Transcript Introduction to Molecular Pathology

Molecular Pathology
 Testing of nucleic acids within a clinical context
 Helpful
 Hereditary disorders
 Oncology
 Infectious diseases
Molecular Pathology
 Specific purposes
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Diagnosis
Prognosis
Prenatal testing
Pharmacotherapy
 Pharmacogenetics
 Pharmacogenomics
Watson and Crick
 The structure of DNA was described by British
Scientists Watson and Crick as long double helix shaped
with its sugar phosphate backbone on the outside and
its bases on inside; the two strand of helix run in
opposite direction and are anti-parallel to each other.
The DNA double helix is stabilized by hydrogen bonds
between the bases

Doctortvrao’s ‘e’ learning series
Watson and Crick discovers DNA /
th
Feb 28 1953
Watson and Crick Builds a Model
th
DNA 7 March 1953
First Document on DNA published in
Nature 25th April 1953
Watson and Crick - Awarded Nobel Prize in
1962
DNA
 A molecule contains two polynucleotide strands that form
an an antiparallel double helix.
 Nucleotides:
 Nitrogenous base (AT GC,U)
 Deoxyribose
 Phosphate
DNA - Structure
 The nucleotide, however,
remains as the
fundamental unit
(monomer) of the nucleic
acid polymer. There are
four nucleotides: those
with cytosine (C), those
with guanine (G), those
with adenine (A), and
those with thymine (T).
DNA
A purine always links with a
pyrimidine base to
maintain the structure of
DNA.
Adenine ( A ) binds to Thymine
( T ), with two hydrogen
bonds between them.
Guanine ( G ) binds to Cytosine
( C ), with three hydrogen
bonds between them.
Chemical structure of DNA
DNA is Endless structure
 The rungs of the ladder
can occur in any order
(as long as the basepair rule is followed)
 Those 4 bases have
endless combinations
just like the letters of
the alphabet can
combine to make
different words.
DNA
 Example
 First strand
 Second strand
GGGTTTAAACCC
CCCAAATTTGGG
Central Dogma of Molecular Biology
DNA makes a Copy of Self
 Replication is the process
where DNA makes a copy
of itself. Why does DNA
need to copy? Simple: Cells
divide for an organism to
grow or reproduce, every
new cell needs a copy of
the DNA or instructions to
know how to be a cell. DNA
replicates right before a cell
divides.
DNA Replication
 DNA replication is
semi-conservative.
That means that when
it makes a copy, one
half of the old strand is
always kept in the new
strand. This helps
reduce the number of
copy errors.
 So we remained what
we were ?
Transcription
 RNA polymerase II mediates transcription and generates a
precursor ss-mRNA identical to the sense (coding) stand
except for U for T.
 Precursor ss-mRNA is processed in nucleus by
spliceosomes that catalyze intron removal and exon
ligation with the regulation by exonic and intronic
enhancers and silencers with production of different pre
m-RNA that go across nucleus
DNA to RNA
creates functional translations
 DNA remains in the
nucleus, but in order for it
to get its instructions
translated into proteins, it
must send its message to
the ribosome's, where
proteins are made. The
chemical used to carry this
message is Messenger
RNA

Doctortvrao’s ‘e’ learning series
DNA – RNA – DNA
a never ending cycle
 RNA has the job of taking the
message from the DNA to the
nucleus to the ribosome's.
 Transcription - RNA is made
from DNA
 Translation - Proteins are
made from the message on
the RNA

