Comparative Genomic Hybridization:

Uses and Limitations

PeterLichter, St&n Joos,Murtim Bentz; und St$an Lmnpel

Comparative genomic hybridization (CGH) has contributed significantly to the current knowledge of genomic
alterations in hematologic malignancies. Characteristic patterns of genomic imbalances not only have
confirmed recent classification schemes in non-Hodgkin’s lymphoma, but they provide a basis for the
successful identification of genes with previously unrecognized pathogenic roles in the development of
different lymphomas. Based on its technical limitations, there is little reason to apply CGH to chromosomes of
metaphase cells in routine diagnostic settings. However, the new approach of CGH to DNA microarrays, a
procedure termed matrix-CGH, overcomes most of the limitations and opens new approaches for diagnostics
and identification of genetically defined leukemia and lymphoma subgroups. Current efforts to develop
leukemia specific matrix-CGH DNA chips, which are designed to meet the clinical needs, are presented and
discussed.
Semin Hematol37:348357. Copyright 0 2000 by W.B. Saunders Company.

T HE DEVELOPMENT of comparative ge-

nomic hybridization (CGH) wasdriven by the
need for tools that allow genome-widescreeningfor
chromosomealterations,independentof the availabil-
ity of metaphasecellsin the specimento be investi-
gated. For many tumors, dividing cellsare difhcult if
not impossible to obtain. For CGH analysis of
tumors, whole genomesof tumor cells and control
cells are used as probes for fluorescence in situ
hybridization (FISH) against chromosomesof nor-
mal metaphasecells.The method allowsthe detection
of gainsand losses of chromosomalregionswithin the
tumor genome by the comparison of hybridization
signalintensities.20~33-35The potential for a genome-
wide screeningwithout prior knowledge of specific
regionsof interest triggeredmultiple CGH studiesof
tumors.22,40,41,75Initially CGH was considered as
tool for the analysis of solid tumors rather than
hematogic malignancies, mainly for two reasons.
First, cytogenetic analysisof leukemiasand lympho-
masby chromosomebanding and FISH wasalready
well advanced.Therefore, many investigatorsdid not
anticipate that sufficiently novel data would be ob-
tained from CGH analysis of these entities. In
accordancewith this view, the large body of data on
chromosome aberrations in cancer revealed by the
analysisof banded chromosomesis basedmainly on
hematologic malignancies,48 whiie only one tenth of
the published CGH studies concern leukemiasor
lymphomas.22,40,41,75 Second, for many leukemias
and lymphomas, highly characteristic chromosome
translocations and inversions have been observed,
which appearto play a pivotal role in diseaseetiology.
As most of these aberrations are balanced-not
accompaniedby lossor gain of material-they arenot
detectedby CGH.
However, asoutlined below,CGH hascontributed
greatly to our current knowledge of genomic alter-
ations in leukemiasand lymphomas. It has not only
provided new evidence for the classificationof non-
Hodgkin’slymphomasbut alsoallowedelucidation of
diseasemechanismsby defining previously unrecog-
nized recurrent genomic imbalances,ultimately lead-
ing to the identification of a number of genesthat
play a pathogenic role in the respective type of
neoplasia.

From the Abteilung Organisation komplacer Genome, Deutscbes

Krebsforscbungxzentrum,Heideberrg, Germany; and the Abteilung
Innere Medizin III, Universit& Ulm, Urn, GernunJ
Addressreprint requeststo Peter Lichter, PbD, Abt. Ovganisation
komphwr Genome, Deutxbes Krebsforscbungszentm, Im Neuen-
beimer Feti280, D-69120 Heidelberg, Germany.
Copyright 0 2000 b WB. Saunbs Company
0037-1963/00/3704-0004$10.00/0
doi:l 0.1053/bem.2000.16594
Technical Aspects
Apart from the optimization of the CGH proto-
col 5~18~19,35,38)45*7
two relevant technical problems
had to be addressed,which areequally important with
respectto the analysisof solid tumors or hematologic
neoplasias:application of CGH (1) to small samples
and (2) to formalin-fixed paraffin-embeddedspeci-

