Comparitive genomic hybridisation

 Human DNA has 6 million nucleotides
packaged into 2 sets of 23 chromosomes.

Large scale
1. Amplifications
2. Deletions
3. Translocations
4. Interstitial deletions
5. Inversions
6. Loss of heterozygosity
Small scale
1. Point mutations/SNIPS
2. Insertions
3. Deletions

 Copy-number variations (CNVs) are alterations
of the DNA of a genome that results in the cell
having an abnormal number of copies of one or
more sections of the DNA.
 Large regions of the genome have been deleted
(fewer than the normal number) or duplicated
(more than the normal number) on certain
chromosomes.
 For example, the chromosome that normally has
sections in order as A-B-C-D might instead have
sections A-B-C-C-D (a duplication of "C") or A-B-
D (a deletion of "C").

 Amplifications and deletions can contribute
to tumorigenesis
 Amplification is the most common change
seen in malignancies
 Detection and mapping provides an
approach to associate an aberration with a
disease phenotype and localising critical
genes-Biomarkers
 Prognosis and therapeutics

 Resistance and susceptibility to disease
Eg: HIV and SLE
 Mental retardation, developmental delay and
seizure disorders
 Dysmorphic features and multiple congenital
anomalies
 Schizophrenia and autism spectrum disorder

 Flourescent in situ hybridization,
 Comparative genomic hybridization,
 Array comparative genomic hybridization,
 Virtual karyotyping with SNP arrays.
 Next-generation sequencing.

 Comparative genomic hybridization (CGH)
or Chromosomal Microarray Analysis (CMA)
is a molecular-cytogenetic method for the
analysis of copy number
changes (gains/losses) in the DNA content of
a given subject's DNA and often
in tumor cells.
 First described in 1993 by Kallioniemi et al.

 DNA from subject tissue and from normal
control tissue (reference) are each labeled
with different tags
 Hybridised to metaphase chromosomes or,
for array- or matrix-CGH
 Regional differences in the fluorescence ratio
of gains/losses vs. control DNA can be
detected and used for identifying abnormal
regions in the genome.

After extraction of test DNA (i.e.
from a tumor sample) and normal
DNA (i.e. from peripheral blood),
the samples are differentially
labeled with discernable
fluorochromes (i.e. tumor DNA
with FITC [green] and control
DNA with TRITC [red]).

 The genomes are combined with an excess of cot
1 DNA and hybridised to metaphase
chromosomes.
 Background hybridization due to repetitive DNA
sequences is a common problem in assays.
 Cot-1 DNA blocking reagent blocks repetitive
DNA sequences and prevents nonspecific
hybridization.

.

 Images of metaphase spreads are then
acquired with a (charged coupled
device) CCD camera and fluorochrome-
specific optical filter sets to capture the
FITC and TRITC fluorescence
Differences in fluorescence intensity
values between tumor and control DNA
represent gains and losses of specific
chromosomes or chromosomal regions .

 A gain of a chromosomal region in the test
sample would result in an increased intensity of
green fluorescence
 A loss within a chromosomal region in the tumor
would be indicated by a shift towards red
intensities.
 CGH analysis software measures fluorescence
intensity values along the length of the
chromosomes and translates the ratios into
chromosome profiles.
 The ratio of green to red fluorescence values is
used to quantitate genetic imbalances in tumor
samples.

 Chromosomal CGH is capable of detecting
loss, gain and amplification of the copy
number at the levels of chromosomes.
 To detect a single copy loss the region must
be at least 5–10 Mb in length.
 Detection of amplifications (e.g. tens or
hundreds of copies of one or few neighboring
genes) is known to be sensitive down to less
than 1 Mb.

Array-comparative genomic
hybridization (also CMA, Chromosomal
microarray analysis, microarray-based
comparative genomic hybridization, array
CGH, a-CGH, aCGH) is a technique to detect
genomic copy number variations at a higher
resolution level than chromosome-
based comparative genomic
hybridization (CGH). .

 Array CGH, or simply aCGH—uses slides arrayed
with small segments of DNA as the targets for
analysis.
 These microarrays are created by the deposit
and immobilization of small amounts of DNA
(known as probes) on a solid support, such as a
glass slide, in an ordered fashion.
 Probes vary in size from oligonucleotides
manufactured to represent areas of interest (25–
85 base pairs) to genomic clones such as
bacterial artificial chromosomes (80,000–200,000
base pairs).