Doctortvrao’s ‘e’ learning series
Translation
 m-RNA directs protein synthesis.
 Occurs in ribosomes (rRNA+proteins)
 Codons (three bases) are read by transfer (tRNA)
 There are 64 possible codons, therefore most of the 21
aminoacids are specified by more than 1 codon
RNA = Ribonucleic acid.
 RNA is similar to DNA
except:
It has one strand
instead of two strands.
Has uracil instead of
thymine
3.Has Ribose instead of
Deoxyribose
Gene Expression
 DNA level expression control
 Transcriptional
 Post-Transcriptional
 Epigenetics
 DNA methylation
 Histone modification
Gene Expression
 DNA level expression control
 Transcriptional
 House keeping genes
 Always on
 Transcription factors
 Usually lie upstream in the promoter region
 Enhancer and silencer elements
Gene Expression
 Post transcriptional
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Export of mRNA out of nucleus
Alternative splicing
mRNA stabilization
mRNA degradation
RNA interference or silencing
 miRNA and siRNA
Gene Expression
 DNA level expression control
 Transcriptional
 Post-Transcriptional
 Epigenetics
 DNA methylation
 Histone modification
What is Gene
 The gene, the basic
units of inheritance; it
is a segment within a
very long strand of
DNA with specific
instruction for the
production of one
specific protein.
Genes located on
chromosome on it's
place or locus.
Modes of inheritance
 Mutations of single genes
 Patterns: autosomal dominant, autosomal recessive, Xlinked, mitochondrial
 Anticipation
 Increased severity of a certain diseases in successive familiar
generations associated with triple repeats
 Mosaicism
 At least two cell lines derived from a single zygote
Modes of inheritance
 Genomic imprinting
 Different expression of alleles depending on parent origin
 Uniparental Disomy
 Both copies inherited from one parent
 Environmental influence
 Chronic diseases
Mutations and Polymorphisms
 Mutation: change in DNA sequence
 Polymorphism: non disease causing change in DNA or a
change found at a frequency of ≥ 1% in population
 When evaluating changes in DNA sequence use neutral
terms: sequence variant, sequence alteration or allelic
variant. There may be:
 Missense, nonsense, deletions, insertions, frame shifts,
duplications, amplifications, trinucleatide repeats.
Single Nucleotide Polymorhisms
and Haplotypes
 SNPs are single base differences in the DNA of individuals
 There are ~10 million SNPs in the human genome
 IMPORTANCE: Pharmacogenetics
 Ex. CYP (cP450)
 Alleles of SNPs that are close together tend to be
inherited together.
 Haplotype: a set of associated SNPs alleles in a region of a
chromosome
Overview of Molecular
Techniques and Instrumentation
 Standard or usual specimen flow
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Specimen collection (blood, tissue)
Nucelic acid isolation (DNA or RNA)
Nucleic acid quantification (optional)
Nucleic acid storage
Nucleic acid amplification (or other)
Test interpretation
Quality control
Nucleic acid isolation (DNA or
RNA)
 Manual vs. automated
 Cell lysis
 Dependent of specimen type, nucleic acid being isolated for,
desired purity and application to be used in
 FFPE yields ~200 pairs
 Purification
 Organic: phenol-chloroform
 Non organic: silica, anion exchange chromatography and magnetic
particles
 DNA or RNA Isolation
 RNA rapidly degrades…
Methods
 DNA sequencing
 Southern Blot
 PCR
 RT-PCR
 Real Time PCR
 Methylation-Specific PCR
 In-situ PCR
 Protein Truncation Test
 Transcription-Mediated
Amplification
 Strand Displacement
Amplification
 Nucleic Acid SequenceBased Amplification
 Signal amplification
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Branching DNA
Hybrid Capture
Invader
FISH
DNA arrays and chips
Gene sequencing
 Determining the exact sequence of the four bases in a
given DNA template
 Two methods
 Maxam-Gilbert
 Chemical degradation
 Sanger
 Chain termination
 Radiolabeled, Dye-prime or Dye-terminator (cycle sequencing)
 Pyrosequencing
 Sequnces a short length of DNA (~30-60 bases)
Applications
of Direct DNA sequences
Clinical condition
Gene
HIV drug resistance
HIV-protease, RT
Cystic fibrosis
CFTR gene
Beta thalassemia
Beta globin
Cancer predisposition
• breast
BRCA1
•Hereditary non polyposis colon
cancer
TP53
•MEN
PTEN Ret proto-oncogene
Congenital hearing loss
Connexin 26
HCV genotyping
5’UTR
Array-based Comparative
Genomic Hybridization
 Comparative Genomic Hybridization is done in
metaphases in classical cytogenetics (M-CGH)
 Resolution 5 Mb
 Bacterial Artificial Chromosome (BAC) maps the human
genome therefore an Array based-CGH can be created (ACGH). Different resolutions up to 32,000 (45 kb)
 cDNA-CGH
 Oligonucleotide-CGH
 Can detect Single Nucleotide Pleomorphisms (SNPs) [Gene
Chip]
Methods
 DNA sequencing
