COMPARITIVE GENOMIC HYBRIDISATION

Namratha R

HUMAN GENOME

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

MUTATIONSLarge scale1. Amplifications2. Deletions3. Translocations4. Interstitial deletions5. Inversions6. Loss of heterozygositySmall scale 7. Point mutations/SNIPS8. Insertions9. Deletions

COPY NUMBER VARIATION 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").

IMPORTANCE OF COPY NUMBER VARIATIONS 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

IMPORTANCE OF COPY NUMBER VARIATIONS 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

DETECTION OF COPY NUMBER VARIATIONS Flourescent in situ hybridization,  Comparative genomic hybridization,  Array comparative genomic

hybridization, Virtual karyotyping with SNP arrays. Next-generation sequencing.

COMPARITIVE GENOMIC HYBRIDISATION 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.

PRINCIPLE OF CGH 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.

PROCEDURE-CGH:STEP 1 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]).

PROCEDURE-CGH-STEP 2

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.

.

PROCEDURE-CGH-STEP 3 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 .

PROCEDURE-CGH-STEP 4 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.

LIMITATIONS OF CGH 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.

MICRO ARRAY BASED CGH 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). .

OVERCOMING LIMITATIONS OF CHG 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).

STRENGTHS OF ARRAY CGH 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

LIMITATIONS Balanced chromosomal translocations

cannot be detected Triploidy cannot be detected.

MAJOR USES OF MICROARRAYCancer applications Tumour specific genetic imbalances

(diagnosis) Progression imbalances (prognosis and

treatment) Novel imbalances(new therapeutic

targets)

CANCER APPLICATIONS-DIAGNOSIS 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

CGH-HEPTOCELLULAR 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.

CGH IN HEPATOCELLULAR CARCINOMA 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.

HEPATOCELLULAR CARCINOMA 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.

CGH IN MEDULLOBLASTOMA 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.

CGH IN MEDULLOBLASTOMA 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.

CANCER APPLICATIONS-PROGNOSIS 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

PNET-CGH IN DIAGNOSIS Comparative genomic hybridization

detects many recurrent imbalances in central nervous system primitive neuroectodermal tumours in children

H Avet-Loiseau et al

CGH IN PNET 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.

CANCER APPLICATIONS-THERAPEUTICS 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

CGH-THERAPEUTICS OF CA PROSTATEAmplification 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.

OTHER APPLICATIONS Molecular classification of different

types of tumours.Eg:Differences in genetic alterations in

pharyngeal,laryngeal and oral SCCs Tumour progressionEg-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

OTHER APPLICATIONS IN CANCER-MILTIPLE TUMOUR LOCALISATIONS To differentiate between a second

primary and metastases To identify primary in case of

metastases To differentiate between two

synchronous primary tumoursM M Weiss et al,Journal of clinical

pathology,2003.

CASE 1 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

CASE 2 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.

CASE 3 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

A TOOL FOR REPRODUCTIVE PATHOLOGY 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.

ADVANTAGES Culture not required-faster results Automated-more objective assessment Better resolution-Detection of

submicroscopic rearrangements.

LIMITATIONS OF CGH 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

INDICATIONS Evaluation of ultrasound abnormalities Evaluation of stillbirths In cases of typical pre natal indications-

after normal karyotype5-6% have abnormal copy number

variation1-1.5% CNV of uncertain significanceSavage 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

RECOMMENDATIONS 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

OTHER USES 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. 

PROCEDURE-CGH 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.

PRINCIPLE OF CGH 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.

IMPORTANCE OF COPY NUMBER VARIATIONS 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

DETECTION OF COPY NUMBER VARIATIONS 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

DETECTION OF COPY NUMBER VARIATIONS 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.

COMPARITIVE GENOMIC HYBRIDISATION 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.

PRINCIPLE OF CGH 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.

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.

METASTASIS OR SECOND PRIMARY 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

CASE 2 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.

PROCEDURE CGH (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 37�C. (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.

ANALYSIS OF REPRODUCTIVE PATHOLOGY SPECIMENS Detection of aneuploidy in placental and

foetal tissues. Predominantly trisomy and monosomy

of X chromosomes

TAKE HOME MESSAGE 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 37�C. (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.

LIMITATIONS OF CGH 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.

OVERCOMING LIMITATIONS OF CHG 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.

PRINCIPLE OF ARRAY 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

MICRO ARRAY CGH 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 BASED CGH 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.

PROCEDURE-ARRAY CGH 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.

APPLICATIONS As a supportive tool in diagnostic

pathology To differentiate between metastasis and

second primary To identify primary tumour location

CASE 1 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.