 Can detect smaller imbalances than with
traditional karyotyping or FISH analysis-as
small as 40-80K bps
 Can detect microdeletions/duplications in a
single experiment
 Can detect rearrangements that might
implications in genetic counselling

 Balanced chromosomal translocations cannot
be detected
 Triploidy cannot be detected.

Cancer applications
 Tumour specific genetic imbalances
(diagnosis)
 Progression imbalances (prognosis and
treatment)
 Novel imbalances(new therapeutic targets)

 A strong correlation between EGFR copy
number on chromosome 7 and GBM
 A gain on chromosome 17q and a loss on
chromosome 17p seen in PNET
 Deletions on 20q seen in haematopoeitic
malignancies(MDS,MPN,AML)
 Cyclin D1 as a target Oncogene at 11q13.3 in
Nasopharyngeal Carcinoma

 Recurrent chromosome alterations in
hepatocellular carcinoma detected by
comparative genomic hybridization.
Guan XY et al.
 Department of Clinical Oncology, Queen Mary
Hospital, University of Hong Kong, Hong
Kong in october 2000.

 Studying global gene expression patterns in HCC
using microarrays.
 Analysis of genomic DNA copy number among 49
HCC samples using BAC array-based comparative
genomic hybridization (CGH). We observed
recurrent and characteristic chromosomal
aberrations, including frequent DNA copy number
gains of 1q, 6p, 8q and 20q, and losses of 4q, 8p,
13q, 16q and 17p.
 High expression of Jab1 in HCC significantly
correlated with DNA copy number gain at 8q.

 Functional analysis in HCC cell lines
demonstrated that Jab1 may regulate HCC
cell proliferation, thereby having a potential
role in HCC development.
 In conclusion, this study shows that array-
based CGH provides high resolution mapping
of chromosomal aberrations in HCC, and
demonstrates the feasibility of correlating
array CGH data with gene expression data to
identify novel oncogenes and tumor
suppressor genes.

 Childhood Medulloblastoma by Comparative
Genomic Hybridization-David A. Reardon et
al at St Jude’s children’s
hospital,Memphis.S,1997.
 Primary medulloblastoma, using comparative
genomic hybridization to evaluate
chromosomal regions for significant gain or
loss of genomic DNA.
 c-myc oncogenes amplification in
medulloblastomas. Evidence of particularly
aggressive behavior of a tumor with c-myc
amplification.

 c-myc amplification was investigated in 27
medulloblastomas.
 Unusual rapidly aggressive course with
massive cerebrospinal fluid dissemination
unresponsive to intrathecal chemotherapy.
 C-myc amplification, may provide a growth
advantage for medulloblastoma cells in vivo,
favoring their rapid dissemination.
 Medulloblastomas with c-myc activation may
represent a subgroup of tumors with a more
aggressive behavior.

 C-myc overexpression due to amplification in
medulloblastomas –large cell/anaplastic type
and poor prognosis
 CNVs on ch.4 associated with lymph node
metastasis in colorectal carcinoma
 Loss of SOCS6 associated with poor prognosis
in lung squamous cell carcinoma
 Amplification of SKP2-aggresiveness in
myxofibrosarcoma
 Gain in 8q region in Ca prostate-poor
prognosis

 Comparative genomic hybridization detects
many recurrent imbalances in central
nervous system primitive neuroectodermal
tumours in children
H Avet-Loiseau et al

 A series of 23 children with primitive
neuroectodermal tumours (PNET) were analysed
with comparative genomic hybridization (CGH).
 Multiple chromosomal imbalances have been
detected in 20 patients. The most frequently
involved chromosome was chromosome 17, with
a gain of 17q (11 cases) and loss of 17p (eight
cases). Further recurrent copy number changes
were detected
 These recurrent chromosomal changes may
highlight locations of novel genes with an
important role in the development and/or
progression of PNET.

