorescence in situ hybridization (FISH) studies
investigating interphase cells have shown
the presence of
cell clones carrying chromosomalaberrations in cases where
no abnormalities were found by banding analysis: the incidence of both trisomy 12”” and deletions of part of the long
arm of chromosome 1 3I4.l5is substantially higher than found
by banding techniques. However, interphase FISH depends
on the preknowledge of candidate regions and on the availability of suitable DNA probes. Furthermore, only very few
From the Deutsches Krebsforschungszentrum, Abt. “Organisation
komplexer Genome”, Heidelberg: and the Medizinische Klinik und
Poliklinik V, Universirat Heidelberg, Heidelberg, Germany.
Submitted November IO, 1994; accepted January 26, 1995.
Supported by grants fromthe Fritz Thyssen Stifung and the European Community (GENE-CT 930055).
Address reprint requests to Martin Bentz, MD, Deutsches Krebsforschungszentrum, Abt.“Organisation komplexer Genome”, Im
Neuenheimer Feld 280, 69120 Heidelberg, Germany.
The publication costsof this article were defrayedin part b y page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8512-0015$3.00/0
3610
chromosomal regions can be examined in a single experiment.
With the recently developed methodof comparative genomic hybridization (CGH),I6 tumor genomes can be rapidly
tested for the presenceof chromosomal imbalances (such as
partial or complete monosomies and trisomies).“”*
Differentially labeled tumor and genomic control DNA are
cohybridized to normal metaphase chromosomes under suppression
conditions and detected using different fluorochromes. The
ratios of the fluorescence intensities generated by tumor and
control DNAindicate regions with a normalgenomic content
as well as overrepresented and underrepresented sequences
within the tumor DNA.
We used CGH to identify copy number changes of chromosomal regions in 28 patients with chronic B-cell leukemias. The results were compared with those obtained by Gbanding analysis and interphase cytogenetics.
PATIENTS AND METHODS
Patient Samples
Twenty-eight patients with chronic B-cell leukemias [25 with BCLL and three with B-cell prolymphocytic leukemia (B-PLL): patients 6 , 9, and 28) were examined. In 26 cases, blood specimens
were obtained, and in two patients (patients 5 and 1 l), cells from
splenectomy samples were obtained. An aliquot of each sample was
used for short-term culture and subsequent G-banding analysis. A
and genomic DNA was prepared
second aliquot was stored -70°C
at
K digestionandphenol-chloroformextraclaterusingproteinase
tion.’” Patients were included in this study depending on the availability of frozen cell material.
G-Banding Analysis
Mononuclear cells were purified from a Ficoll gradient and cultured at a concentration of1O6 cells per milliliterusing the following
mitogens: antihuman immunoglobulin M (IgM; Jackson Immunoresearch Laboratories, West Grove, PA), B-cell growth factor (10%;
Cellular Products Inc. Buffalo, NY), and phorbol-l2-myristate-l3acetate (1 to IO ng/mL) or calcium ionophore ( l pmoVL; Calbiochem, La Jolla,CA). Harvesting, slide preparation, and banding were
performed as published previou~ly.’~
Between 10 and30 metaphases
(median, 20) were analyzed per case.
Comparative Genomic Hybridization
Hybridization was performed as
d e ~ c r i b e d . ” ~Bri
~ ”efly, normal
human genomic DNA (control DNA) was labeled with digoxigeninBlood, Vol 85, No 12 (June 15), 1995: pp 3610-3618
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361 1
CGH IN CHRONIC B-CELL LEUKEMIAS
11-deoxyuridine triphosphate (dUTP; Boehringer Mannheim, Mannheim, Germany), and tumor DNA was labeled with biotin-16-dUTP
(Boehringer Mannheim) by a standard nick translation reaction;
DNase I concentration was adjusted to result in an average fragment
size of 300 to 1,OOO bp. One microgram of labeled tumor DNA, 1
pg of labeled control DNA, and 70 pg of human Cot I DNA (BRL
Life Sciences, Gaithersburg, MD) were cohybridized to slides with
metaphase cells prepared from blood of a healthy donor. After hybridization for 2 to 3 days and posthybridization washes, test and
control DNA were detected via fluorescein isothiocyanate (FITC)
and rhodamine, respectively. Chromosomes were counterstained
with 4,6-diamidino-2-phenylindole(DAPI; 200 ng/mL), resulting in
a Q banding-like pattern that was used for chromosome identification.
