Somatic mutation webinar

qBiomarker™ Somatic Mutation PCR Arrays

Cancer Mutation Analysis Made Easy

Shankar Sellappan, Ph.D
Global Product Manager
Shankar.Sellappan@QIAGEN.com

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Sample & Assay Technologies

Legal Disclaimer

• SABiosciences products are intended for molecular biology applications.
These products are not intended for the diagnosis, prevention, or treatment
of a disease.
• For up-to-date licensing information and product-specific disclaimers, see
the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks
and user manuals are available at www.QIAGEN.com or can be requested
from QIAGEN Technical Services or your local distributor.

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Welcome to SABiosciences: Who We Are

• SABiosciences is now a

Company
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Table of Contents

• DNA Mutations Overview
• What is a qBiomarker Somatic Mutation PCR Array?
How to Analyze Samples for DNA Mutations
• Workflow for qBiomarker Somatic Mutation PCR Arrays
• How qBiomarker Somatic Mutation PCR Assays Work
• Data Analysis
• How to Order

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What is a Somatic DNA Mutation?
Definition
Alterations in DNA that can occur in any of the cells of the
body except germ cells
– NOT passed on to children
Single Point Mutation or Multiple Mutations Possible within
a Gene

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Somatic Mutations vs Single Nucleotide Polymorphisms (SNPs)

• Somatic Mutations occur in cells that are NOT passed down
to children
• SNPs occur in Germline cells (that ARE passed down)
• What is important in SNP analysis is the Allele Frequency
• Which means how often it occurs

Note: qBiomarker Somatic Mutation PCR Arrays & Assays
only show the Presence of a Mutation
Qualitative, not Quantitative Information
Quantitative may come in future.
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Physiological Implications of DNA Mutations

Changes in a single or multiple nucleotides within
a gene, or across related genes in a pathway,
may alter the resulting protein, either by activity
level or functional ability, resulting in:
Cancers
Cardiovascular diseases (Arrhythmia)
Blood/hematologic disorders

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Implications of DNA Mutations in Drug Development

Drug compounds may behave differently in a
similar profile of sample, based on Somatic
Mutation profile.
Ex. 30% of Breast Cancers overexpress HER2.
–However, not all respond to HER2 therapy
(i.e. Herceptin®)
• This may indicate a different HER2
mutation occurring in samples, thereby
yielding different outcomes.

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How do DNA Mutations Occur?
• Copying Errors
• as part of Replication Process
• Estimated Error Rate of DNA Polymerase: 1 in 108 bases
• Human Genome = 2.9 billion bases (2.9 x 109 bases)
• DNA Damage
• Resulting from Environmental Agents, such as:
• Sunlight (UV)
• Cigarette Smoke
• Radiation

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What Kinds of DNA Mutations are There?

• Point Mutation
•A

G

• Frame Shift Mutation / Insertion / Duplication
• ACGTACGTC
• ACGATACGTC

• ACGTACGTC

• ACGTACGTC

• ACGAGGTACGTC

• ACGTACTACGTC

• Deletion
• ACGTACGTC

From 1 – 100s of bases

• ACGACGTC
• Inversion
• ACGTACGTC
• ACTGACGTC

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How many DNA Mutations have been identified?
• ~ 140,000 DNA Mutations Identified
• Most DNA Mutations NOT Functionally Relevant at the Protein Level
• Multiple Consortiums
• COSMIC: Catalog of Somatic Mutations in Cancer: Sanger Institute
• HGMD: The Human Gene Mutation Database at the Institute of Medical
Genetics in Cardiff
• dbRBC: Blood Group Antigen Gene Mutation Database: NCBI

• EXPECTATIONS

IMPORTANT POINT TO REMEMBER

• Cells under study may contain one to a few mutations as assayed for on a
pathway-focused qBiomarker Somatic Mutation PCR Array

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Table of Contents

• Data Analysis
• How to Order

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What is a Somatic Mutation PCR Array?
Somatic Mutation PCR Array
• Definition: Collection of mutation specific assays for multiple mutations
in a single gene, as well as mutations present in genes in downstream
signaling activities
• Example: EGFR Pathway Somatic Mutation PCR Array
• EGFR Mutations: 29 Different mutations present in the EGFR gene
• AKT1 Mutations: 1
• BRAF Mutations: 12
• KRAS Mutations: 18
• HRAS Mutations: 11
• NRAS Mutations: 12
• MEK1 Mutations: 4
• PI3K Mutations: 7
• PTEN Mutations: 6
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Why Study Somatic Mutations in a Pathway Array?
EGFR Mutations
AKT1 Mutations
BRAF Mutations
KRAS Mutations
HRAS Mutations
NRAS Mutations
MEK1 Mutations
PI3K Mutations
PTEN Mutations

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What is the Layout of a qB Somatic Mutation PCR Array?