 Overexpression of Her2 on chromosome 17-
Trastuzumab
 Amplification of androgen receptor gene-
resistance to androgen deprivation therapy
 Gains of 1q21–q22 and 13q12–q14 Are
Potential Indicators for Resistance to
Cisplatin-based Chemotherapy in Ovarian
Cancer Patients1
 Gefitinib in non small cell carcinoma lung
with overexpression of EGFR

Amplification of the androgen receptor gene and
progression of human prostate cancer
 Tapio visakorpi Kallioniemi1 1Laboratory of Cancer
Genetics, Tampere University Hospital and Institute
of Medical Technology, Tampere, Finland
 Overexpression of amplified genes is often
associated with the acquisition of resistance to
cancer therapeutic agents
 Comparative genomic hybridization shows that
amplification of the Xq11−q13 region (the location),
is common in tumours recurring during androgen
deprivation therapy.
 AR amplification emerges during androgen
deprivation therapy by facilitating tumour cell
growth in low androgen concentrations.

 Molecular classification of different types
of tumours.
Eg:Differences in genetic alterations in
pharyngeal,laryngeal and oral SCCs
 Tumour progression
Eg-65% of grade III show loss of long arm of
chromomsome 16 in contrast to 16% of grade
I carcinomas
 Genomic changes at various stages

 To differentiate between a second primary
and metastases
 To identify primary in case of metastases
 To differentiate between two synchronous
primary tumours
M M Weiss et al,Journal of clinical
pathology,2003.

 66 year old woman with polypoidal lesion in
the gastric cardia
 Diagnosed as poorly diferentiated
adenocarcinoma arising in a villous adenoma
 Two years later, flat lesion in the distal
oesophagus
 Histopathology showed superficially invasive
squamous cell carcinoma
 SCC-15 aberrations, gastric tumour-4
aberrations which were mutually exclusive
 Generally SCCs show more complex
chromosal aberrations than adenocarcinomas

 76 year old lady underwent lumpectomy for
ductal carcinoma breast with sentinel lymph
node positive.
 Three years later, undifferentiated carcinoma in
the bladder.
 Both were positive for CK,CAM5.2 and
progesterone receptor.
 CGH showed 25 aberrations in the breast tumour
and nine aberrations in the bladder
 Amplifications were at different regions.
 Excludes the possibility of common origin of
the tumours.

 58 year old woman-synchronous endometroid
carcinomas of ovary and endometrium
 Both diploid by flow cytometry
 Three aberrations in the uterus and two in
the ovary none of which were shared
 Separate primary tumours

 Evaluation of foetal anomalies and stillbirths.
 Can identify chromosome abnormalities 100x
smaller than by karyotyping
 Submicroscopic deletions can be detected.
 Tissues from patients with subtle
rearrangements involving telomeric regions
have been analyzed by CGH, and with
extreme care, even the variation in the
telomeric regions can be detected by this
technique.

 Culture not required-faster results
 Automated-more objective assessment
 Better resolution-Detection of
submicroscopic rearrangements.

 Cannot differentiate between diploid,triploid
and tetraploid complements-because the
relative gene content is balanced.
 Cannot identify balanced structural
chromosomal translocations
 Cannot distinguish low levels of mosaicism
from diploid
 Cannot distinguish high levels of moaicism
from trisomy

 Evaluation of ultrasound abnormalities
 Evaluation of stillbirths
 In cases of typical pre natal indications-after
normal karyotype
5-6% have abnormal copy number variation
1-1.5% CNV of uncertain significance
Savage et al,Curr opinion obg gyn,2011.
 Karyotyping did not find any abnormality
that aCGH did not.
 Hillman et al,US ob gyn,2011.S

 Array CGH with genetic counselling as an
adjunct tool in pre natal cases with
anatomical abnormalities with normal
phenotype
 Fetal demise-unable to demonstrate a
conventional phenotype

 To detect cryptic translocations in cases of
idiopathic mental retardation,developmental
delay, seizure disorder and autism spectrum
disorder

 Mucinous tubular and spindle cell carcinoma (MTSCC) has
recently been integrated into the World Health Organization
classification. Although MTSCC is generally a low-grade
carcinoma, MTSCC with high-grade morphology has been recently
reported. We present the first case of high-grade MTSCC
withcomparativegenomichybridization findings. A 60-year-old
Japanese man presented with weight loss and general fatigue. He
underwent radical nephrectomy because of the clinical diagnosis
of renal cancer. Histologic examination of renal tumor showed
findings of high-grade
MTSCC.Comparativegenomichybridization analysis showed gain
of chromosomes 1q, 7, 16, 19q, and Y and loss
of chromosomes 1p, 6p, 8p, 11q (del(11)(q23)), and 13. G-band
karyotype showed gain of chromosomes 2, 3, 5, 7, 12, 16, and 20
and loss of chromosome 15. Results of our molecular genetic
analysis support the idea that high-grade MTSCC is a real
counterpart of low-grade MTSCC. There is no evidence to
designate such tumors as unclassified renal cell carcinoma.