348 Seminars in Hematology, Vo137, No 4 (October), 2000:~~ 348-357

 Usesand Limitations of CGH 349

mens. Because CGH relies on the presence of at least Table 2. Genes in Amplicons Originally Identified by CGH

35% tumor cell~,‘~ many studies require physical Region Gene Hematologic Disease
enrichment by cell sorting or by microdissection of
tissue sections, both methods yielding only small 2p12-p16 REL DLBCL (including stom-
ach, PMBCL)
numbers of cells that can be used for the investigation.
2p23-p24 MYCN MCL, Burkitt’s lymphoma
Hod&i&/Reed-Sternberg cells scattered throughout 3q26-q27 BCL6 MZBCL
a tissue section serve as a particularly demanding 8q24 MYC AML, DLBCL, FL, MCL
example, where due to the genetic heterogeneity the 91324 JAK2 HD, PMBCL
9P IFNBl, CDKN2A AML
analysis should be performed ideally at the single cell llq23-qter ETSl, FL/l, AML
level.31,55 In order to perform CGH from small SRPR, NFRKB,
samples, genomic DNA needs to be amplified by a KCNJ.5
protocol that maintains sequence representation as 12~13 CCNDZ CLL
12q12-q14 GLI, CDK4, DLBCL
much as possible. The universal polymerase chain
MDM2
reaction (PCR) protocol, termed degenerate oligo- 12q13-q14 GLI FL
nucleotide-primed PCR (DOP-PCR),70 was opti- 14q31-q32 IGH CLL
mized and has been widely used for this pur- 18q12 BCL2 DLBCL, MZBCL, MC

