Mehmet Gunduz, MD, PhD

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PRACTICE OF GENE ANALYSIS IN HUMAN CANCER
Mehmet Gunduz, MD, PhD Department of Oral
Pathology and Medicine Graduate School of
Medicine, Dentistry and Pharmaceutical Sciences,
Okayama University
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The Human Genome

• 25,000 Genes
• ?? 200,000 Proteins
• a) alternative mRNA splicing
• post-translational modifications (2-7 millions?
  )
• -Phosphorylation
• -Acetylation
• -Sumolation
• -Ubiquitination

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Basic Gene Structure
Promoter
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The Role of Oncogene and Tumor Suppressor Gene
(TSG) in Cancer
Oncogene
TSG
Normal Cell
Loss of the balance
Cancer Cell
x
x
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Methods Commonly Used in Gene Analysis of Cancer

• Polymerase Chain Reaction (PCR)
• Real-Time PCR
• Loss of Heterozygosity (LOH)
• Single Strand Confirmation Polymorphism (SSCP)
• Sequencing

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Polymerase Chain Reaction(PCR)

• Target site amplification (ability to rapidly and
  exponentially amplify a particular DNA sequence)
• Rapid
• Sensitive
• Specific

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Some Facts about PCR

• 300,000,000 US were paid for the patent (1991)
• Nobel Prize 1993
• Included in the title or abstract of more than
  100,000 (!) scientific publications.
• Real-time PCR is becoming the most common use of
  PCR

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Components of the PCR
AGATCGCTTA
CGCTTCAAAG
Primer Antisense
Primer Sense
Template DNA
T
C
DNA polymerase (Thermostable)
T
C
A
A
Taq
G
dNTPs
G
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Thermocycling Profile
100 80 60 40 20
Denature 94C
Temp C
Extend 72 C
Anneal 55 C
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Analysis of PCR Products
-
M 1 2 3 4

Gel Electrophoresis

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RT-PCR Analysis RNA(cDNA) is used as template
instead of genomic DNA to examine mRNA expression
of a specific gene
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RT-PCR
Gunduz et al. Oncogene 2002
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Multiplex RT-PCR
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ING3
Actin
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Real-Time PCR Real-time PCR monitors the
fluorescence emitted during the reaction as an
indicator of amplicon production at each PCR
cycle (in real time) as opposed to the endpoint
detection
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BioRad iCycler
Roche LightTyper LightCycler
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The five-fold dilution series seems to plateau at
the same place even though the exponential phase
clearly shows a difference between the points
along the dilution series. This reinforces the
fact that if measurements were taken at the
plateau phase, the data would not truly represent
the initial amounts of starting target material.
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• Real-time PCR advantage
• not influenced by non-specific amplification
• amplification can be monitored real-time
• no post-PCR processing of products
• ultra-rapid cycling (30 minutes to 2 hours)
• wider dynamic range of up to 1010-fold
• requirement less RNA than conventional assays
• detection is capable down to a 2-fold change
• confirmation of specific amplification by melting
  point analysis
• most specific, sensitive and reproducible