Digital Image Analysis
Image acquisition, processing, and evaluation were performed as
described previo~sly.''.~'Images were acquired using an epifluorescence microscope (Axioplan; Zeiss, Oberkochen, Germany)
equipped with a cooled CCD camera (Photometrics, Tucson, AZ).
FITC and rhodamine images of the tumor and control DNA were
used for quantitative analysis. Ratio profiles of each individual chromosome were calculated using a dedicated software?' For each case,
the mean ratio profiles of between 4 and 10 metaphase cells were
computed. Thresholds for the identification of imbalances were defined as 0.75 (lower threshold) and1.25 (upper threshold). These
are the theoretical values that are expected in a diploid tumor cell
population for a monosomy or a trisomy of a certain chromosomal
region in 50% of the test cells. Chromosomes or chromosomal regions with fluorescence ratios outside of this interval were considered to be over- or underrepresented, respectively. In studies comparing CGH results with data obtained by other cytogenetic methods,
these thresholds were proven to provide robust diagnostic criteria,2I-23
Interphase Cytogenetics
The following probes were used for interphase cytogenetics: chromosome-specific plasmid libraries pBS7 and pBS8-; cosmid clones
cos-myc72 (8q24)25; a yeast artificial chromosome (YAC) clone
mapping to chromososomal band 11q2326;chromosome 12-specific
alphoid probe D12Z3 (Oncor Inc, Gaithersburg, MD); phage contig
spanning the whole RBI tumor suppressor gene (13q14), provided
by Thaddeus Dryja, Cambridge, MAZ7;and four overlapping cosmid
clones containing the p53-encoding genomic region (17~13).~*
Hybridization was performed on methanoUaceticacidfixed cells as
de~cribed.'~
With the exception of one case where only 150 cells
were analyzed (hybridization with cos-myc72 in case 6), in all other
instances at least 200 cells were evaluated. In case ofthe YAC
probe, Ah-polymerase chain reaction (FTR) amplification of the
insert was performed as described el~ewhere.~'When interphase
analysis was performed with cosmid or phage probes to look for
deletions, a further cosmid probe was included as an internal control
for hybridization effi~iency.'~
CGH, chromosomal banding analysis,
and interphase cytogenetics using probes for chromosomes 12,
13q14, and 17q13 were performed in a blind fashion.
RESULTS
G-Banding Analysis
G-banding analysis was performed in all 28 patients. In
five patients, no metaphase cells were found. Thirteen cases
exhibited normal karyotypes. In each of the remaining 10
patients with clonal abnormalities, chromosomal imbalances
were present. The complete karyotypes are listed in Table l.
CGH Analysis
In 9 of the 28 patients, no chromosomal gains or losses
were found by CGH analysis. In the other 19 patients, the
following chromosomal imbalances were identified in more
than one case: loss of chromosomal material on 17p (nine
cases), 1lq (four cases), 6q (three cases), 13q (three cases),
and 18p (two cases); and gains of chromosomal material on
8q (three cases, one of whichwas an amplification), 3q
(two cases), 12p (two amplifications at different sites of this
chromosome arm in a single patient; Fig 1A through D),
trisomy 12 (two cases), 15q (two cases, one of which exhibited an overrepresentation of the whole chromosome 15), and
17q (two cases). The following imbalances of chromosomal
regions were detected only once: overrepresentations of 3p,
12q, 18q, and 22q, as well as whole chromosome 19; underrepresentations of 3p, 4q, 7q, 8p, lOq, 15q, and 16p. The
complete data for all patients are shown in Table 1.
Comparison of CGH and Banding Data
Additional Aberrations Found by CGH
In 13 of the 28 cases, additional imbalances were detected
by CGH (Table 1). These discrepancies can be classified in
four different categories. (1) Although normal karyotye was
found on banding analysis, clonal abnormalites were found
by CGH (six cases: 2, 8, 18, 23, 24, and 27). (2) No metaphase cells were obtained after short-term culture (three
cases: 10, 14, and21). (3) By banding analysis, complex
karyotype abnormalities were found. The chromosomal origin of some of these aberrations (eg, marker chromosomes)
could not be identified by banding analysis in three cases
(cases 6, 22, and 28), and in two of these cases, DNA amplifications were detected by CGH. (4) The imbalanced chromosomal region was small and involved in a cryptic translocation (one case, 5). Interphase analysis using specific DNA
probes directed against chromosomal regions with discrepant
findings was performed in 8 of these 13 cases. The results
were as follows.