pose. 43,64,67Nevertheless, careful evaluation uncovered Abbreviations: DLBCL, diffuse large B-cell lymphoma;
the risk of false-positive CGH data when DOP-PCR PMBCL, primary mediastinal B-cell lymphoma; MCL, mantle
is applied to only one or a very few cells. A recently zone lymphoma; MZBCL, marginal zone B-cell lymphoma;
published amplification procedure39 promises to over- AML, acute myeloid leukemia; FL, follicular lymphoma; HD,
Hodgkin disease.
come this problem, since single-cell CGH has been
successful using this protocol in carefully controlled aberrationsdetectedby other means.However, several
studies. It is a common problem of CGH studies that studiesuncovered an unexpected frequency of high-
many tissue samples are formalin-fixed and parafhn- level amplifications. For example, the existence of
embedded, yielding genomic DNA of suboptimal double-minute chromosomes,a cytogenetic hallmark
quality, especially from archived specimens are investi- for gene amplification, in a subsetof acute myeloid
gated. Fixation conditions have been tested exten- leukemiashasbeenknown but there wasno evidence
sively in many laboratories,29J46,67resulting in practi- for geneamplification in chronic lymphocytic leuke-
cal recommendations, in particular to use only buffered mia (CLL). This phenomenon is particularly striking
formalin and to restrict the duration of the fixation for non-Hodgkin’s lymphomas: while the catalog of
process to 1 day. chromosome aberrations basedon banding studies
lists only 19 cytogenetic markersfor gene amplifica-
Analysis of Hematologic Neoplasias tion in 3,500 non-Hodgkin’s lymphoma c~e.s,~*
comprehensiveanalysesby CGH discoveredamplifi-
Publications of CGH of hematologic malignancies cations in some 10% of the these lymphomas.41~72
are listed in Table 1. At first glance, in most leukemias, Focal high-level amplificationsareidealfor the identi-
the observed losses and low copy number gains are in fication of amplified genes,becausethe number of
agreement with previously reported chromosome possiblecandidatesparticipating in the amplicon is
small,thus allowing a rapid testof the copy number of
Table 1. Publications of CGH in Hematologic Neoplasias candidate genesby Southern blot analysis,quantita-
tive PCR, or FISH. To assess the pathogenicrole of an
Disease References
amplified gene, it is mandatory to perform subse-
Myeloid leukemia, myelodys- 8, 15,21, 23,28,49, quent expressionanalysisat the RNA and protein
plastic syndrome 53, 69, 73 levels, as well as to determine possible effects on
Acute lymphoblastic leukemia 24,37, 44, 56, 74 factors downstream or upstream in the biochemical
Chronic B-cell leukemia 9,36,54, 72
Myeloma, plasmacytoma 1,3,14
pathways in which the respectivefactors function.
Non-Hodgkin’s lympoma 2,4,6, 7,10,12, 13, Table2 listsall oncogenesidentified sofar asamplified
16,27,32,50-52, genesin hematologic malignancies,basedon CGH
60-62,65, 71, 72 study and subsequentverification of their participa-
Hodgkin’s disease 31,55
tion in the amplicon by other means.
350 Lichter et aL
Many of these genes were previously not known to
play a role in the respective tumor type. For example,
there had been no indication of the involvement of
the MYCN oncogene in any lymphoma, although it
is well known to be highly amplified in advanced
neuroblastoma and a few other tumors. Furthermore,
interesting details of the mechanisms involved in the
development of non-Hodgkin’s lymphomas were re-
vealed. For example, it is well known that the
translocation t(14;18) in folk&r lymphoma results
in the juxtaposition of the BCL2 gene on chromo-
some band 18q2 1 and the enhancer of the immuno-
globulin heavy chain gene locus, by which BCL2
becomes aberrantly overexpressed or constitutively
expressed. In difI&e large B-cell lymphoma, BCL2 is
overexpressed by an alternative mechanism, increase
of the gene copy number through amplification.50
Interestingly, in a recent CGH study of diflke large
B-cell lymphoma at different locations in the body,
high level amplifications of distal 18q were identified,
particularly in the central nervous system, but BCL2
does not participate in the amplicons.71 The great
potential of CGH studies to identify genes with
oncogenic capacity is not restricted to amplified
regions. Our own studies in mantle cell lymphoma
might serve as an example: genome-wide screening by
CGH revealed a set of unbalanced chromosome
regions, which were the basis for subsequent compre-
hensive interphase FISH analyses.lO Fine mapping of
a recurrent deletion on chromosome 1 lq identified
the ATM gene as a possible candidate,6* which was
subsequently verified by detailed mutation analyses.63
Classification of non-Hodgkin’s lymphomas has
been an area of clinical importance. The most recently
revised European American lymphoma (REAL) clas-
sification25 integrates molecular genetic data with
morphologic and immunologic criteria for the first
time. In this classification, a number of non-
Hodgkin’s lymphoma types are listed with the indica-
tion that they are likely composed of several subenti-
ties. Wtth regard to diffuse large B-cell lymphoma
(DLBCL), the primary mediastinal B-cell lymphoma
(PMBL) was already recognized as a subentity in this
classification scheme. CGH analysis of a series of
PMBL provided striking evidence for the distinction
of this lymphoma entity; for example, the gain of
chromosome 9p is present in about 50% of the cases
while this alteration is extremely rare in any other
non-Hodgkin’s lymphoma type,31 (Bentz M, Barth
TFE, Brtiderlein S, et al, submitted). However, more
recently this lesion has also been found in a CGH
study of Hodgkin disease (HD) . Despite the genomic
imbalances detected by CGH in PMBL and HD, the
genetic make-up of both neoplasmas seems- to be
sirr~ilar.~~An emerging picture from the summaries of
CGH studies is that not only individual imbalances
but patterns of commonly occurring changes are
characteristic for a particular tumor type. Several
studies have addressed the question whether such
patterns might even be associated with the localization
of the lymphoma in the body. For example, the
heterogeneous group of DLBCL is believed to con-
tain, in addition to the already recognized PBML,25
more distinct subgroups. Characteristic imbalances
identified in primary central nervous system lympho-
mas might point to such a subgroup within the