 Southern Blot
 PCR
 RT-PCR
 Real Time PCR
 Methylation-Specific PCR
 In-situ PCR
 Protein Truncation Test
 Transcription-Mediated
Amplification
 Strand Displacement
Amplification
 Nucleic Acid SequenceBased Amplification
 Signal amplification
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Branching DNA
Hybrid Capture
Invader
FISH
DNA arrays and chips
Southern Blot
 Edwin M Southern, 1974
 DNA extracted
 DNA cut into pieces (Restriction Endonucleases)
 Electrophoresis and size separated
 Blot (transferred) to a membrane
 Anealed with labeled (radioactive, fluorescence,
chemiluminescent) probe
Southern Blot
working protocol
Uses of Southern Blotting
 Southern blots are used in gene discovery and mapping,
evolution and development studies, diagnostics and
forensics.
In regards to genetically modified organisms, Southern
blotting is used as a definitive test to ensure that a
particular section of DNA of known genetic sequence has
been successfully incorporated into the genome of the
host organism.
 Used in prognosis of cancer and in prenatal diagnosis of
genetic diseases
Methods
 DNA sequencing
 Southern Blot
 PCR
 RT-PCR
 Real Time PCR
 Methylation-Specific PCR
 In-situ PCR
 Protein Truncation Test
 Transcription-Mediated
Amplification
 Strand Displacement
Amplification
 Nucleic Acid SequenceBased Amplification
 Signal amplification
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Branching DNA
Hybrid Capture
Invader
FISH
DNA arrays and chips
PCR
 Kary B. Mullis 1983
 Target amplification
 Single oligonucletide
 Multiplexed
 Mimics the natural process of DNA replication, therefore,
requires:
 DNA template, DNA polymerase, dNTPs, buffer, Mg++, two
primers to flag the target sequence
 Thermal cycler
 Denaturation ~95°C
 Annealing ~45-60°C
 Extension ~72°C
PCR
 Denaturation
 Breaks the hydrogen bonds between the ds-DNA
 Anealing
 Binding to oligonucleotide sequence (probe)
 Extension
 DNA polymerase (heat stable, Taq [Thermophilus aquaticus])
replicates the selected DNA sequence
 Xn = X0 × (1 + E)n E= 0 - 1
RT-PCR
 To detect or quantify RNA transcripts or viral RNA
 RNA is converted to DNA
 Reverse transcriptase (Avian Myeloblastosis Virus and
Moloney Murine Leukemia virus)
 Isothermal reaction with primers: oligo dT, random
hexamer primers, or target specific primers
 One step vs. two steps
PCR or RT-PCR
 Product analysis / detection
 Real Time
 Hybridization
 Membrane bound
 Reverse line blots
 Liquid Bead Array with Flow Cytometry
 Electrophoresis
 Agarose
 Capillary
 Cycle sequencer
Multiplexed – PCR and ELISA
Protein Expression Profiling
Cancer Markers
Cardiac Markers
Cellular Signaling
Cytokines, Chemokines, and Growth
Factors
Endocrine
Isotyping
Matrix Metalloproteinases
Metabolic Markers
Neurobiology
Transcription Factors/Nuclear Receptors
Genetic Disease
Cystic Fibrosis
Cytochrome p450
Immunodiagnostics
Allergy Testing
Autoimmune Disease
HLA Testing
Infectious Disease
Vaccine Testing
Newborn Screening
Biodefense/Environmental
Genomic Research
FlexmiR® v2 Custom microRNA Assay
FlexmiR microRNA Panels
Gene Expression Profiling
Genotyping
Luminex
Real Time - PCR
 Amplifies and detects PCR product fluorescently in each
well of PCR plate
 Don’t have to run gel afterwards
 Use for endpoint detection
 Examples
 Fast PCR screening without gels
 Locate clone or mutant of interest
 Genotyping SNPs
 Genotype individuals using allele specific primers
Real Time - PCR
 The crossing threshold or
cycle threshold (Ct) is the
amplification cycle
number at which
fluorescence is obtained
 Ct is proportional to the
amount of staring
template (interrogated
sequence) in the sample
 Excellent for Q-PCR
PCR
Advantages
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Sensitivity
Specificity
Speed
Versatility
Automated
No need for intact
DNA/RNA
Disadvantages
 Target sequence needs to be
known
 Target needs to be conserved
among individuals
(polymorphisms)
 Oligonucleotide length
 Can fail in the detection of
chromosomal abnormalities like
translocations, inversions, large
addition or deletions
 Contamination (F+)
Methods
 DNA sequencing
 Southern Blot
 PCR
 RT-PCR
 Real Time PCR
 Methylation-Specific PCR
 In-situ PCR
 Protein Truncation Test
 Transcription-Mediated
Amplification
 Strand Displacement
Amplification
 Nucleic Acid SequenceBased Amplification
 Signal amplification
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
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Branching DNA
Hybrid Capture
Invader
FISH
DNA arrays and chips
Branched DNA
makes the complicated matters simple
 The technology uses variety
of branched DNA ( bDNA )
probes and signal
amplification reporter
molecules
 And generate
Chemiluminescent signal.
 The signal correlates with
target nucleic acid