 The CGH technique is advantageous for the analysis
of reproductive pathology specimens since tissue
culture failure, culture artifacts, and maternal-cell
contamination associated with traditional cytogenetic
analysis are eliminated, and at the same time, the
whole chromosome complement is
examined.Limitations of the CGH technique include
its inability to determine ploidy, identify balanced
rearrangements, distinguish low levels of mosaicism
from diploid, and distinguish high levels of mosaicism
from complete trisomy. A combination of
appropriately selected, additive techniques, such as
FISH, traditional cytogenetics, and flow cytometry,
can assist in diagnosing any cytogenetic abnormalities
in reproductive specimens.

 After extraction of test DNA (i.e. from a tumor sample) and normal DNA (i.e.
from peripheral blood), the samples are differentially labeled with discernable
fluorochromes (i.e. tumor DNA with FITC [green] and control DNA with TRITC
[red]). The two genomes are combined with an excess of human Cot-1 DNA
and then hybridized to normal metaphase chromosomes . Images of
metaphase spreads are then acquired with a (charged coupled device) CCD
camera and fluorochrome-specific optical filter sets to capture the FITC and
TRITC fluorescence . Differences in fluorescence intensity values between
tumor and control DNA represent gains and losses of specific chromosomes or
chromosomal regions . For example, a gain of a chromosomal region in the
test sample would result in an increased intensity of green fluorescence. A loss
within a chromosomal region in the tumor would be indicated by a shift
towards red intensities. Specialized CGH analysis software measures
fluorescence intensity values along the length of the chromosomes and
translates the ratios into chromosome profiles . The ratio of green to red
fluorescence values is used to quantitate genetic imbalances in tumor
samples.

 Cancer progresses through a series of histopathological
stages. Progression is thought to be driven by the
accumulation of genetic alterations and consequently gene
expression pattern changes. The identification of genes
and pathways involved will not only enhance our
understanding of the biology of this process, it will also
provide new targets for early diagnosis and facilitate
treatment design. Genomic approaches have proven to be
effective in detecting chromosomal alterations and
identifying genes disrupted in cancer. Gene expression
profiling has led to the subclassification of tumors. In this
article, we will describe the current technologies used in
cancer gene discovery, the model systems used to validate
the significance of the genes and pathways, and some of
the genes and pathways implicated in the progression of
preneoplastic and early stage cancer.


DNA from subject tissue and from normal
control tissue (reference) are each labeled with
different tags for later analysis and
hybridized to
normal metaphase chromosomes or, for array- or
matrix-CGH, to a slide containing hundreds or
thousands of defined DNA probes.
Using epifluorescence microscopy
and quantitative image analysis, regional
differences in the fluorescence ratio of
gains/losses vs. control DNA can be detected and
used for identifying abnormal regions in the
genome.

CNVs have been associated with susceptibility or
resistance to disease. Gene copy number can be
elevated in cancer cells. For instance, the EGFR copy
number can be higher than normal in non-small cell
lung cancer. In addition, a higher copy number
of CCL3L1 has been associated with lower
susceptibility to HIV infection, and a low copy
number of FCGR3B (the CD16 cell surface
immunoglobulin receptor) can increase susceptibility
to systemic lupus erythematosus and similar
inflammatory autoimmune disorders.Copy number
variation has also been associated
with autism,schizophrenia, and idiopathic learning
disability.


The human genome is comprised of 6 billion chemical bases (or nucleotides) of DNA packaged into two sets of 23
chromosomes, one set inherited from each parent. The DNA encodes roughly 27,000 genes. It was generally thought
that genes were almost always present in two copies in a genome. However, recent discoveries have revealed that
large segments of DNA, ranging in size from thousands to millions of DNA bases, can vary in copy-number. Such copy
number variations (or CNVs) can encompass genes leading to dosage imbalances. For example, genes that were
thought to always occur in two copies per genome have now been found to sometimes be present in one, three, or
more than three copies. In a few rare instances the genes are missing altogether (see figure).