DLBCL.“*a71 It will be interesting to see to what
extent patterns detected by CGH allow further differ-
entiation of subentities within the current non-
Hodgkin’s lymphoma classification and whether the
anatomical site can be correlated with the genetic
make-up of the respective tumor cells.
Many human leukemias and lymphomas exhibit
characteristic chromosome rearrangements (transloca-
tions or inversions), which can serve as critical param-
eter for their classification. The known chromosome
aberrations in hematologic neoplasias have been com-
piled: chromosome translocations and inversions,
such as balanced rearrangements which are character-
istic for a lymphoma entity, are primary, while
evidence of genomic imbalances are additional alter-
ations.3o Many of the secondary changes are associ-
ated with clinical tumor progression. In agreement
with this observation is the finding that several of the
translocations do not lead to increased tumor inci-
dence in transgenic mouse models; additional genetic
changes must occur for the development of a fully
malignant phenotype. In the light of this concept,
with increasing amount of CGH data on non-
Hodgkin’s lymphomas, it will be interesting to see
whether genomic imbalances ultimately correlate with
clinical parameters and have prognostic relevance.
Limitations
Because CGH can measure only differences in copy
number, rearrangements not associated with loss or
gain of material are not detectable. To uncover such
alterations, it is mandatory to apply alternative meth-
ods (described in this issue). In this regard, application
 Usesand Limitaticms of CGH
of chromosome banding and multicolor chromo-
somepainting by FISH is limited to thosecasesfrom
which adequatemetaphasecell preparations can be
obtained. While this problem can be overcome by
interphasecytogeneticsor molecularanalysisof break-
points, such approachesdepend on prior knowledge
of the potential rearrangementsto be investigated.
It is important to stressthat CGH data provide an
averageof imbalances:they are representativeof a
major proportion of the cell population under investi-
gation. The advantage is that the risk of analyzing
only a small cell clone which may not be representa-
tive of the tumor is minimized, but there is no
measureof the degree of heterogeneity within the
tumor, and a subclone-ne with additional genomic
alterations giving rise to metastasis-may escape
analysis.
While a fay laboratoriesconfirm their chromo-
some analysisin acute lymphoblastic leukemia by
CGH, the method has not entered the domain of
routine diagnostics. The procedure is technically
demandingand providesonly limited resolution;with
standardprotocols, low copy number gainsand losses
suchastrisomiesand deletionsaredetectedonly when
the unbalancedregion is at least 10 Mb.1’z35A more
labor-intensive alternative protocol reportedly results
in resolution of up to 3 Mb.38 For the detection of
high-level amplifications, 2 Mb (product of oopy
number and amplicon size) are required.33J8Thus,
even the smallestpossibleunbalancedregion detected
by chromosomalCGH consistsof a DNA segmentof
considerablesize harboring many genespotentially
playing a pathogenic role in the diseaseunder investi-
gation.
Matrix-CGH
The restrictionsof CGH are due mainly to the useof
metaphase chromosomes as hybridization targets.
Individual chromosomesneed to be identified before
measurement of fluorescence intensity ratios. Al-
though there are computerized algorithms for the
identification of chromosomes,electronically gener-
ated karyotypes still need to be carefully reassessed
by
well-trained personnel. This necessity restricts the
potential for automation of CGH and prohibits its
application in high-throughput analyses.As outlined
above, the limited resolutionisalsoinherent to the use
of metaphasechromosomesas the read-out due to
their degreeof DNA condensation.
351
To circumvent both problems,it wasimperative to
replace metaphasechromosomesas targets for the
comparative in situ hybridization. Thus, a chip-based
CGH approach termed “matrix-CGH” has been
developedG6producing not only a higher resolution
but alsoproviding a basisfor automated analysesof
genomic imbalances.In this method, the chromo-
some targets are substituted by well-defined DNA
fragments cloned in various vectors, which are then
immobilized on a glasssurfacein order to generatea
microarray with each clone at a well-defined posi-
tion.57J9a66 To obtain reproduciblepositioning of the
respective DNA clone, a robotic device is used.
Currently, a variety of arraying robots areavailablefor
the generationof microarraysfrom ink jets to pin and
capillary-basedprinting systems.The robot delivers
dropletsof DNA solutionwith a volume of lessthan 1
nL. Although ink-jetting systemsare accurate, they
aregenerallyslowerand might therefore be lesssuited
for large-scalemicroarrays. Furthermore, the fine
needles of such systems may clog when high-
molecularweight DNA, suchasP1 artificial chromo-
some(PAC) or bacterialartificial chromosome(BAC)
DNA, is usedfor arraying. More recently, a number
of robust arraying robots have becomecommercially
available,which rely on the commonly usedsplit pin
systemsof printing. While the market situation is
constantly changing, useful systemscan be found via
compilations on internet pages (see, for example,
http://www. mpiz-koeh. mpg.de/- weissbaa/Adis/
DNA-arrdy--links. htm I).
Evaporation of the dropletsshould be allowed at a
humidity that facilitatesdelayed drying. This proce-
dure results in spots typically 100 to 300 E;Lmin
diameter, although with specializedpins smallerspots
may be obtained. Following the arraying procedure,
the DNA is fixed and denatured. The chips are
hybridized simultaneouslywith differentially labeled
test and control DNAs (Fig 1) followed by stringent
washing.Evaluation of the fluorescencesignalson the
chips is performed by image analyses.For this pur-
pose,imagesfrom the entire DNA-chip areacquired,
using either confocal laser scannerdevices or high
apertureopticswith a chargedcoupled device (CCD)
camera. While at first custom-built, a few good
systemshave recently becomecommercially available
(seeweb addressabove).The important criteria for the
suitability of read-out devicesinclude (1) the quality
of the separationof the fluorescencesignalsof differ-
ent wavelengths (separation of the two channels
352 Lid&r et al