Doctortvrao’s ‘e’ learning series
Capture Extenders (CEs), Label Extenders (LEs), and Blocking Probes (BLs)
Branched DNA applications
 Detection HIV, HBV,
and HCV
 Measures viral loads
 Less sensitive than PCR

Doctortvrao’s ‘e’ learning series
Hybrid Capture
 Qiagen
 Signal amplification technique
 Denaturated DNA gets hybridized to complimentary unlabeled
RNA sequences (if DNA sequence is present)
 Antibody bound to the well is attracted to RNA:DNA hybrids
 A second conjugated anti RNA:DNA hybrid antibody is added
 Chemiluminescent signal is generated in proportion of target
DNA present
Product Overview – Update
 CervistaTM HPV HR is an FDA approved test that screens for the
presence of 14 high-risk HPV types
 100% detection of CIN3+ and 99.1% NPV for CIN2+
 Only FDA approved HPV screening test with
an internal control
 Reduces patient call backs
 Limits QNS (only 2 ml sample volume required)
 <1% indeterminate rate
 No equivocal zone for interpretation
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HPV HR Indications
Indications for use:
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•
To screen patients with atypical squamous cells of undetermined
significance (ASC-US) cervical cytology results to determine the
need for referral to colposcopy
•
In women 30 years and older the CervistaTM HPV HR test can be
used with cervical cytology to adjunctively screen to assess the
presence or absence of high-risk HPV types. This information,
together with the physician’s assessment of cytology history,
other risk factors, and professional guidelines, may be used to
guide patient management
© 2009 Hologic, Inc. All right reserved.
B0043-0309 RevA
Product Design & Chemistry
®
Invader Chemistry

A technology protected by 128 issued U.S. patents

Structure-specific recognition and cleavage with Cleavase® enzyme

Signal amplification

Isothermal reactions: no thermal cycling needed

Fluorescence detection
Probe
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Repeating Process
Amplifies Signal
Cleavase®
Enzyme
© 2009 Hologic, Inc. All right reserved.
B0043-0309 RevA
Cervista HPV HR Test Design
TM
 Developed from phylogenetic
tree of A-superfamily HPV
strains, based on L1 region
 Cervista HPV is specific for
high-risk types selected from the
A5/A6, A7 and A9 virus groups:
A7
A5
A9
A6
A5/A6 51, 56, 66
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A7
18, 36, 45, 59, 68
A9
16, 31, 33, 35, 52, 58
© 2009 Hologic, Inc. All right reserved.
B0043-0309 RevA
®
Secondary reaction
(Simultaneous)
Primary
reaction
Invader Chemistry Overview - Summary
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Signal amplification is
typically ~107 per molecule
of target sequence.
© 2009 Hologic, Inc. All right reserved.
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®
Invader HPV Biplex Reaction Format
HPV-specific targets
Human DNA-specific target
Cleavage Site
Cleavage Site
Probe
Probe
C
A
Invader® Oligo
Invader® Oligo
G
T
Released 5´ Flap
Released 5´ Flap
C
A
Cleavage
Site
Cleavage
Site
F1
F2
Q
Q
C
A
FRET Cassette 2
FRET Cassette 1
F2
F1
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Cervista™ HPV HR Test Workflow
MAGNET
Pellet Cells
Read & Analyze
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Genfind Extraction
Incubation
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Reaction
Setup
B0043-0309 RevA
Cervista™ HPV HR Test Process
Full 4-hour walk-away time enhances productivity.
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Interpretation of Results
User-friendly
Data Analysis Software