Why are CNVs important?
Differences in the DNA sequence of our genomes contribute to our uniqueness. These changes influence most traits
including susceptibility to disease. It was thought that single nucleotide changes (called SNPs) in DNA were the most
prevalent and important form of genetic variation. The current studies reveal that CNVs comprise at least three times
the total nucleotide content of SNPs. Since CNVs often encompass genes, they may have important roles both in
human disease and drug response. Understanding the mechanisms of CNV formation may also help us better
understand human genome evolution.

How does the new CNV map help?
The new global CNV map will transform medical research in four areas. The first and most important area is in
hunting for genes underlying common diseases. To date, attempts to identify these genes have not really considered
the role CNVs may play in human health. Second, the CNV map is being used to study familial genetic conditions.
Third, there are thousands of severe developmental defects caused by chromosomal rearrangements. The CNV map is
being used to exclude variation found in unaffected individuals, helping researchers to target the region that might
be involved. The data generated will also contribute to a more accurate and complete human genome reference
sequence used by all biomedical scientists.


 The gene copy number (also "copy number
variants" or CNVs) is the number of copies of
a particular gene in the genotype of an
individual. Recent evidence shows that the
gene copy number can be elevated in cancer
cells.


 Amplifications and deletions can contribute
to tumorigenesis
 Basic understanding of cancer and diagnosis
 Detection and mapping provides an approach
to associate an aberration with a disease
phenotype and localising critical genes

Copy number variation can be discovered
by cytogenetic techniques such
as fluorescent in situ
hybridization, comparative genomic
hybridization, array comparative genomic
hybridization, and by virtual
karyotyping with SNP arrays. Recent
advances in DNA sequencing technology have
further enabled the identification of CNVs
by next-generation sequencing.


Why are CNVs important?
Differences in the DNA sequence of our genomes
contribute to our uniqueness. These changes
influence most traits including susceptibility to
disease. It was thought that single nucleotide
changes (called SNPs) in DNA were the most
prevalent and important form of genetic
variation. The current studies reveal that CNVs
comprise at least three times the total
nucleotide content of SNPs. Since CNVs often
encompass genes, they may have important roles
both in human disease and drug response.
Understanding the mechanisms of CNV formation
may also help us better understand human
genome evolution

 The human genome is comprised of 6 billion chemical
bases (or nucleotides) of DNA packaged into two sets of 23
chromosomes, one set inherited from each parent. The
DNA encodes roughly 27,000 genes. It was generally
thought that genes were almost always present in two
copies in a genome. However, recent discoveries have
revealed that large segments of DNA, ranging in size from
thousands to millions of DNA bases, can vary in copy-
number. Such copy number variations (or CNVs) can
encompass genes leading to dosage imbalances. For
example, genes that were thought to always occur in two
copies per genome have now been found to sometimes be
present in one, three, or more than three copies. In a few
rare instances the genes are missing altogether (see
figure).


 Alteration in DNA copy number is one of the many
ways in which gene expression and function may be
modified. Some variations are found among normal
individuals, others occur in the course of normal
processes in some species and still others participate
in causing various disease states. For example, many
defects in human development are due to gains and
losses of chromosomes and chromosomal segments
that occur before or shortly after fertilization, and
DNA dosage-alteration changes occurring in somatic
cells are frequent contributors to cancer. Detecting
these aberrations and interpreting them in the
context of broader knowledge facilitates the
identification of crucial genes and pathways involved
in biological processes and disease. .


 Copy number analysis usually refers to the
process of analyzing data produced by a test
for DNA copy number variation in patient's
sample. Such analysis helps detect
chromosomal copy number variation that
may cause or may increase risks of various
critical disorders.Copy number variation can
be detected with various types of tests such
as fluorescent in situ hybridization
, comparative genomic hybridization and
with high-resolution array-based tests based
on array comparative genomic hybridization
(or aCGH) and SNP array technologies

 Alteration of gene expression
 Identification of critical genes involved in
disease processes

 Copy number variation can be discovered
by cytogenetic techniques such
as fluorescent in situ
hybridization, comparative genomic
hybridization, array comparative genomic
hybridization, and by virtual
karyotyping with SNP arrays. Recent
advances in DNA sequencing technology have
further enabled the identification of CNVs
by next-generation sequencing.

 Comparative genomic hybridization (CGH)
or Chromosomal Microarray Analysis (CMA)
is a molecular-cytogenetic method for the
analysis of copy number
changes (gains/losses) in the DNA content of
a given subject's DNA and often
in tumor cells.