measuredsignalsand, thus, affecting the ratios of

fluorescencesignals;and (3) repetitive DNA present
in the probe solution still contributes to a small
“background” despite the nearly complete suppres-
sion of their hybridization in standardprotocols. To
facilitate an accurate classificationof the representa-
tion of a certain DNA sequence,we usea statistical
procedure in which independent measurementsfor
each target are analyzed. Following an automated
statistical procedure for the assessmentof control
Figure 1. Schematic illustration of the principles of chromo- signals,normalization, ratio calculation and classifica-
somal CGH and of matrix-CGH. For matrix-CGH, the meta- tion have been automated resulting in the label
phase chromosomes as targets are replaced by a matrix of
“underrepresented, ” “overrepresented,”or “balanced”
microarrayed DNA fragments. A simultaneous hybridization
of test and control DNA results in fluorescence intensity for each target-DNA on the final print-out (Lampel
ratios, indicating gains and losses of the respective genomic S, Gottel D, Lichter P,et al, in preparation).
material within the test genome.

required to be read-out); and (2) sensitivity and Current Status

linearity of the detection systemsto allow accurate
quantitative measurements of the fluorescencein both
channels. The latter criterion applies especially to
matrix-CGH, sinceit is more demanding than other
chip techniquesassubtlefluorescenceratio differences
need to be reliably scored.In theory, the maximum
ratio differencesfor low copy number changesis 0.5
(0.5 for a deletion and 1.5 for a trisomy comparedto
1.0 for a balanced state). In practice, these ratio
differencesare considerably smaller because(1) the
test specimen rarely consistsof a cell population
which is homogeneousregarding genomic imbal-
ances; (2) the hybridization procedure inherently
resultsin backgroundfluorescencecontributing to the
The resolution of matrix-CGH is defined by the
complexity of the selectedtarget DNAs. In an initial
setof experiments,we analyzedtumors that contained
deletionsthat were presentby interphasecytogenetics
but were too small to be detected by chromosomal
CGH.G6 Applying matrix-CGH with a contig of
DNAs from the region of interest (13q14) cloned
into PAC vectors, the genomic imbalancescould be
found reliably (Fig 2). Thus, by matrix-CGH, a
resolution is achievedwhich is at leasttwo orders of
magnitude better than routine chromosomalCGH.
Low copy number gainsand lossesare reliably scored
with genomic DNA fragments in the size range of
fragmentscloned in Pl , PAC, or BAC vectors (about