Greek Symbol = Gene
# = Mutation

• For Normalization
• Assays detect nonvariable region (not ARMSbased design)
• Designed to control
differences in sample input
and quality
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Array Layout: Example: EGFR Pathway

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qBiomarker Cancer Somatic Mutation PCR Arrays
Available Pathways

• EGFR pathway (lung cancer, colorectal cancer, glioblastoma multiforme)
• ERBB2 (HER-2/neu) pathway (breast cancer, lung, gastric cancers)
• PDGFR pathway (GIST, glioblastoma, prostate cancer)
• FLT3 pathway (acute meyloid leukemia (AML))
• RTK I & II (96-well) or RTK-HT (384-well)
• KIT pathway (leukemia, gastrointestinal stromal, testicular germ cell tumors)
• MET pathway (kidney, liver, stomach, breast and brain cancers)
• FGFR pathway (leukemia and multiple myeloma)
• Upcoming:
•PI3K pathway (one of the most commonly activated pathways in cancer)
• P53-Rb pathways (associated with resistance to multiple chemo-therapeutic drugs)
• β-Catenin/CTNNB1-APC pathway (colon, prostate, uterine cancers)
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Table of Contents

• Data Analysis
• How to Order

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Analysis Methods for DNA Mutations

Method

Benefits

Drawbacks

Point Mutation Analysis

Exact

Time Consuming

Quantify DNA Targets
Real-time PCR

Fast
Reliable
High-throughput

Mass Spectrometry

Exact

Time Consuming
Need Instrument/Training

High Resolution Melt

Inexpensive
Fast
Simple

Base change identity not
obvious

Microarray

Large Coverage

Sensitivity
Protocol complex

Next-Generation
Sequencing

Exact

Time Consuming

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Sensitivity of Mutation ID Technologies / Principles

Measurement of Sensitivity:
In a population of 100 total cells, sensitivity of:
• RT-PCR (PCR Arrays): 1-2%
• 1-2 mutant DNA + 98-99 wild-type DNA
• Pyromark Sequencing: 5%
• 5 mutant DNA + 95 wild-type DNA
• Method of DNA sequencing based on the “sequencing by
synthesis principle”
• Sequenom/Microarray: 10%
• 10 mutant DNA + 90 wild-type DNA

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qBiomarker Somatic Mutation Assay Detection Limit

WGA = Whole
Genome Amplification

Assay detection limit test for qBiomarker Braf V600E assay. A series of 10ng
genomic DNA samples, which contain genomic DNA from A375 cell line mixed with WT
genomic DNA at different ratios, were tested on qBiomarker Somatic Mutation Assay for
Braf V600E with or without whole genome amplification. Mutation detection limit for this
assay is determined to be ~1%.

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Table of Contents

• Data Analysis
• How to Order

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DNA Sample Requirement

• If less than 5 ng DNA available from Fresh frozen samples, Whole
Genome Amplification recommended.

NOTE: WGA NOT recommended for FFPE samples

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How do qBiomarker Somatic Mutation Arrays Work?
Fresh/frozen samples: 200 – 500 ng gDNA
FFPE samples: 200 – 500 ng gDNA

Fresh/frozen samples: 5 – 10 ng gDNA

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Table of Contents

• Data Analysis
• How to Order

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How do qBiomarker Somatic Mutation Assays Work?

.

Hydrolysis Probe Assays + ARMS

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Definition: Hydrolysis Probes & Assays

.

Relies on the 5’ – 3’ exonuclease activity of the Taq activity of Taq Polymerase
The Exonuclease Activity degrades a hybridized non-extendable DNA probe during
the extension step of the PCR.
DNA Probe: Hybridizes to a region within the amplicon and is dual-labeled with a
reporter dye and quenching dye.

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Definition: Allele Specific Amplification with ARMS®

ARMS: Amplification Refractory Mutation System
Strategy in which Primers Discriminate Among Templates
Differing by a Single Nucleotide
Oligos with a mismatched 3’-residue WILL NOT FUNCTION
as Primers in a PCR Reaction
But with the 3’-residue matching at the exact spot of the
mutation

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Primer Elongation & Probe Activation with ARMS®

Common Region

F

F

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Q

Q


Key to qBiomarker Somatic Mutation Assay Design
Additional mismatch used
Mutation Position & Additional Mutation
– 1-3 bp near mutation site (location is proprietary)
Increases specificity

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Allele Specific Amplification with ARMS®
ARMS: A Closer Look

}

Will Work
ARMS Mutation Primer
Template: Mutation

TCAGTAAA
AGTCAGTT

Mutation Locus:
C is correct for WT

Why this works: Internal Mismatches: Enzyme Requirement Less Stringent

}

Won’t Work

Add’l Mutation:
Location Varies

ARMS Mutation Primer
Template: Wild-Type

TCAGTAAA
AGTCAGTC

ARMS Mutation

Mutation Locus:
C is correct for WT
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Why ARMS + Hydrolysis Probe?
Why not just Hydrolysis Probe?