 After extraction of test DNA (i.e. from a tumor sample) and normal DNA
(i.e. from peripheral blood), the samples are differentially labeled with
discernable fluorochromes (i.e. tumor DNA with FITC [green] and control
DNA with TRITC [red]) (Figure 3A). The two genomes are combined with
an excess of human Cot-1 DNA and then hybridized to normal metaphase
chromosomes . Images of metaphase spreads are then acquired with a
(charged coupled device) CCD camera and fluorochrome-specific optical
filter sets to capture the FITC and TRITC fluorescence . Differences in
fluorescence intensity values between tumor and control DNA represent
gains and losses of specific chromosomes or chromosomal regions . For
example, a gain of a chromosomal region in the test sample would result
in an increased intensity of green fluorescence. A loss within a
chromosomal region in the tumor would be indicated by a shift towards
red intensities. Specialized CGH analysis software measures fluorescence
intensity values along the length of the chromosomes and translates the
ratios into chromosome profiles . The ratio of green to red fluorescence
values is used to quantitate genetic imbalances in tumor samples.


 59 year old woman with clear cell tumour of
ovary
 Underwent nephrectomy 7 yrs ago for renal
cell carcinoma
 11 aberrations in the ovarian tumour and 25
in the renal cell carcinoima

 76 year old lady underwent lumpectomy for
ductal carcinoma breast with sentinel lymph
node positive.
 Three years later, undifferentiated
carcinoma in the bladder.
 Both were positive for CK,CAM5.2 and
progesterone receptor.
 CGH showed 25 aberrations in the breast
tumour and nine aberrations in the bladder
Amplifications were at different regions.

 (A) CGH begins with the isolation of both (1) genomic tumor DNA and (2) DNA from
an individual with a normal karyotype (reference or control DNA). The two genomes
are differentially labeled such that, for instance, the tumor DNA can be detected
with a green fluorochrome (FITC) and the control DNA with a red fluorochrome
(TRITC). (3) The differentially labeled genomes are then combined in the presence
of excess Cot-1 DNA.
(B) Both the probe and karyotypically normal target metaphase chromosomes are
heat denatured prior to hybridization for a 24-72 hour period at 37ï¿½C.
(C) Following a series of detection steps, metaphase chromosomes are imaged by
epifluorescence microscopy with DAPI, FITC and TRITC filters consecutively.
(1) The differences in fluorescence intensities along a chromosome are a reflection
of the actual copy number changes in the tumor genome relative to the normal
reference. The result of the hybridization shows gains and losses; in the event that a
specific chromosome region is lost in the tumor, the color of that region is shifted to
red. A gain would be represented by an increased intensity of the green
fluorescence. (2) A minimum of 5 metaphases (or 10 copies of each chromosome)
are analyzed to determine an average ratio profile. A ratio of 1 represents an equal
copy number in the tumor and the reference genome. The vertical lines to the left
and right of the chromosome represent a loss (< 0.8) and a gain (>1.2), respectively.


 Detection of aneuploidy in placental and
foetal tissues.
 Predominantly trisomy and monosomy of X
chromosomes

 Differentiation between metastasis and
secondary tumours
 Identification of the primary tumour location
in case of metastasis

 Conventional karyotyping remains the principal cytogenetic tool
in prenatal diagnosis.
 Targeted array CGH, in concert with genetic counseling, can be
offered as an adjunct tool in prenatal cases with abnormal
anatomic findings and a normal conventional karyotype, as well
as in cases of fetal demise with congenital anomalies and the
inability to obtain a conventional karyotype.
 Couples choosing targeted array CGH should receive both pretest
and posttest genetic counseling. Follow-up genetic counseling is
required for interpretation of array CGH results. Couples should
understand that array CGH will not detect all genetic pathologies
and that array CGH results may be difficult to interpret.
 Targeted array CGH may be useful as a screening tool; however,
further studies are necessary to fully determine its utility and its
limitations.
 References