Case 2. In this patient, banding analysis showed a normal karyotype in 15 metaphases. By CGH, a loss of chromosomal material on 1 l q (bands 1lq14 to 1lq25) was detected.
Interphase analysis using a YAC probe mapping to chromosomal band 1lq23 showed only one signal in 80.4% of cells
(Fig 2F).
Case 5. By G-banding, a de1(7)(q32)was found asthe
sole chromosomal imbalance. In contrast, CGH analysis
showed an overrepresentation of chromosomal material on
8q (bands 8q23 to qter). Furthermore, the terminal deletion
on chromosome 7 extended up to chromosomal band 7q31
by CGH analysis. The overrepresentation of the terminal
part of chromosome 8 was confirmed by FISH analysis with
the cosmid probe cos-myc72 mapping to chromosomal band
8q24: three hybridization signals wereseenin69.4%
of
interphase cells. Dual color FISH using chromosome-specific
DNA plasmid librariesfor chromosomes 7 and 8 resolved both
discrepanciesseenin this case:onthefewmetaphasecells
From www.bloodjournal.org by guest on April 29, 2017. For personal use only.
BENTZ ET AL
3612
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From www.bloodjournal.org by guest on April 29, 2017. For personal use only.
3613
CGH IN CHRONIC B-CELL LEUKEMIAS
assessable after hybridization,the additional chromosome 8
material was translocated to the terminal part of the derivative
chromosome 7 (Fig 2A). The resulting banding pattern of this
derivativechromosome[der(7)t(7;8)(q31;q23)]isindistinguishable from a terminal deletion with breakpoiit
the
in 7q32.
Case 6. In this case, complex karyotype abnormalities
were found by banding analysis. In many of the imbalances,
the chromosomal origin of the imbalanced material was not
identified. By CGH, overrepresentation of the whole chromosome15and material on 12qand 17q, as well as an
amplificationmapping to chromosomalband 8q24, were
seen.By interphase analysis with the cosmid probe containing the MYC gene, an amplification was detected in 5%
of cells as a large, disperse hybridization signal. Inmore
than 90%of cells, two focal hybridization signals were seen.
A low copy numberamplificationwasalsosuspected
by
Southern blot analysis (data not shown).
Case 8. This patient exhibited anormal karyotyp on
banding analysis (21 metaphases analyzed). CGH analysis
showed additional material on chromosomes 8 (8q13 to
8qter) and 17q, as well as a loss of material on 16p, 17p,
and 18p. Interphase analysis using the cosmid contig spanning the p53 gene on chromosome 1 7 ~ 1 3demonstrated a
single hybridization signal in 88.8% of cells.
Case 10. In three instances, metaphase preparation was
not possible for banding analysis in this patient. CGH analysis showed a deletion on chromosome 13 (bands 13q14 to
q21). This wasconfirmed by interphase analysis with the
phage contig spanning the RBI gene on chromosome band
13q14: 67% of cells exhibited only one hybridization signal
with this probe.
Case 21. No metaphase cells were available for banding
analysis. By CGH, loss of chromosomal material on l l q
and 13q, as well as a trisomy 12 and overrepresentation of
chromosomal material on 22q, were found. By interphase
cytogenetic analysis, both the trisomy 12 (82% of cells exhibiting three signals) and the deletion on 13q (48.7%of cells
showing one signal with the RBI probe) were confirmed.
Case 22. In this case, a complex karyotype with a marker
chromosome andahomogeneously
staining regionwas
found by banding analysis. CGH showed a number
of imbalances, including two amplification sites (Fig 1). The loss of
FISH:
chromosomal material on 17p wasconfirmedby
86.9% of cells exhibited one signal after hybridization with
the probe spanning the p53 gene.
Case 23. G-banding analysis showedanormalkaryotype. By CGH,losses of chromosomal material on 4q(4q22q28), 6q(6q15-q22), and 17p were found. Again, hybridization was performed withthe p53 probe: 90.1% of the nuclei
exhibited a single hybridization signal.
Case 27. On G-banding, a normal karyotype was found.
CGH showed interstitial deletions on 1lq and 13q. The latter
was confirmedby FISH using the phage contig spanning the
RBI gene; in 8 1.6% of interphase cells, only one hybridization signal was seen.