13q14 -
D13S273
II I 413525 ----
II
PAC9SLWMO PAfZSSM62 PAt.X%&M PActme9xsz
PAWxe9S PAW3SM.4
PAc273P3 - PACs

Figure 2. Example of a fine mapping analysis of a deletion using matrix-CGH. Individual DNA fragments of a PAC-contig were
arrayed as target for a CGH experiment. Signal ratios obtained in 2 tumor cases (indicated by light and dark shades) are
displayed. Ratios of ~1.0 indicate a balanced situation in the tumor DNA, as expected for the disomic side of the breakpoint.
Across the deleted region the fluorescence intensity ratios measured are ~0.6 indicating loss of genomic sequences. The
resolution of deletion detection in this particular experiment is defined by the smallest PAC clone, which is 75 kb.
 Usesand Limitations of CGH
100 kb or greater),57~66 while high-level amplification
is detectablewith targetsonly severalkilobasesin size.
The use of severalsmaller fragments from one ge-
nomic region astargetsalsoallowsa reliablescoringof
low copy number differencesby applying statistical
means.Recently, an impressiveresolution has been
reported by the successfulapplication of cDNA arrays
for the assessment of DNA copy numbers.59
DNA chipswith arrayedfragmentsapproximately
100 kb in size, derived from a set of contiguously
mapped DNAs, can be usedto study microdeletions
or overrepresentations.Such arrayed contigs are an
ideal tool to facilitate gene hunting efforts in tumor
genetics;hybridization of a seriesof tumors contain-
ing a recurrent deletion or amplification allowsidenti-
fication the smallestcommon region of gain or loss
rapidly. The feasibility of this approach has recently
beendemonstratedusingthe variablehigh-levelampli-
fications on 20q13.2 in breastcancer,for example.57
Thus, matrix-CGH might become the method of
choice for bridging the gap between chromosome
regionsmore than 10 Mbp in size, identified by loss
of heterozygosity or cytogenetics to be crucial in
tumor pathogenesis,and positional cloning strategies,
which are feasiblewhen the region of interest is
confined to a segmentof approximately 1 Mbp or
smaller.
Prospective Chip Designs
With the expectedcompletion of the physicalmap of
the human genomeon the level of fragmentscloned
in cosmid, Pl, PAC, and BAC vectors, it seems
reasonableto predict the development of a whole
human genomematrix-CGH chip. A first generation
of such a chip likely will consist of more than 500
target fragments to achievea resolution that is about
the standard of Giemsa banded chromosomes.The
analysisof a cell population containing a homoge-
neously stained region (HSR) or double-minute
chromosomes,both cytogenetic hallmarks for gene
amplification, would immediately revealthe genomic
sequences participating in the amplicon. Conversely,
deletionswould be detectedwith high resolution by a
singlehybridization. Thus, matrix-CGH with awhole
genome chip would be an ideal tool to screenfor
regions of microdeletions or small gains previously
unidentified, particularly useful for the analysisof
caseswith suspectedchromosomalaberrationsbut a
normal karyotype. Sincethe cryptic aberrationsinvolv-
353
ing telomeric bands in families with unexplained
mental retardation or dysmorphic featuresare associ-
atedwith gain and lossof genomicmaterial, it may be
of interest to usematrix CGH asa screeningtool and
asan alternative to FISH approachesunder develop-
ment. Additionally, matrix-CGH might be a suitable
technique to study the extent of genomicduplications
and deletions that have occurred during evolution
and that seem to exist as polymorphisms within
populations.
In the near future, we will likely seethe develop-
ment of genomic chips “a la carte,” designedto meet
specific diagnostic needsfocusing on selectedsubre-
gions of the human genome. Arrayed DNA frag-
ments of contiguousgenomic DNA will allow identi-
fication of critical unbalancedregionsto support gene
hunting efforts; they may alsobe targeted to analyze
suspectedmicrodeletion syndromes.Alternatively, tar-
get DNA sequencescould be selected which are
known to be associatedwith the development or
progressionof certain tumor types. If they are of
prognostic relevance,they could become important
tools in the designof treatment strategies.
Matrix-CGH Chips Specific for Hematologic
Diseases
As outlined above, the analysisof genomic alterations
in hematologic neoplasia has revealed numerous
specific changesin copy number of chromosome
bands, arms, or of entire chromosomes.These find-
ings support identification of subgroupswithin cer-
tain types of tumors or, if the changesareof predictive
value, have the potential to contribute to the individu-
alization of therapy. For example, in B-cell chronic
lymphocytic leukemia (B-CLL), the recurrent aberra-
tions are losseswithin bands 6q21, 1lq22-q23,
13q14, and 17~13, aswell asgains of bands 3q26,
8q24, and 12q13-q21, some of which are closely
associatedwith the clinical course, particularly re-
sponse to therapy and survival.‘7 Comprehensive
clinical studieswill be required to assess the value of
thesegenetic markersfor the stratification of patient
groupssubjectedto di&rent treatment protocols. It is
therefore mandatory to develop a rapid and reliable
diagnostic test to analyze all relevant genomic alter-
ations of B-CLL. We have focused on the develop-
ment of matrix-CGH DNA chipsfor the diagnosisof
the most recurrent genomic imbalancesin hemato-
logic malignanciessuch as B-CLL. Genomic DNA
 Licbter et al