 (A) CGH begins with the isolation of both (1) genomic tumor DNA and (2) DNA from an individual
with a normal karyotype (reference or control DNA). The two genomes are differentially labeled
such that, for instance, the tumor DNA can be detected with a green fluorochrome (FITC) and the
control DNA with a red fluorochrome (TRITC). (3) The differentially labeled genomes are then
combined in the presence of excess Cot-1 DNA.
(B) Both the probe and karyotypically normal target metaphase chromosomes are heat denatured
prior to hybridization for a 24-72 hour period at 37ï¿½C.
(C) Following a series of detection steps, metaphase chromosomes are imaged by epifluorescence
microscopy with DAPI, FITC and TRITC filters consecutively.
(1) The differences in fluorescence intensities along a chromosome are a reflection of the actual
copy number changes in the tumor genome relative to the normal reference. The result of the
hybridization shows gains and losses; in the event that a specific chromosome region is lost in the
tumor, the color of that region is shifted to red. A gain would be represented by an increased
intensity of the green fluorescence. (2) A minimum of 5 metaphases (or 10 copies of each
chromosome) are analyzed to determine an average ratio profile. A ratio of 1 represents an equal
copy number in the tumor and the reference genome. The vertical lines to the left and right of the
chromosome represent a loss (< 0.8) and a gain (>1.2), respectively.
 Further modification of the CGH technique includes the replacement of metaphase chromosomes
with unique DNA sequences spotted in arrays on a glass slide. Fluorescence intensities in both test
and reference DNA hybridizations to the immobilized sequences on the array (i.e. cDNA, BAC or
oligos) are averaged and normalized, and can be used to calculate an increase or decrease in copy
number (Pinkel et al., 1998; Hyman et al., 2002). This array CGH allows for higher resolution of
closely spaced genomic aberrations as well as the detection of microdeletions.


Gene amplification is the most frequently
observed type of genetic change associated
with cancer. The mechanisms of gene
amplification are not well understood, but
better understanding of the role played by
gene amplification in cancer may lead to
new cancer biomarkers and improved
prognostic and diagnostic indicators of
cancer progression.

 Figure 2. Detection of gene amplification by comparative
genomic hybridization (CGH) and fluorescent in situ hybridization
(FISH). (a) Fluorescence images of gene amplification
by CGH. Fluorescein isothiocyanate (FITC),
tetramethylrhodamine (Rhod.) and 4,6-diamidino-2-phenylindole
dihydrochloride (DAPI) images from the same metaphase
chromosome spread are shown. Each image was acquired using a
charge-coupled device camera and a fluorescence microscope.
The green-to-red fluorescence ratios were measured along each
chromosome. (b) Detection of 2p24–p24 amplification in
neuroblastoma. A neuroblastoma with MYCN amplification shows
gain at 2p24–p24. (c)MYCN amplification in neuroblastoma
using FISH. MYCN amplification is detected as multiple spots in
interphase nuclei with two centromeric spots. Chromosome 2 is
identified by the tetramethylrhodamine-labeled pericentromeric
probe, and the MYCN is identified with an FITC-labeled probe.


 Chromosomal CGH is capable of detecting loss,
gain and amplification of the copy number at the
levels of chromosomes. However, it is considered
that to detect a single copy loss the region must
be at least 5–10 Mb in length. Detection of
amplifications (e.g. tens or hundreds of copies of
one or few neighboring genes) is known to be
sensitive down to less than 1 Mb. Therefore, one
must take into consideration that while CGH is
sensitive to specific types of copy number gains,
the resolution of regional deletions is more
limited.

Instead of using metaphase chromosomes, this
method—which is known as array CGH, or simply
aCGH—uses slides arrayed with small segments of
DNA as the targets for analysis. These microarrays are
created by the deposit and immobilization of small
amounts of DNA (known as probes) on a solid support,
such as a glass slide, in an ordered fashion. Probes
vary in size from oligonucleotides manufactured to
represent areas of interest (25–85 base pairs) to
genomic clones such as bacterial artificial
chromosomes (80,000–200,000 base pairs). Because
probes are several orders of magnitude smaller than
metaphase chromosomes, the theoretical resolution
of aCGH is proportionally higher than that of
traditional CGH.

 Regardless of the type of probe, the basic methodology for aCGH
analysis is consistent. First, DNA is extracted from a
test sample (e.g., blood, skin, fetal cells). The test DNA is then
labeled with a fluorescent dye of a specific color, while DNA from
a normal control (reference) sample is labeled with a dye of a
different color. The two genomic DNAs, test and reference, are
then mixed together and applied to a microarray. Because the
DNAs have been denatured, they are single strands; thus, when
applied to the slide, they attempt to hybridize with the arrayed
single-strand probes. Next, digital imaging systems are used to
capture and quantify the relative fluorescence intensities of the
labeled DNA probes that have hybridized to each target. The
fluorescence ratio of the test and reference hybridization signals
is determined at different positions along the genome, and it
provides information on the relative copy number of sequences in
the test genome as compared to the normal genome.