Intuitive user interface

Screen-by-screen walk- through of
process steps
Flexible

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Multiple reporting options
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Clinical Performance
Cervista™ HPV Clinical Trial Overview
 Screened >50,000 women; enrolled approximately 4,000
 Samples collected from 89 sites in 23 states, providing diversity
 Included both ASC-US and normal cytology arms
 ASC-US: (>1,300 women)
 Primarily intended to show that women with abnormal Pap test results and
negative HPV results have < 1% probability of cervical disease (CIN2/3), or
conversely >99% NPV
 Also intended to show a sensitivity ≥ 90% for detecting cervical disease
 Normal cytology (NILM) arm: (>2,000 women)
 Intended to establish that HPV-positive subjects are more likely than HPVnegative subjects to develop cervical disease (CIN2+) over a 3-year period
 All clinical objectives achieved
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Clinical Performance
CIN3+ detection:
 Cervista™ HPV HR versus Colposcopy/Consensus Histology results
(CIN3+) among women with ASC-US cytology
Cervista™ HPV HR
‡No CIN,
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Colposcopy/Consensus Histology
Positive
Negative‡
Total
Positive
22
747
769
Negative
0
563
563
Total
22
1310
1332
100%
detection
CIN1 or CIN2 by Central Histology or Colposcopy without Central Histology.
© 2009 Hologic, Inc. All right reserved.
B0043-0309 RevA
Clinical Performance
CIN2+ detection:
 Cervista™ HPV HR versus Colposcopy/Consensus Histology results
(CIN2+) among women with ASC-US cytology
Cervista™ HPV HR
‡ No
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Colposcopy/Histology
Positive
Negative‡
Total
Positive
64
705
769
Negative
5
558
563
Total
69
1263
1332
93%
detection
CIN or CIN1 by Central Histology or Colposcopy without Central Histology
© 2009 Hologic, Inc. All right reserved.
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Clinical Performance
Comparison of Cervista™
HPV HR clinical trial and ALTS
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ASC-US/LSIL Triage Study (ALTS)
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Organized and funded by the NCI

Included over 5,000 patients

Benchmark ASC-US triage study
a Cervista™
HPV HR multicenter clinical trial, 2006–2008. Clinical and analytical data on file, Hologic, Inc.
Immediate colposcopy arm of ALTS.
c Number of subjects with known disease status and Cervista™ HPV HR results.
d Referral rate for women 30 years of age and older was 43%.
b
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Cervista HPV HR Benefits
TM
Confidence of an Internal Control
Only FDA approved HPV screening test with an internal control

