Comparative genomic hybridization is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells
SNP (Single Nucleotide Polymorphic), SNP mapping, SNP profile, SNP types, SNP analysis by gel electropherosis and by mass spectrometry, SNP effects, single strand conformation polymorphism, SNP advantages and disadvantages and application of SNP profile in drug choice
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
complete Single Nucleotide Polymorphiitsm Detection methods with Advance techniques with its applications
Single nucleotide polymorphisms are single base variations between genomes within a species.
There are at least 10 million polymorphic sites in the human genome.
SNPs can distinguish individuals from one another
Denaturing Gradient Gel Electrophoresis
Chemical Cleavage Of Mismatch
Single-stranded Conformation Polymorphism (SSCP)
MutS Protein-binding Assays
Mismatch Repair Detection (MRD)
Heteroduplex Analysis (HA)
Denaturing High Performance Liquid Chromatography (DHPLC)
UNG-Mediated T-Sequencing
RNA-Mediated Finger printing with MALDI MS Detection
Sequencing by Hybridization
Direct DNA Sequencing
Single-feature polymorphism (SFP)
Invader probe
Allele-specific oligonucleotide probes
PCR-based methods
Allele specific primers
Sequence Polymorphism-Derived (SPD) markers
Targeting induced local lesions in genomes (TILLinG)
Minisequencing primers
Allele-specific ligation probes
A micro-array is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern.
This was made by me while I was in Masters. I have made few animations. I hope it makes understanding better.
The content is made by searching through internet and referencing books. I do not claim any content in whole presentation except the animations made on the subject.
SNP (Single Nucleotide Polymorphic), SNP mapping, SNP profile, SNP types, SNP analysis by gel electropherosis and by mass spectrometry, SNP effects, single strand conformation polymorphism, SNP advantages and disadvantages and application of SNP profile in drug choice
Fluorescent in situ hybridization (FISH) is a cytogenetic technique that can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes.
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
complete Single Nucleotide Polymorphiitsm Detection methods with Advance techniques with its applications
Single nucleotide polymorphisms are single base variations between genomes within a species.
There are at least 10 million polymorphic sites in the human genome.
SNPs can distinguish individuals from one another
Denaturing Gradient Gel Electrophoresis
Chemical Cleavage Of Mismatch
Single-stranded Conformation Polymorphism (SSCP)
MutS Protein-binding Assays
Mismatch Repair Detection (MRD)
Heteroduplex Analysis (HA)
Denaturing High Performance Liquid Chromatography (DHPLC)
UNG-Mediated T-Sequencing
RNA-Mediated Finger printing with MALDI MS Detection
Sequencing by Hybridization
Direct DNA Sequencing
Single-feature polymorphism (SFP)
Invader probe
Allele-specific oligonucleotide probes
PCR-based methods
Allele specific primers
Sequence Polymorphism-Derived (SPD) markers
Targeting induced local lesions in genomes (TILLinG)
Minisequencing primers
Allele-specific ligation probes
A micro-array is a tool for analyzing gene expression that consists of a small membrane or glass slide containing samples of many genes arranged in a regular pattern.
This was made by me while I was in Masters. I have made few animations. I hope it makes understanding better.
The content is made by searching through internet and referencing books. I do not claim any content in whole presentation except the animations made on the subject.
Micro array based comparative genomic hybridisation -Dr Yogesh DDr.Yogesh D
This is a brief introduction to the technique and principle of Array Comparative Genomic Hybridization. Array CGH is a powerful tool for genetic testing and has been enormously useful in cancer cytogenetics, prenatal genetic testing etc.
Define karyotype and FISH
Describe the procedure of karyotyping and FISH
Explain chromosomal abnormalities through karyotyping and FISH
Describe the principles of FISH
Presented by-
Dr. Subarna Das
Resident, MS Anatomy
Phase-A, Year-1, Block-2
Guided by-
Prof. Laila Anjuman Banu
Chairman
Department of Anatomy, BSMMU
NSA Diagnostic Laboratory has been operating since 1958, founded by Prof. Nasseh Amin. NSA is considered as one of the most advanced labs in Egypt. Maintaining personalized services for its stakeholders, as well as the main role of the lab "Diagnosis"
NSA Diagnostic Laboratory operates through two different segments.
Firstly, a group of stand-alone labs located at prime locations all over Egypt, with the latest and up to date equipments.
Secondly, being the backbone of well reputed hospitals and some polyclinics where NSA is the lab that is responsible for all medical testing there, serving all our patients with class A quality.
Our main focus is delivering quality care and with Cost-value return. NSA plays a key role in improving the health of many Egyptians, by providing access to quality service for more than 200,000 patients annually.