Imbalances Found by Banding Analysis, But Missed by
CGH
In two cases, imbalances that had been detected by banding analysis were not diagnosed by CGH.
Case 12. In this case, an unbalanced whole arm translocation der(l7; 18)(qlO;qlO) resulting in the loss of one copy
of 17p and 18p was diagnosed on banding analysis. By CGH,
the ratio profile of the short arm of chromosome 18 was
shifted towards underrepresentation; however, the threshold
for underrepresentation was not reached. By visual inspection, a less intensive FITC fluorescence of 18p was clearly
visible. For explanation, see Discussion.
Case 20. By banding analysis, metaphases with a normal
karyotype and two clones with aberrations were found both
had losses of 17p and 18p. In addition, one of these clones
(5 of 21 metaphases) had a loss of 3p (whole arm), and the
other clone (4 of 21 metaphases) had a loss of 3q
(whole
arm, two copies of 3p present). By CGH, the profile for
chromosome 3was
shifted towards underrepresentation
without exceeding thethreshold. Interphase analysis with
the p53 probe demonstrated that only 55% of cells belonged
to one of the aberrant clones. As only part of the aberrant
cells had losses of material on 3p or 3q, the percentages of
cells carrying the respective imbalances were too low to be
detected by CGH.
Comparison of CGH With Interphase Cytogenetics
In all patients, interphase cytogenetic analysis for the presence of a trisomy 12 as well as for allelic losses of the RBI
and TP53 tumor suppressor genes wasperformed. These
data have been published p r e v i ~ u s l y . ' ~ ~ ' ~ ~ ' ~ ~ ~ ~
Trisomy 12
In two of the 28 patients, a trisomy 12 had been diagnosed
by interphase cytogenetic analysis (Table 1). In both cases,
the overrepresentation of the whole chromosome 12was
detected by CGH.
Loss of the RB1 Gene
In 9 of the 28 patients, a loss of one copy of the RB1
tumor suppressor gene had been detected in between 48.7%
and 98.1% (median 79%) of interphase cells. By CGH, a
loss of chromosomalmaterial involving band 13q14 was
detected inonlythree patients (patients 10, 21,and 27).
Interestingly, in each of the other six patients with an RBI
deletion (patients 2, 7, 17, 23, 24, and 25), the ratio profiles
exhibited a shift towards underrepresentation without reaching the diagnostic thresholds. This indicates that deletions
involving 13q14 in CLL are often small.
Loss of the TP53 Gene
Of the 28 patients, 9 had anallelic loss of the TP53 gene in
percentages of cells ranging from 24.5% to 90.4% (median,
64.5%; cases 6, 8, 9, 12, 14,20,22,23, and 28). In all cases
with the exception of case 14, wherethe deletion was present
in only 24.5% of cells, a loss of chromosomal material on
17p was detected by CGH. As shown by banding analysis,
in most of these cases, the whole short arm of chromosome
17 was deleted.
DISCUSSION
CGH has recently been developed and has proven to be
a powerful tool for the detection of chromosomal gains and
From www.bloodjournal.org by guest on April 29, 2017. For personal use only.
3614
losses in avarietyof tumors.16"X~Z2~'~~""X Using CGH, we
identified numerous gains andlosses, as well as three amplification sites, in 28 patients with chronic B-cell leukemias.
Many of theimbalances detected with CGHare well
known to occur in this typeof leukemia': losses of chromosomal material on 6q, l l q , and 13q as well as gainsof
material on chromosome 12. A loss of material on 17p has
been found by our group in 17 of 100 patients.2x However,
another imbalance, a gain of chromosomal material on 8q,
which was detected in 3 of our 28 patients (Fig 2B), had
not been described as a frequent structural aberration before.
In two of these three cases, the presence of the additional
chromosome 8 material was confirmed by other methods. In
the third case, no material for further analysis was available.
Interestingly, in all cases, the overrepresentation of chromosome 8q was not detected by banding analysis.