Confirms the presence of adequate cellular material for testing

Confirms that no inhibitory substances are present

Minimizes false-negatives due to insufficient sample cellularity
Test Contains an Internal Control
Cervista HPV HR: The only FDA-approved HPV
test with an internal control
TM
Example Invader Call ReporterTM Output
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Minimizes False Positives
 Cross-reactivity to these common low-risk HPV types causes
false-positive results, which can lead to unnecessary colposcopies
1Hybrid
Capture® 2 High-Risk HPV DNA Test® package insert #L00665, Rev. 2, 2007
Solomon D., et al. A Comparison of Two Methods to Determine the Presence of High-Risk HPV Cervical Infections.
Am J Clin Pathol 2008;130:401-408.
2Castle PE,
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Substantially Reducing Patient Call Backs
 Requires only half the sample
volume of other HPV tests
(2 ml vs. 4 ml)
Minimum Sample amount Required to Perform Test1
4 ml
2 ml
 Increases the likelihood of
useful sample volume
remaining for additional testing
hc2
 Providing clear results without
an equivocal (gray) zone
Indeterminate Rate
4.7%
 Reduces the indeterminate
rate to <1%, compared with
4% or more for other HPV
tests1
<1%
hc2
1Solomon
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et al, JNCI, 2001.
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Cervista™ HPV vs. hc2 ® and RCS ®
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Summary
Key Factor
Internal control
Minimum TPPT
sample vol. required
Cross-reacts to common
low-risk HPV types?
Equivocal zone for interpretation?
Maximum walk-away time
(manual process)
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CervistaTM HPV HR
Yes
Limits false negatives
hc2®
None
2 ml
4 ml
Limits QNS
More QNS
Yes
No
Types 6,11,42,43,44,53
No
Yes
Not required
(1.0 to 2.5 RLUs/CO)
4 hours
1 hour
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Methods
 DNA sequencing
 Southern Blot
 PCR
 RT-PCR
 Real Time PCR
 Methylation-Specific PCR
 In-situ PCR
 Protein Truncation Test
 Transcription-Mediated
Amplification
 Strand Displacement
Amplification
 Nucleic Acid SequenceBased Amplification
 Signal amplification
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Branching DNA
Hybrid Capture
Invader
FISH
DNA arrays and chips
In Situ Hybridization
 Probe types:
 Centromeric or CEP (chromosome enumeration probe)
 Whole chromosome probes or paints [metaphase only]
 Locus specific probe or identifier (LSI)
 Section pretreatment.
 The labeled probe is first denatured (by heating or under
alkaline conditions) into single DNA strands
 Hybridized to the target DNA (~Southern blotting) or RNA
(~northern blotting) immobilized on a membrane (blotting) or
in situ.
 Metaphase and Interphase cells
ISH
Advantages
 Interphase nuclei
 Archive material
 Can detect anomalies
hidden to other methods
 Detects polysomy,
losses, amplifications,
translocations
Disadvantages
 Only provides information
of the specific target
 Work together with classic
cytogenetics
 Minimal Residual Disease
 Do not provide allelespecific information
 Do not detect small
changes
In Situ Hybridization
 More in use Chromosomal translocations
 Useful in CMV, HSV, VZV
 Sub types of papilloma virus
 Useful in Mycobacteria, fungi and parasites
 Helicobacter pylori from gastric biopsies
 Legionella pneumophila
 Pneumocystis jiroveci
 Tests done on paraffin embedded specimen
 Need applications in Infectious diseases