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Real-Time RT-PCR for ING4 mRNA Expression Analysis
Gunduz et al. Gene 2005
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Gunduz et al. Gene 2005
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Quantitation by RT-PCR
Conventional
Real-time
RNA extraction
RNA extraction
Reverse Transcription
Reverse Transcription
cDNA to PCR rx
cDNA to PCR rx
PCR reaction
Electrophoresis/ Dot Blots/imaging
Image analysis
Real-time PCR
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Microarrays
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Mechanism of Metastasis
Highly metastatic primary tumor
Metastasis
Gene signature of metastasis
Gene related with metastasis
Gene related with resistance to metastasis
Non-metastatic primary tumor
No metastasis
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mRNA expression in the primary tumors with
metastatic lymph nodes
mRNA expression in the primary tumors without
metastatic lymph nodes
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mRNA expression in the primary tumor and their
metastatic foci
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LOH in candidate metastasis suppressors
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Possible Future Clinical Application
Advanced cancer
Current method
vessel
ELISA Low sensitivity
Detection of cancer antigens such as Scc, CEA,
AFP by ELISA
No gross metastasis (early stage)
Future method
vessel
Genetic Daignosis High sensitivity
Highly sensitive molecular diagnosis
Cancer cell
Free DNA
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Inactivation Mechanism of Tumor Suppressor Gene
2nd hit (mutation or decreased mRNA expression)
1st hit (LOH, deletion)
Normal
Susceptible carrier
Cancer
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LOH at 19p13 (BRG1 Location)
Gunduz et al. Int J Oncol 2005
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LOH at 13q34 (ING1 Location)
Gunduz et al. Cancer Res 2000
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LOH at 7q31 (ING3 Location)
Gunduz et al. Oncogene 2002
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LOH at 12p13 (ING4 Location)
Gunduz et al. Gene 2005
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LOH with Gene-Scan Method
Beder and Gunduz et al. J Cancer Res and Clin
Oncol 2005
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Beder and Gunduz et al. Lab Invest 2003
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Beder and Gunduz et al. Lab Invest 2003
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Genome-Wide LOH Analysis
Beder and Gunduz et al. Lab Invest 2003
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Chromosome LOH Gene
17p13 9p21 13q14 5q21 13q34 7q31 12p13 19p1
3
88 86 60 54 68 48 66 64
P53 P16 Rb APC ING1 ING3 ING4 BRG1
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SSCP-DNA Sequencing

• SSCP single strand conformation polymorphism
• sequencing nucleotide sequences of DNA or RNA
• - direct sequencing of genomic DNA (PCR)
• or by cloning such regions and sequencing from
  the cloned fragment

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SSCP-Sequencing Analysis
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SSCP Analysis
Gunduz et al. Cancer Res 2000
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P53 exon 8 SSCP
Shifted Band
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P53 exon 8, Codon 285 GAG AAG (Glu
Lys)
Normal
Mutant
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• Mutation types
• 1. Missense
• 2. Nonsense
• 3. Frameshift (deletion or insertion)

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missense mutation- a mutation that results in a
change (substitution) of one aa for another
A
A
Reading frame 1 ATT- ile mutation--ATG- met
To determine whether it is a missense mutation,
we need to know the frame (shown in green)
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A
A
Reading frame 2 TTA- Leu mutation--TGA-
stop nonsense mutation-mutation that generates a
stop
Nonsense mutations usually lead to premature
translation termination resulting in a shortened
protein.
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Frame-shift mutation
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His-Ser-Pro-Val-Pro
His-Ala-Pro-Val-Pro
His-His-Leu-Tyr-Gln
His-Val-Thr-Cys-Thr
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Detection of Polymorphism by SSCP P53 exon 4,
Codon 72, ccc cgc (Pro Arg)
Patterns 1 2 3
Arg
Pro
Arg/Pro
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Hetero
P53 exon 4, Codon 72, ccc cgc (Pro Arg)
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RFLP Method
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p21 polymorphism
Codon 31 (Arg)
p21 pol S
CTGCCGT GACGGCA
p21 polymorphism C/C
P21 pol AS
300 bp
Codon 31 (Ser)
126 bp
C TGCA GT G ACGT CA
p21 polymorphism A/A
p21 pol AS
174 bp
300 bp
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Result 1 p21 Codon 31 SNP
Size marker
Heterozygote Polymorphism (C/A)
Homozygote Polymorphism (A/A)
Homozygote Polymorphism (C/C)
300 bp
300 bp
174 bp
174 bp
126 bp
126 bp
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p16 polymorphism
p16 polymorphism C/C
p16 polymorphism G/G
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Result 2 p16 codon570 SNP
Size marker
Homozygote Polymorphism (C/C)
Heterozygote Polymorphism (C/G)
Homozygote Polymorphism (G/G)
144 bp
144 bp
109 bp
109 bp
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THANK YOU
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After gel check sequencing PCR was done using the
following protocol