In twopatients, amplifications ofchromosomal subregions
were identified (Figs 1 and 2B). Whereas double minutes or
homogeneously staining regions, the cytogenetic hallmarks
of gene amplification, have been described in approximately
2% to 3% of acute myeloid leukemias," only a few cases
of chronic B-cell leukemias with such abnormalities have
been rep~rted.~'
In one of our patients (patient 6), the c-myc
protooncogene was amplified. This gene is most commonly
involved in amplifications of myeloid leukemias:' and amplification of c-myc has been described in one case of BC L L b e f ~ r e . ~In' another patient (patient 22) of our series,
two amplificationswere found, both mapping to the short
arm of chromosome 12 (12~11-p12 and 12~13;
Fig I). Overrepresentation of material on chromosome 12, namely a trisomy 12, is a very common chromosome abnormality in BCLL. Although the chromosomal region 12q13 to q22 has
considered
been
pathogenetically
thismost
finding suggests that genes on 12pmight also play a significant role in the leukemogenesisof B-cell disorders. On chromosomal band 1 2 ~ 1 3several
,
candidate genes arelocalized:
these include a cyclin gene (cyclin D2), fibroblast growth
factor 6, lymphocyte activation gene 3, a G-protein, and the
tumor necrosis factor receptor 1," as wellas the recently
described ETS-like gene TEL, which is rearranged in some
cases of chronic myelomonocytic l e ~ k e m i a . ~Band
'
12~12
is the chromosomal locus
of the KRAS protooncogene, which
BENT2 ET AL
is known to be
mutated in some casesof acute lymphoblastic
leukemia."
Comparison of CGH data and interphase cytogenetic results for trisomy 12and losses of chromosomearm 17p
corresponded well. With the exception of one case showing
a 17p deletion in only 24.5% of interphase cells, all abnormalities found by interphase analysiswere detected by CGH.
The discrepancy found in this case is due to the thresholds
used as diagnostic criteria (see above): if the genomes of
less than 50% of cells harbor the imbalances, the respective
profiles are likely not to reach the thresholds for under- and
overrepresentation, respectively (see also case20). Although
RBI deletions were found in percentages ranging between
48.7% and 98.1 %, six of nine allelic losses of this chromosomal regiondetected
by interphase cytogenetics were
missed by CGH analysis. This is most likely due to the small
size of the respectivedeletions that may bebeyond the spatial
resolution of the CGH analysis. With chromosome preparation techniques and evaluationprocedurescurrentlyused,
the size required for the detection of an imbalance may be
in the range of the short arm of chromosome 18: a deletion
of this region was detected in two of our cases and missed
in another case(case 12). Preparation of more elongated
chromosomes may increasethespatialresolution.
Other
strategies to achieve this have been discussed in detail before.*'
Comparison of the CGH data with the data obtained by
banding analysis showed a high proportion of cases (1 3 of
28) where additional imbalances were found by CGH analysis. Amongthese, there weresix patients that had no chromosome aberrations on banding analysis. Other discrepancies
were based on a failure of preparing metaphase cells; complexkaryotypes with markerchromosomes, in whichthe
additional or deleted chromosomalregions could be mapped
to specific regions of the genome; and one casewith a small
overrepresentedregionthat
was not detected by banding
analysis. In the majority of cases (8 of 13). at least one of
the discrepancies was tested
by FISH using specific DNA
probes, and in each of thesecases,the
CGH resultwas
confirmed. These dataindicate that in a considerable proportion of CLL cases, additional abnormalities can be found by
CGH. In 6 of 13 patients exhibiting a normal karyotype on
P
Fig 1. Comparative genomic hybridization in CLL. (A) Complete average ratio profile of -sa 22 (seealso B through D). The ratios of FITC
to rhodamine fluorescence are plotted along each single chromosome. The mode of the intensity ratios of this case (central line) and the
thresholds for overrepresentation (right line) and undarrepresentation (left line) are shown. Underrepresentations ofpart of chromosome 3p
as well as an overrepresentation of chromosomalmaterial on 12p are diagnosed.Note the two sharp
and 8p. the proximal part of 15q and 17p
peaks of the profile on 12p. Underrepresentation of the whole chromosome X is due to the sexes of test (male) and control (female) DNA.