Hydrolysis Probes
Measures just target strand abundance
ARMS (Amplification Refractory Mutation System)
Preferentially amplifies the mutant alleles
– We are trying to detect as low as 1-2% somatic
mutation in a wild-type DNA background
- ARMS – designed primers will preferentially
amplify the mutant sequence

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qBiomarker Somatic Mutation Array
Detection of BRAF, EGFR, and KRAS Mutations
• Samples: Asian, Female, non-smoker origin; Lung Adenocarcinoma
• Expected: 30% Mutation in EGFR
EGFR

BRAF
KRAS

∆Ct

FFPE
Samples

Mutations
Notice how each FFPE sample harbors only 1 or two mutations
So we are NOT looking for spikes everywhere
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qBiomarker Somatic Mutation Assay Validation
Pyrosequencing Confirmation of KRAS & EGFR Mutations

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Table of Contents

• Data Analysis
• How to Order

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Somatic Mutation PCR Array Data Analysis
• Purpose: Qualitative assessment of samples
• Is the DNA Mutation Present and if so, which one?

• Data Analysis Methods depends on:
• 1. Availability of wild-type sample
• 2. Sample type/quality
• Cell lines
• Fresh or frozen samples
• FFPE samples
• 3. How many samples?

• NOTE: Compare Presence of Same Mutant Assay Across Samples
• NOTE: Each sample harbors only a single or at most few mutations
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qBiomarker Somatic Mutation PCR Array Data Analysis
Average Ct Method
Recommended for experiments using:
• FFPE samples
• Large #s of samples (> 4)
• Wide range of sample quality
• Wild-type controls not necessary
• Assumes that for a given locus, mutation occurs in small % of samples.
average

∴Ct

= Mutation Assay Background in Wild-Type Sample
sample

Compare Ct

sample

If Ct

average

to Ct

<< Ct

average

= ∆Ct
Sample has Mutation

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Average Ct method examples

.

FFPE cell line samples
Lung Cancer & Colon Cancer cell lines
MDA-MB-231 as a positive control
G464V Braf and G13D Kras mutation status gathered from
COSMIC database and confirmed by:
– DxS Kras PCR test
– Braf Pyrosequencing assay

.

9 FFPE lung cancer samples, 1 placenta FFPE sample
Sample origin
Adenocarcinoma
Expected EGFR mutation rate: ~10%

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Average Ct: Example Data: Collaboration: β-Test
Mutation Profiling of FFPE Samples on EGFR PCR Array

l
Cel
cer
Can

∆Ct

es
L in

Conclusion of the β-test
The two known mutations in MDA-MB-231 cell line FFPE sample were correctly called:
2 Kras and 1 EGFR mutation (~10%) called in 3 lung cancer FFPE samples.
Mutation

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Average Ct: Example Data: In-House Data
Mutation Profiling of FFPE Samples on EGFR PCR Array
Tested 10 FFPE adenocarcinoma samples
Expect high % (>30%) EGFR activating mutations (point mutation & deletions)
Paez et al. Science. (2004) 304:1497-500

c
ocae
Aden

∆Ct

a
rinom

Mutation

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∆∆Ct Method
Recommended for experiments using:
• Small #s of samples (< 4)
• Basic Cell Line Experiments….Looking for Mutation in just a couple of cell line
samples
• Results affected by sample quality (Wild-type and Test Samples)
• Wild-type controls NECESSARY
• Wild-type controls MUST be of high-quality
• Considers Copy # Changes
• NORMALIZES (∆Ct)
• Caveats
• Penalizes good quality samples (False Negatives)
• Rewards bad quality samples (False Positives)

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∆∆Ct Method

* A raw Ct cutoff for Ct
(sample locus X) will
be applied (usually
35); If sample Ct >
cutoff Ct, the sample
will NOT be called
“mutant” for that locus

Normalized Ct For Allele/Mutation Site = ∆Ct = CtMUT – CtCNA
Mutation Assay

CNA

∆Ct

WT

40

28

12

Mutant

30

27

3

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∆∆Ct = 9


Canc
er

Cell L
ines

∆∆C
∆∆ t Method: Example
EGFR Pathway: Cancer Cell Lines (200 ng gDNA)

Mutation

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Two Methods: ∆∆Ct vs Average Ct: How to Choose
Data Analysis Method Selection Summary

• ∆∆Ct method
• Must have wild-type control
• Works well for projects using:
• Fresh/frozen samples
• Smaller (≤4) number of samples
• i.e. sample quality similar

• Average Ct method
• Works well for projects using:
• FFPE samples
• Large number of samples
• i.e. sample quality can be in a wide range
• Without Wild-type controls

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Data Analysis: Excel Files: Pathway-Specific

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Table of Contents

• Data Analysis
• How to Order

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qBiomarker Somatic Mutation: Product Availability
337021: qBiomarker Somatic Mutation PCR Arrays

• Please visit http://sabiosciences.com/somaticmutationarray.php for the most up-todate list of Pathway/Disease Focused Somatic Mutation PCR Arrays

337011: qBiomarker Somatic Mutation PCR Mutation Assays
100 reactions
•NOTE: Corresponding qBiomarker Probe Master Mixes are included with ALL ORDERS.
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Additional Resources

E-Learning Center:
Recorded Seminars
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qBiomarker™ Somatic Mutation PCR Arrays & Assays

Shankar Sellappan, Ph.D.
Global Product Manager
Shankar.Sellappan@QIAGEN.com

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