 Studies of subtelomeric rearrangements illustrate how aCGH has
revealed an unprecedented amount of information about the
complexity of the human genome. Present on all but the short
arms of acrocentric chromosomes 13, 14, 15, 21, and 22,
subtelomeric regions have been the subject of a great deal of
study because they are relatively gene-rich and are prone to
rearrangement by a number of mechanisms . Moreover,
rearrangement of subtelomeric regions has been suggested to
represent a high proportion of abnormalities in individuals with
idiopathic mental retardation. Interestingly, recent large-scale
prospective studies using aCGH on similar populations show that
interstitial deletions (which are caused by two breaks in
the chromosome arm, the loss of the intervening segment, and
the rejoining of the chromosome segments) are two to three
times more frequent than terminal imbalances in subtelomeric
regions

In microarray CGH, the substrate is not a
normal metaphase spread, but an array of
DNA fragments (100 bp to 100 kb), and the
precise chromosomal locus of each is known
(fig 1). In this way, by using an array of
approximately 5000 spots, a genome wide
analysis for gains and losses at a 1 Mb
resolution is possible.6

Array-comparative genomic
hybridization (also CMA, Chromosomal
microarray analysis, microarray-based
comparative genomic hybridization, array
CGH, a-CGH, aCGH) is a technique to detect
genomic copy number variations at a higher
resolution level than chromosome-
based comparative genomic
hybridization (CGH). It can be used to create
a virtual karyotype.


DNA from a test sample and normal reference
sample are labelled differentially, using
different fluorophores, and hybridized to
several thousand probes. The probes are derived
from most of the known genes and non-coding
regions of the genome, printed on a glass slide.
The fluorescence intensity of the test and of the
reference DNA is then measured, to calculate
the ratio between them and subsequently the
copy number changes for a particular location in
the genome.

 As a supportive tool in diagnostic pathology
 To differentiate between metastasis and
second primary
 To identify primary tumour location

 66 year old woman with polypoidal lesion in
the gastric cardia
 Diagnosed as poorly diferentiated
adenocarcinoma arising in a villous adenoma
 Two years later, flat lesion in the distal
oesophagus

 Papillary thyroid carcinoma (PTC) is the most common well-differentiated thyroid
cancer. Although the great majority of the cases exhibit an indolent clinical course,
some of them develop local invasion with distant metastasis, and a few cases
transform into undifferentiated/anaplastic thyroid carcinoma with a rapidly lethal
course. To identify gene copy number alterations predictive of metastatic potential
or aggressive transformation, array-based comparative genomic hybridization (CGH-
array) was performed in 43 PTC cases. Formalin-fixed and paraffin-embedded
samples from primary tumours of 16 cases without metastasis, 14 cases with only
regional lymph node metastasis, and 13 cases with distant metastasis, recurrence or
extrathyroid extension were analysed. The CGH-array and confirmatory quantitative
real-time PCR results identified the deletion of the EIF4EBP3 and TRAK2 gene loci,
while amplification of thymosin beta 10 (TB10) and Tre-2 oncogene regions were
observed as general markers for PTC. Although there have been several studies
implicating TB10 as a specific marker based on gene expression data, our study is
the first to report on genomic amplification. Although no significant difference
could be detected between the good and bad prognosis cases in the A-kinase anchor
protein 13 (AKAP13) gene region, it was discriminative markers for metastasis.
Amplification in the AKAP13 region was demonstrated in 42.9% and 15.4% of the
cases with local or with distant metastasis, respectively, while no amplification was
detected in non-metastatic cases. AKAP13 and TB10 regions may represent potential
new genomic markers for PTC and cancer progression.


 e present study was performed to provide
direct evidence on copy number changes
during progression from chronic phase (CP)
to blastic phase (BP) in chronic myeloid
leukemia (CML) through a longitudinal
follow-up study. Matched CP and BP
samples in three patients were analyzed
using high-resolution array comparative
genomic hybridization (aCGH) chips. During
blastic transformation, loss of large
genomic segments including 6q14.1-q22.31,
chromosome 7 and 9p13.2-p21.3 were
noted.

Comparitive genomic hybridisation

Comparitive genomic hybridisation

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