The gray shaded areasindicate heterochromatic chromosome regionswith a high content of repetitive sequences that are suppressad bytha
Cot l-DNA in the hybridization solution. Consequently, RTC and rhodamine fluorescence intensities are very low in these regions. Minute
absolute variations of fluorescence intensities within these regions may result in gross alterations of the ratio profiles. Therefore, they are
excluded from evaluation. The same applies to the Y chromosome. (B and C) Normal metaphase spread hybridized with DNA from case 22
detected via FlTC (B, green) and a control DNA detected via rhodamine (C, red). Two very strong band-like hybridization signals are seen on
the short arms of chromosomes 12, indicating the presence of amplified DNA sequencesmapping to two different loci on 12p. Weakerstaining
X is clearly visible. The corresponding chromosomes are indicated
in panel B. (D)FlTC to rhodamine
of chromosomes 3p.8p.17p.and
in the tumor genome
fluorescence intensity ratio image of the same metaphase spreadas in B and C. Chromosomal regions overrepresented
are shown in green, whereas underrepresented regions areshown in red. The loss of a chromosome in 100% of cells (X chromosome in this
male patient) results in a yellow color in the fluorescence ratio image. (E) Average ratio profiles for chromosomes 17 of the other seven cases
exhibiting a deletion of 17p and, in two casas, an overrepresentstion of 17q.
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3615
CGH IN CHRONICB-CELLLEUKEMIAS
A
1
2
3
-.6 .. . 7
8
13
15
14
20
19
9
10
16
21
4
5
11
12
17
18
Bi
22
X
E
case 6
Case 12
Case 8
Case 20
Case 9
Case 23
Case 26
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BENTZET AL
3616
B
case 8
Case 6
Case 5
a
'1
C
a
8
Case 14
Case 23
8
Case 4
6
I)
D
Case 2
11
E
Case 27
11
11
11
Case 10
13
13
13
Fig 2. Average ratio profiles ofchromosomes frequently affected by gains or losses. (A, B)In case 5, a cryptic translocationof the overreprechromosome 7 was mapped
sented chromosome 8 material to thedeleted chromosome 7 was present. By banding analysis, the breakpoint on
more distal than by CGH (see average ratio profile of this case in panel B). A FISH experiment using chromosome-specific DNA libraries for
chromosomes 7 (red) and 8 (green), respectively, demonstrates that a part of chromosome 8 is translocated t o the deleted chromosome 7
homolog. Two furthercases showed overrepresentation of chromosome 8 indicated in panel B. (C) Average ratio profiles of cases 4,14, and
23 exhibiting deletions on6q. (D, F) Average ratio profiles of the fourcases with deletions on chromosome l l q . On banding analysis, case 2
had a normal karyotype. Interphase cytogenetic analysis using a YAC probe mapping t o l l q 2 3 was performed. (F) Only one hybridization
signal is visible in the cells, indicating a deletion of chromosome l l q . (E) Average ratio profiles of three cases (cases 10,21, and 27) with
deletions on 13q.
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CGH IN CHRONIC B-CELL LEUKEMIAS
banding analysis, clonal aberrations were found by CGH. In
these cases, the analysis of metaphase cells clearly did not
reflect the chromosomal changes present in the malignant
clone. Such discrepancies between chromosome changes
present in the interphase cell fraction but not inthe proliferating cells have been detected in cases of trisomy 12 and
deletions of chromosomal band 13q14.9“’In these FISH
studies, higher incidences of the respective chromosomal
aberrations were found than suspected by banding analysis.
In the CGH study presented here, screening for all types of
chromosomal imbalances was performed, and almost half of
the cases exhibited additional chromosome aberrations that
had been missedby banding analysis. This implies that,
in cytogenetic studies relying on banding analysis in CLL,
possibly relevant karyotype changes are missed in a considerable proportion of cases. In another study, we compared
banding and CGH data in 10 cases of myeloid leukemias
not showing dis~repancies.~’
In contrast to CLL, in myeloid
leukemias, banding data in general reflect the chromosomal
changes present in the malignant clone. This may be an
explanation for the identification of prognostically relevant
chromosomal changes in acute leukemias, whereas such clinically important specific aberrations are still a matter of discussion in CLL. Delineation of frequent chromosomal gains
and losses by CGH in CLL may be an important contribution
of this new technique to the understanding of this disease.
The data obtained by CGH will provide the basis for further
analyzing a larger number of cases by FISH using specific
DNA probes mapping to the chromosomal regions mostfrequently affected.
ACKNOWLEDGMENT
We gratefully acknowledge Sandra Weitz and Daniela Diehl for
excellent technical assistance and Thaddeus Dryja (Cambridge, MA)
for providing the phage contig spanning the RBI gene.
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1995 85: 3610-3618
Comparative genomic hybridization in chronic B-cell leukemias
shows a high incidence of chromosomal gains and losses
M Bentz, K Huck, S du Manoir, S Joos, CA Werner, K Fischer, H Dohner and P Lichter
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