Somatic mutation webinar

Sample & Assay Technologies- 1 -

qBiomarker ™ Somatic Mutation PCR Arrays

Cancer Mutation Analysis Made Easy

Shankar Sellappan, Ph.DGlobal Product Manager

[email protected]


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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


What is a Somatic DNA Mutation?

� Definition� Alterations in DNA that can occur in any of the cel ls of the

body except germ cells– NOT passed on to children

� Single Point Mutation or Multiple Mutations Possibl e within a Gene


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.


�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

Physiological Implications of DNA Mutations


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.


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


What Kinds of DNA Mutations are There?

• Point Mutation

• A � G

• Frame Shift Mutation / Insertion / Duplication

• ACGTACGTC

• ACGATACGTC

• Deletion

• ACGTACGTC

• ACGACGTC

• Inversion

• ACGTACGTC

• ACTGACGTC

• ACGTACGTC

• ACGAGGTACGTC

• ACGTACGTC

• ACGTACTACGTC

From 1 – 100s of bases


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


Table of Contents






• Data Analysis

• How to Order


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 g enes in downstream signaling activities

• Example: EGFR Pathway Somatic Mutation PCR Array

• EGFR Mutations: 29 Different mutations present in t he 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


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


What is the Layout of a qB Somatic Mutation PCR Arra y?

Greek Symbol = Gene

# = Mutation

• For Normalization

• Assays detect non-variable region (not ARMS-based design)

• Designed to control differences in sample input and quality


Array Layout: Example: EGFR Pathway


qBiomarker Cancer Somatic Mutation PCR ArraysAvailable 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)

• ββββ-Catenin/CTNNB1-APC pathway (colon, prostate, uterine cancers)


Table of Contents






• Data Analysis

• How to Order


Analysis Methods for DNA Mutations

SensitivityProtocol complexNeed Instrument/Training

Large CoverageMicroarray

Time ConsumingNeed Instrument/Training

ExactMass Spectrometry

Base change identity not obvious

InexpensiveFastSimple

High Resolution Melt

Time ConsumingNeed Instrument/Training

ExactNext-Generation Sequencing

FastReliableHigh-throughput

Quantify DNA Targets� Real-time PCR

Time ConsumingExactPoint Mutation Analysis

DrawbacksBenefitsMethod


Sensitivity of Mutation ID Technologies / Principle s

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


qBiomarker Somatic Mutation Assay Detection Limit

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%.

WGA = Whole Genome Amplification


Table of Contents






• Data Analysis

• How to Order


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


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


Table of Contents






• Data Analysis

• How to Order


How do qBiomarker Somatic Mutation Assays Work?

. Hydrolysis Probe Assays + ARMS


. 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.

Definition: Hydrolysis Probes & Assays


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 FUNCTIONas Primers in a PCR Reaction

� But with the 3’-residue matching at the exact spot of the mutation


Primer Elongation & Probe Activation with ARMS ®

F Q

F Q

Common Region


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


Allele Specific Amplification with ARMS ®

ARMS: A Closer Look

AGTCAGTCTemplate: Wild-TypeMutation Locus:

C is correct for WT

TCAGTAAAARMS Mutation Primer

}Won’t WorkARMS Mutation

Add’l Mutation: Location Varies

AGTCAGTTTemplate: MutationTCAGTAAAARMS Mutation Primer

Mutation Locus:

C is correct for WT

}Will Work

Why this works: Internal Mismatches: Enzyme Requirement Less Stringent


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


Mutations

FFPE Samples

KRAS∆Ct

Notice how each FFPE sample harbors only 1 or two mutations

�� So we are NOT looking for spikes everywhere

qBiomarker Somatic Mutation ArrayDetection of BRAF, EGFR, and KRAS Mutations

• Samples: Asian, Female, non-smoker origin; Lung Adenocarcinoma

• Expected: 30% Mutation in EGFR

BRAF

EGFR


qBiomarker Somatic Mutation Assay ValidationPyrosequencing Confirmation of KRAS & EGFR Mutations


Table of Contents






• Data Analysis

• How to Order


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


qBiomarker Somatic Mutation PCR Array Data AnalysisAverage C t 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.

∴∴∴∴Ctaverage

= Mutation Assay Background in Wild-Type Sample

�Compare C tsample

to C taverage

= ∆Ct

� If Ctsample

<< Ctaverage

�� Sample has Mutation


Average C t 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%


Average C t: Example Data: Collaboration: ββββ-TestMutation Profiling of FFPE Samples on EGFR PCR Arra y

Conclusion of the ββββ-testThe 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.

∆Ct

Mutation

Cancer C

ell Lines


� Tested 10 FFPE adenocarcinoma samples

� Expect high % (>30%) EGFR activating mutations (point mutation & deletions)

� Paez et al. Science. (2004) 304:1497-500

Ad

enocaecrinoma

Mutation

∆Ct

Average C t: Example Data: In-House DataMutation Profiling of FFPE Samples on EGFR PCR Arra y


qBiomarker Somatic Mutation PCR Array Data Analysis∆∆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)


qBiomarker Somatic Mutation PCR Array Data Analysis∆∆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 – Ct

CNA

27

28

CNA

3

12

∆Ct

30Mutant

40WT

Mutation Assay

�� ∆∆Ct = 9


∆∆∆∆∆∆∆∆Ct Method: ExampleEGFR Pathway: Cancer Cell Lines (200 ng gDNA)

Mutation

Can

cer

Cel

l Lin

es


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

qBiomarker Somatic Mutation PCR Array Data AnalysisTwo Methods: ∆∆Ct vs Average C t: How to Choose


Data Analysis: Excel Files: Pathway-Specific


Table of Contents






• Data Analysis

• How to Order


qBiomarker Somatic Mutation: Product Availability

337021: qBiomarker Somatic Mutation PCR Arrays

337011: qBiomarker Somatic Mutation PCR Mutation Assays100 reactions

•NOTE: Corresponding qBiomarker Probe Master Mixes are included with ALL ORDERS.

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


E-Learning Center:Recorded Seminars

Additional Resources


qBiomarker™ Somatic Mutation PCR Arrays & Assays

Shankar Sellappan, Ph.D.Global Product Manager

[email protected]

Somatic mutation webinar

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