Low level somatic variant detection by Sanger sequencing of formalin-fixed pa...Thermo Fisher Scientific
Deleterious sequence variants play an important role in the initiation and progression of many different cancer types. The detection of germline variants by the gold standard Sanger sequencing has been well established, however, the detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a lower level, has been more challenging.
DNA Amplification is a Ubiquitous Mechanism of Oncogene Activation in Lung an...Shryli Shreekar
Chromosomal translocation is the best-characterized
genetic mechanism for oncogene activation. However, there
are documented examples of activation by alternate
mechanisms, for example gene dosage increase, though
its prevalence is unclear. Here, we answered the fundamental question of the contribution of DNA amplification
as a molecular mechanism driving oncogenesis. Comparing
104 cancer lines representing diverse tissue origins
identified genes residing in amplification ‘hotspots’ and
discovered an unexpected frequency of genes activated by
this mechanism. The 3431 amplicons identified represent
B10 per hematological and B36 per epithelial cancer
genome. Many recurrently amplified oncogenes were
previously known to be activated only by disease-specific
translocations. The 135 hotspots identified contain 538
unique genes and are enriched for proliferation, apoptosis
and linage-dependency genes, reflecting functions advantageous to tumor growth. Integrating gene dosage with
expression data validated the downstream impact of the
novel amplification events in both cell lines and clinical
samples. For example, multiple downstream components of
the EGFR-family-signaling pathway, including CDK5,
AKT1 and SHC1, are overexpressed as a direct result of
gene amplification in lung cancer. Our findings suggest that
amplification is far more common a mechanism of
oncogene activation than previously believed and that
specific regions of the genome are hotspots of amplification.
Developing a framework for for detection of low frequency somatic genetic alt...Ronak Shah
Cancer is a complex, heterogeneous disease of the genome. Most cancers result
from an accumulation of multiple genetic alterations that lead to dysfunction of cancer-associated
genes and pathways. Recent advances in sequencing technology have enabled comprehensive
profiling of genetic alterations in cancer. We have established a targeted sequencing platform
(IMPACT: Integrated Mutation Profiling of Actionable Cancer Targets) using hybridization capture and
next-generation sequencing (NGS) technology, which can reveal mutations, indels and copy number
alterations involving 340 cancer related genes.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
2. Human DNA has 6 million nucleotides
packaged into 2 sets of 23 chromosomes.
3. 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
4. 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").
5.
6. 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
7. 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
8. Flourescent in situ hybridization,
Comparative genomic hybridization,
Array comparative genomic hybridization,
Virtual karyotyping with SNP arrays.
Next-generation sequencing.
9. 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.
10. 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.
11.
12. 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]).
13.
14. 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.
.
15.
16. 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 .
17.
18. 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.
19.
20.
21.
22. 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.
23. 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). .
24. 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).
25.
26.
27.
28.
29.
30. 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
31. Balanced chromosomal translocations cannot
be detected
Triploidy cannot be detected.
32. Cancer applications
Tumour specific genetic imbalances
(diagnosis)
Progression imbalances (prognosis and
treatment)
Novel imbalances(new therapeutic targets)
33.
34. 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
35. 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.
36. 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.
37. 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.
38. 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.
39. 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.
40. 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
41. Comparative genomic hybridization detects
many recurrent imbalances in central
nervous system primitive neuroectodermal
tumours in children
H Avet-Loiseau et al
42. 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.
43. 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
44. 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.
45. 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
46. 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.
47. 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
48. 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.
49. 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
50. 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.
51. Culture not required-faster results
Automated-more objective assessment
Better resolution-Detection of
submicroscopic rearrangements.
52. 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
53. 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
54. 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
55. To detect cryptic translocations in cases of
idiopathic mental retardation,developmental
delay, seizure disorder and autism spectrum
disorder
56.
57. 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.
58. 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.
59. 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.
60. 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.
61. 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.
62. 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.
63.
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.
64. 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.
65. 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
66. 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.
67.
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
68. 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).
69. 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. .
70. 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
71. Alteration of gene expression
Identification of critical genes involved in
disease processes
72. 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.
73. 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.
74. 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.
75.
76.
77.
78.
79.
80. 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.
81. 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
82.
83. 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.
84. (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.
85. Detection of aneuploidy in placental and
foetal tissues.
Predominantly trisomy and monosomy of X
chromosomes
86. Differentiation between metastasis and
secondary tumours
Identification of the primary tumour location
in case of metastasis
87.
88.
89.
90. 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
91. (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.
92.
93. 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.
94. 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.
95. 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.
96. 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.
97. 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.
98. 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
99. 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
100. 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.
101. 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.
102.
103. As a supportive tool in diagnostic pathology
To differentiate between metastasis and
second primary
To identify primary tumour location
104. 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
105. 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.
106.
107.
108. 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.