Doctortvrao’s ‘e’ learning series
ISH - PNA probes
 Traditionally either cloned probes or synthesized oligonucleotide
probes have been used for hybridization.
 Peptide nucleic acid (PNA) probe, a nucleotide analogue capable of
binding to DNA/RNA in a sequence-specific manner obeying the
Watson-Crick base pairing rules.
 In PNA, the sugar phosphate backbone of DNA/RNA has been
replaced by a synthetic peptide backbone keeping the distances
between bases exactly the same as in DNA/RNA.
 Further, the PNAs are very stable molecules. Experiments have shown
virtually no degradation by DNases, RNases, proteinases or
peptidases.
 The PNA probes are labeled with fluorescein and detected using a
sensitive PNA ISH Detection Kit (colorimetric).
Clinical Uses for ISH
ISH - Examples
 Genotyping of Neoplasms
 Polysomy and other gains
 Trisomy 12 in B-CLL
 Losses
 del 1p / del 19q
 Amplification
 HER2/neu
 Translocations
 t(9,22)(q34;11) BCR/ABL in CML
ISH - Examples
 Constitutional Molecular Genetics
 Sex chromosome enumeration
 Gender
 Polysomy and other gains
 Trisomy 21 in Down’s
 Losses
 del(22q11.2) in DiGeorge syndrome
ISH - PNA probes
 EBER
 EBV lytic
 Kappa and Lambda
ISH
Urinary Cytopathology
Urovision™
 >60,000 new cases of bladder cancer
 Recurrence of Urothelial carcinomas 50-80%
 Follow up cytoscopy and urine cytology (sensitivity ~48%)
 Aneuploidy 3, 7, 17 and loss of 9p21 (CDKN2A p16)
 Sensitivity 96% in HG UCs
 Interpretation: 35 abnormal cells
 ≥4 cells with aneuploidy in ≥2 chromosomes
 Loss of 9p21 in ≥12 cells
ISH
Solid Tumors
1p / 19q - Oligodendroglioma
Dual Fusion Translocation Probe
Break Apart Translocation Probe
ISH – Solid Tumors
1p36/19q13 – Oligodendroglioma
panelIncludes:1p36/1q25 (1p36
deletion)19q13/19p13 (19q13 deletion)
Oligodendrogliomas, mixed
oligoastrocytomas
EGFR/CEP7
Colorectal, breast and non-small cell
lung carcinomas, and glioblastoma
multiforme. EGFR gene amplification
by FISH may identify tumors predicting
responsiveness to EGFR-targeted
therapies
EWSR1 (22q12) translocations
(Breakapart)
Clear cell sarcoma, Extraskeletal
myxoid chondrosarcoma, PNET/Ewing
sarcoma, Desmoplastic small round cell
tumor
ISH – Solid Tumors
HER2/CEP17
Identifies the subset of breast
carcinoma patients eligible for
Herceptin™ (trastuzumab) therapy.
MDM-2/SE12
Well-differentiated liposarcoma,
dedifferentiated liposarcoma, atypical
lipomatous tumor, and pleomorphic
lipoma
SS18 (SYT) translocations (Breakapart)
Synovial Sarcoma
TOP2A / CEP17
A predictive biomarker in a subset of
breast carcinomas. TOP2A gene
amplification may predict response to
anthracycline-containing breast
chemotherapy.
ISH - Lymphomas
MALT1 (18q21) translocations
(Breakapart)
Translocations involving the MALT1
gene have been detected in
approximately 20-30% of patients with
extranodal low grade marginal zone Bcell lymphomas of MALT type (i.e.,
MALT lymphomas). Patients with
t(11;18)(q21;q21)-positive gastric MALT
lymphomas do not respond to
Helicobacter pylori eradication therapy,
are associated with more advance stage
disease, and usually do not show
transformation to large cell lymphoma.
t(14;18) IGH/MALT1
Subset of MALT lymphomas (Marginal
zone B cell lymphoma)
t(11;18), MALT1/API2
Subset of MALT lymphomas (Marginal
zone B cell lymphoma)
ISH - Lymphomas
MYC (8q24) translocations (Breakapart) Burkitt lymphoma; MYC translocations
(MYC/IGH, MYC/kappa, MYC/lambda);
t(8;14), t(2;8), t(8;22)
t(11;14) CCND1/IGH
Identifies mantle cell lymphoma and
subset of plasma cell neoplasms.
Patients with multiple myeloma that
have a t(11;14)(q13;q32) have been
reported to have a neutral to slightly
improved clinical course. FISH-based
assays provide the most sensitive and
specific methodology for detecting the
t(11;14)(q13;q32).
ISH - Lymphomas
t(14;18) IGH/BCL2
Identifies follicular lymphoma and
subset of DLBCL with the
t(14;18)(q32;q21), which results in
constitutive overexpression of the BCL2 protein leading to alterations in
programmed cell death (i.e., apoptosis)
and tumor cell proliferation. FISHbased assays provide the most sensitive
and specific methodology for detecting
the t(14;18)(q32;q21).
ISH - Leukemia
t(9;22) BCR/ABL
CML and subset of ALL. CML has been
traditionally diagnosed by detection of a
Philadelphia chromosome (Ph) which has
become the hallmark of this disease and is the
result of a reciprocal translocation between
the BCR gene on chromosome 22 and the ABL
gene on chromosome 9. Detection of the
Philadelphia chromosome by FISH (or other
techniques) helps to confirm and/or monitor
patients with CML or other myeloproliferative
disorder. An alternate translocation involving
BCR and ABL, which is also detected by this
assay, can be seen in acute lymphoblastic
leukemia (ALL).