Figure 3. (A) Architecture of a matrix-CGH chip for the detection of genetic imbalances in B-CLL. Test and control targets are
flanked by control clusters (indicated) monitoring specificity of hybridization and suppression and determining signal noise.
(B) Detection of a high copy number amplification within a tumor sample. Target #85, containingthe human MYCgene, is part
of the amplicon, whereas the adjacent target #83 participates only in part to the amplicon. Note that the MYC amplification is
easily scored by visual inspection, whereas low copy number gains and losses are evaluated by dedicated image analysis
procedures only. The fluorescence images of test (green) and control (red) probe DNA were pseudocolored and overlayed for
illustration purposes.

sequences derived from the respective regions were Automated high-resolution genome-wide screen-
selected, isolated, and arrayed on a dedicated B-CLL ing for chromosomal imbalances can be envisioned as
matrix-CGH chip. While the first generation of this a diagnostic standard supplementing or even partly
chip yielded a specificity and sensitivity of approxi- substituting for current diagnostic procedures relying
mately 95%, a second-generation chip has now been on examination of chromosome preparations. Re-
developed to improve its robustness and reliability To cently, the successful application of cDNA arrays for
this aim, additional targets from the relevant genomic the assessment of DNA copy numbers has been
regions were included to further increase the redun- reported. s9 In principal, this provides the possibility of
dancy of information further. This chip contains using the same chip design for the identification of
more than 80 diierent targets routinely spotted in 10 genomic as well as transcript imbalances. Thus, it
replicas each (Fig 3); its reliability is currently being becomes feasible to gain information of the copy
assessed. number and the activity of the same genes within a
A more precise level of analysis could be reached by given cell population.
using a set of target DNAs representative for all genes
with oncogenic potential-all proto-oncogenes and
tumor-suppressor genes-which might provide a valu- References
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